Patent Publication Number: US-2022220502-A1

Title: Virus compositions with enhanced specificity in the brain

Description:
RELATED APPLICATIONS 
     This Application claims the benefit of and priority to U.S. Provisional Patent Application Ser. No. 62/832,836, filed Apr. 11, 2019, the content of which is incorporated herein in its entirety. 
    
    
     STATEMENT AS TO FEDERALLY SPONSORED RESEARCH 
     This invention was made with government support under NS087949, MH117069, and OD025535 awarded by National Institutes of Health. The government has certain rights in the invention. 
    
    
     SUMMARY 
     Recombinant adeno-associated viruses (rAAVs) are widely used as vectors for gene delivery in basic scientific research and therapeutic applications because of their ability to transduce both dividing and non-dividing cells, their long-term persistence as episomal DNA in infected cells, and their low immunogenicity. These characteristics make them appealing for applications in both basic science and in clinics, such as gene therapy. However, there is a need to significantly improve the performance of existing serotypes to specifically target distinct brain cell-types, upon systemic delivery to a subject. This need is especially acute when the AAV must across the blood brain barrier (BBB) to reach the central nervous system (CNS). 
     Systemic delivery of existing AAV serotypes show limited transduction of certain cell types and organs, and non-specific, overlapping tropisms in others. This leads to several complications in gene therapy applications, including but not limited to off-target effects due to transduction of unimpacted organs and cell types (in particular, the liver), and the necessity for a larger viral dosage to achieve sufficient therapeutic levels in the tissue or organ of interest. 
     Disclosed herein are rAAVs with engineered specificity into the capsid structure through iterative rounds of positive and negative selection, yielding variants with tropisms having an increased specificity and transduction efficiency when measured in the CNS, and in some cases, a decreased specificity and transduction efficiency in an off-target environment, like the liver. The rAAVs described herein achieve widespread transduction to target environment (e.g., target cell types or tissues) in a subject upon systemic delivery (e.g., intravenous injection). 
     Also provided are methods of engineering the rAAVs of the present disclose using a multiplexed Cre recombination-based AAV targeted evolution (M-CREATE) method. The M-CREATE method generates enhanced transduction efficiency and/or specificity by (1) introducing variations in the capsid protein sequence, (2) in vivo unbiased selection and recovery of only those variants that travel to defined cell populations, (3) cross the cell membrane, (4) travel to the nucleus, and (5) unpackage and express their genetic dosage amount. Variant capsids exhibiting the most desirable tropism (e.g., enhanced efficiency and specificity for a particular in vivo environment) are recovered and identified by deep sequencing. Strategies for unbiased selection and analysis include determining variants&#39; enrichment score (by normalizing the target tissue library to starting virus library) and unbiased propagation between rounds of selections through a synthetic library construction (where each variant is represented equally). Also disclosed are detailed characterizations of the resultant libraries from sequencing data which provide useful insights on the selection of variants towards a target. 
     Disclosed herein are AAV capsid libraries generated using M-CREATE. The first library, a 7-mer peptide insertion in AAV9 (7-mer-i library) was built for parallel in vivo selections across different brain cell types—endothelial cells, neurons, and astrocytes —and yielded a large pool of AAV9 variants with enhanced ability to target as well as cross the blood-brain barrier (BBB) and broadly transduce the central nervous system (CNS). The second library, a 3-mer peptide substitution in AAV-PHP.B β-mer-s library), was reinvestigated by incorporating deep sequencing to recover capsids. A pool of AAV-PHP.B variants were discovered including a variant that transduce CNS neurons with greater specificity. The rAAVs of the present disclosure can efficiently target the endothelial cells of the blood-brain barrier, variants that can broadly transduce different cell types in the central nervous system, and a variant exhibiting greater specificity towards transducing neuron. 
     Aspects disclosed herein provide AAV capsids comprising: (a) an AAV capsid protein comprising: (i) a first amino acid sequence that is at least 98% identical to amino acid 217 to amino acid 736 of SEQ ID NO: 1; and (ii) a second amino acid sequence at least 57.1% identical to an amino acid sequence provided in Tables 2-3 or  FIG. 33  inserted at an amino acid position 588_589 within SEQ ID NO: 1, wherein the AAV capsid protein is characterized by at least one of an increased specificity and an increased transduction efficiency when measured in a central nervous system (CNS) in a subject when delivered to the subject systemically, relative to a native AAV capsid protein provided in SEQ ID NO: 1. In some embodiments, the second amino acid sequence is at least 71.4% identical to the amino acid sequence provided in Tables 2-3 or  FIG. 33 . In some embodiments, the second amino acid sequence is at least 86.7% identical to the amino acid sequence provided in Tables 2-3 or  FIG. 33 . In some embodiments, the second amino acid sequence is selected from the group consisting of TALKPFL, TTLKPFL, TLQIPFK, TMQKPFI, SIERPFK, RYQGDSV, and TTLKPFS. In some embodiments, the AAV capsid protein is present in VP1, VP2, and VP3 of the AAV capsid. In some embodiments, the AAV capsid is chimeric. In some embodiments, 60 copies of the AAV capsid protein are assembled into the AAV capsid. In some embodiments, the CNS comprises a cell-type selected from the group consisting of a neuron, an oligodendrocyte, an astrocyte, and a brain vascular cell. In some embodiments, the CNS comprises a tissue that is selected from the group consisting of a brain, a thalamus, a cortex, a striatum, a ventral midbrain, and a spinal cord. In some embodiments, the AAV capsid protein further comprises an amino acid substitution A587D. In some embodiments, the AAV capsid protein further comprises an amino acid substitution Q588G. In some embodiments, the AAV capsid protein further comprises an amino acid substitution comprising A589N. In some embodiments, the AAV capsid protein further comprises an amino acid substitution comprising Q590P. In some embodiments, the second amino acid sequence at the amino acid position 588_589 within SEQ ID NO: 1 is not TLAVPFK, KFPVALT, SVSKPFL, FTLTTPK, MNATKNV, NGGTSSS, TRTNPEA, or YTLSQGW. In some embodiments, the AAV capsid is isolated and purified. In some embodiments, the AAV capsid is formulated as a pharmaceutical formulation for intravenous administration to treat a disease or a condition of the liver, the pharmaceutical formulation further comprising a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical formulation further comprises a therapeutic agent. 
     Aspects disclosed herein provide AAV capsids comprising an AAV capsid protein comprising a seven amino acid insertion (X1 X2 X3 X4 X5 X6 X7) between amino acid 588 and amino acid 589 in an amino acid sequence of the AAV capsid protein provided in SEQ ID NO: 1, wherein X1 is an amino acid selected from the group consisting of E, D, G, R, S and T. In some embodiments, X2 is an amino acid selected from the group consisting of A, G, I, L, M, N, Q, T, and Y. In some embodiments, X3 is an amino acid selected from the group consisting of E, K, L, T, and Q. In some embodiments, X4 is an amino acid selected from the group consisting of G, I, K, L, R, T, and V. In some embodiments, X5 is an amino acid selected from the group consisting of A, D, G, P, L, Q, and V. In some embodiments, X6 is an amino acid selected from the group consisting of F, K, N, P, Q, S, and V. In some embodiments, X7 is an amino acid selected from the group consisting of I, K, L, P, and V. In some embodiments, the seven amino acid insertion is selected from the group consisting of TALKPFL, TTLKPFL, TLQIPFK, TMQKPFI, SIERPFK, RYQGDSV, and TTLKPFS. In some embodiments, the AAV capsid protein is present in VP1, VP2, and VP3 of the AAV capsid. In some embodiments, the AAV capsid is chimeric. In some embodiments, 60 copies of the AAV capsid protein are assembled into the AAV capsid. In some embodiments, the AAV capsid protein is characterized by at least one of an increased specificity and an increased transduction efficiency when measured in a central nervous system (CNS) in a subject when delivered to the subject systemically, relative to a native AAV capsid protein provided in SEQ ID NO: 1. In some embodiments, the CNS comprises a cell-type selected from the group consisting of a neuron, a glial cell, a oligodendrocyte, an ependymal cell, an astrocyte, a Schwann cell, a satellite cell, and an enteric glial cell. In some embodiments, the CNS comprises a tissue that is selected from the group consisting of a brain, a thalamus, a cortex, a striatum, a ventral midbrain, and a spinal cord. In some embodiments, the AAV capsid protein further comprises an amino acid substitution comprising A587D. In some embodiments, the AAV capsid protein further comprises an amino acid substitution comprising Q588G. In some embodiments, the AAV capsid protein further comprises an amino acid substitution comprising A589N. In some embodiments, the AAV capsid protein further comprises an amino acid substitution comprising Q590P. In some embodiments, the seven amino acid insertion is not TLAVPFK, KFPVALT, SVSKPFL, FTLTTPK, MNATKNV, NGGTSSS, TRTNPEA, or YTLSQGW. In some embodiments, the AAV capsid is isolated and purified. In some embodiments, the AAV capsid is formulated as a pharmaceutical formulation for intravenous administration to treat a disease or a condition of the liver, the pharmaceutical formulation further comprising a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical formulation further comprises a therapeutic agent. 
     Aspects provided herein provide AAV capsids comprising: (a) an AAV capsid protein comprising: (i) a first amino acid sequence that is at least 98% identical to amino acid 217 to amino acid 736 of SEQ ID NO: 1; and (ii) a second amino acid sequence at least 57.1% identical to an amino acid sequence provided in Table 4 or  FIG. 35  at an amino acid position 588_589 within SEQ ID NO: 1, wherein the AAV capsid protein is characterized by at least one of an increased specificity and an increased transduction efficiency when measured in a liver in a subject when delivered to the subject systemically, relative to a native AAV capsid protein provided in SEQ ID NO: 1. In some embodiments, the second amino acid sequence is at least 71.4% identical to the amino acid sequence provided in Table 4 or  FIG. 35 . In some embodiments, the second amino acid sequence is at least 86.7% identical to the amino acid sequence provided in Table 4 or  FIG. 35 . In some embodiments, the second amino acid sequence is selected from the group consisting of KAYSVQV, PSGSARS, and RTANALG. In some embodiments, the AAV capsid protein is present in VP1, VP2, and VP3 of the AAV capsid. In some embodiments, the AAV capsid is chimeric. In some embodiments, 60 copies of the AAV capsid protein are assembled into the AAV capsid. In some embodiments, the AAV capsid is isolated and purified. In some embodiments, the AAV capsid is formulated as a pharmaceutical formulation for intravenous administration to treat a disease or a condition of the liver, the pharmaceutical formulation further comprising a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical formulation further comprises a therapeutic agent. 
     Aspects disclosed herein provide AAV capsid proteins comprising: (i) a first amino acid sequence that is at least 98% identical to amino acid 217 to amino acid 736 of SEQ ID NO: 1; and (ii) a second amino acid sequence at least 57.1% identical to an amino acid sequence provided in Tables 2-3 or  FIG. 33  inserted at an amino acid position 588_589 within SEQ ID NO: 1, wherein the AAV capsid protein is characterized by at least one of an increased specificity and an increased transduction efficiency when measured in a central nervous system (CNS) in a subject when delivered to the subject systemically, relative to a native AAV capsid protein provided in SEQ ID NO: 1. In some embodiments, the second amino acid sequence is at least 71.4% identical to the amino acid sequence provided in Tables 2-3 or  FIG. 33 . In some embodiments, the second amino acid sequence is at least 86.7% identical to the amino acid sequence provided in Tables 2-3 or  FIG. 33 . In some embodiments, the second amino acid sequence is selected from the group consisting of TALKPFL, TTLKPFL, TLQIPFK, TMQKPFI, SIERPFK, RYQGDSV, and TTLKPFS. In some embodiments, the AAV capsid protein is present in VP1, VP2, and VP3 of an AAV capsid. In some embodiments, the AAV capsid is chimeric. In some embodiments, 60 copies of the AAV capsid protein are assembled into the AAV capsid. In some embodiments, the CNS comprises a cell-type selected from the group consisting of a neuron, an oligodendrocyte, an astrocyte, and a brain vascular cell. In some embodiments, the CNS comprises a tissue that is selected from the group consisting of a brain, a thalamus, a cortex, a striatum, a ventral midbrain, and a spinal cord. In some embodiments, the AAV capsid protein further comprises an amino acid substitution A587D. In some embodiments, the AAV capsid protein further comprises an amino acid substitution Q588G. In some embodiments, the AAV capsid protein further comprises an amino acid substitution comprising A589N. In some embodiments, the AAV capsid protein further comprises an amino acid substitution comprising Q590P. In some embodiments, the second amino acid sequence at the amino acid position 588_589 within SEQ ID NO: 1 is not TLAVPFK, KFPVALT, SVSKPFL, FTLTTPK, MNATKNV, NGGTSSS, TRTNPEA, or YTLSQGW. In some embodiments, the AAV capsid protein is isolated and purified. In some embodiments, the AAV capsid protein is formulated as a pharmaceutical formulation for intravenous administration to treat a disease or a condition of the liver, the pharmaceutical formulation further comprising a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical formulation further comprises a therapeutic agent. 
     Aspects disclosed herein provide AAV capsids proteins comprising a seven amino acid insertion (X1 X2 X3 X4 X5 X6 X7) between amino acid 588 and amino acid 589 in an amino acid sequence of the AAV capsid protein provided in SEQ ID NO: 1, wherein X1 is an amino acid selected from the group consisting of E, D, G, R, S and T. In some embodiments, X2 is an amino acid selected from the group consisting of A, G, I, L, M, N, Q, T, and Y. In some embodiments, X3 is an amino acid selected from the group consisting of E, K, L, T, and Q. In some embodiments, X4 is an amino acid selected from the group consisting of G, I, K, L, R, T, and V. In some embodiments, X5 is an amino acid selected from the group consisting of A, D, G, P, L, Q, and V. In some embodiments, X6 is an amino acid selected from the group consisting of F, K, N, P, Q, S, and V. In some embodiments, X7 is an amino acid selected from the group consisting of I, K, L, P, and V. In some embodiments, the seven amino acid insertion is selected from the group consisting of TALKPFL, TTLKPFL, TLQIPFK, TMQKPFI, SIERPFK, RYQGDSV, and TTLKPFS. In some embodiments, the AAV capsid protein is present in VP1, VP2, and VP3 of a AAV capsid. In some embodiments, the AAV capsid is chimeric. In some embodiments, 60 copies of the AAV capsid protein are assembled into the AAV capsid. In some embodiments, the AAV capsid protein is characterized by at least one of an increased specificity and an increased transduction efficiency when measured in a central nervous system (CNS) in a subject when delivered to the subject systemically, relative to a native AAV capsid protein provided in SEQ ID NO: 1. In some embodiments, the CNS comprises a cell-type selected from the group consisting of a neuron, a glial cell, a oligodendrocyte, an ependymal cell, an astrocyte, a Schwann cell, a satellite cell, and an enteric glial cell. In some embodiments, the CNS comprises a tissue that is selected from the group consisting of a brain, a thalamus, a cortex, a striatum, a ventral midbrain, and a spinal cord. In some embodiments, the AAV capsid protein further comprises an amino acid substitution comprising A587D. In some embodiments, the AAV capsid protein further comprises an amino acid substitution comprising Q588G. In some embodiments, the AAV capsid protein further comprises an amino acid substitution comprising A589N. In some embodiments, the AAV capsid protein further comprises an amino acid substitution comprising Q590P. In some embodiments, the seven amino acid insertion is not TLAVPFK, KFPVALT, SVSKPFL, FTLTTPK, MNATKNV, NGGTSSS, TRTNPEA, or YTLSQGW. In some embodiments, the AAV capsid protein is isolated and purified. In some embodiments, the AAV capsid protein is formulated as a pharmaceutical formulation for intravenous administration to treat a disease or a condition of the liver, the pharmaceutical formulation further comprising a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical formulation further comprises a therapeutic agent. 
     Aspects provided herein provide AAV capsids proteins comprising: (i) a first amino acid sequence that is at least 98% identical to amino acid 217 to amino acid 736 of SEQ ID NO: 1; and (ii) a second amino acid sequence at least 57.1% identical to an amino acid sequence provided in Table 4 or  FIG. 35  at an amino acid position 588_589 within SEQ ID NO: 1, wherein the AAV capsid protein is characterized by at least one of an increased specificity and an increased transduction efficiency when measured in a liver in a subject when delivered to the subject systemically, relative to a native AAV capsid protein provided in SEQ ID NO: 1. In some embodiments, the second amino acid sequence is at least 71.4% identical to the amino acid sequence provided in Table 4 or  FIG. 35 . In some embodiments, the second amino acid sequence is at least 86.7% identical to the amino acid sequence provided in Table 4 or  FIG. 35 . In some embodiments, the second amino acid sequence is selected from the group consisting of KAYSVQV, PSGSARS, and RTANALG. In some embodiments, the AAV capsid protein is present in VP1, VP2, and VP3 of an AAV capsid. In some embodiments, the AAV capsid is chimeric. In some embodiments, 60 copies of the AAV capsid protein are assembled into the AAV capsid. In some embodiments, the AAV capsid protein is isolated and purified. In some embodiments, the AAV capsid protein is formulated as a pharmaceutical formulation for intravenous administration to treat a disease or a condition of the liver, the pharmaceutical formulation further comprising a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical formulation further comprises a therapeutic agent. 
     Aspects disclosed herein comprise plasmid vectors comprising a nucleic acid sequence encoding the AAV capsids and AAV capsid proteins described herein. In some instances, the plasmid vector is bacterial. In some instances, the plasmid vector is derived from Escherichia coli. In some instances, the nucleic acid sequence comprises, in a 5′ to 3′ direction: (1) a 5′ inverted terminal repeat (ITR) sequence, (2) a Replication (Rep) gene, (3) a Capsid (Cap) gene, and (4) a 3′ ITR, wherein the Cap gene encodes the AAV capsid protein described herein. In some instances, the plasmid vector encodes a pseudotyped AAV capsid protein. In some instances, the Cap gene is derived from the deoxyribose nucleic acid (DNA) provided in any one of SEQ ID NOs: 6-10. In some instances, the nucleic acid sequence comprising the Cap gene is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the DNA sequences provided in U.S. App. Ser. No. 16/582,635, incorporated herein by reference. In some instances, the 5′ ITR and the 3′ ITR are derived from an AAV2 serotype. In some instances, the 5′ ITR and the 3′ ITR are derived from an AAVS serotype. In some instances, the 5′ ITR and the 3′ ITR are derived from an AAV9 serotype. 
     Aspects disclosed herein provide methods of treating a disease or condition in a subject comprising administering a therapeutically effective amount of a pharmaceutical formulation comprising the AAV capsid protein or the AAV capsid of the present disclosure. In some embodiments, the disease or the condition is a disease or a condition of a central nervous system (CNS) or a liver of the subject. In some embodiments, the disease or condition of the liver is selected from the group consisting of Alagille Syndrome, Alcohol-Related Liver Disease, Alpha-1 Antitrypsin Deficiency, Autoimmune Hepatitis, Benign Liver Tumors, Biliary Atresia, Cirrhosis, Crigler-Najjar Syndrome, Galactosemia, Gilbert Syndrome, Hemochromatosis, Hepatic Encephalopathy, Hepatitis A, Hepatitis B, Hepatitis C, Hepatorenal Syndrome, Intrahepatic Cholestasis of Pregnancy (ICP), Lysosomal Acid Lipase Deficiency (LAL-D), Liver Cysts, Liver Cancer, Newborn Jaundice, Non-Alcoholic Fatty Liver Disease, Primary Biliary Cholangitis (PBC), Primary Sclerosing Cholangitis (PSC), Reye Syndrome, Type I Glycogen Storage Disease, and Wilson Disease. In some embodiments, the disease or condition of the CNS is selected from group consisting of Absence of the Septum Pellucidum, Acid Lipase Disease, Acid Maltase Deficiency, Acquired Epileptiform Aphasia, Acute Disseminated Encephalomyelitis, Attention Deficit-Hyperactivity Disorder (ADHD), Adie&#39;s Pupil, Adie&#39;s Syndrome, Adrenoleukodystrophy, Agenesis of the Corpus Callosum, Agnosia, Aicardi Syndrome, Aicardi-Goutieres Syndrome Disorder, AIDS -Neurological Complications, Alexander Disease, Alpers&#39; Disease, Alternating Hemiplegia, Alzheimer&#39;s Disease, Amyotrophic Lateral Sclerosis (ALS), Anencephaly, Aneurysm, Angelman Syndrome, Angiomatosis, Anoxia, Antiphospholipid Syndrome, Aphasia, Apraxia, Arachnoid Cysts, Arachnoiditis, Arnold-Chiari Malformation, Arteriovenous Malformation, Asperger Syndrome, Ataxia, Ataxia Telangiectasia, Ataxias and Cerebellar or Spinocerebellar Degeneration, Atrial Fibrillation and Stroke, Attention Deficit-Hyperactivity Disorder, Autism Spectrum Disorder, Autonomic Dysfunction, Back Pain, Barth Syndrome, Batten Disease, Becker&#39;s Myotonia, Behcet&#39;s Disease, Bell&#39;s Palsy, Benign Essential Blepharospasm, Benign Focal Amyotrophy, Benign Intracranial Hypertension, Bernhardt-Roth Syndrome, Binswanger&#39;s Disease, Blepharospasm, Bloch-Sulzberger Syndrome, Brachial Plexus Birth Injuries, Brachial Plexus Injuries, Bradbury-Eggleston Syndrome, Brain and Spinal Tumors, Brain Aneurysm, Brain Injury, Brown-Sequard Syndrome, Bulbospinal Muscular Atrophy, Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL), Canavan Disease, Carpal Tunnel Syndrome, Causalgia, Cavernomas, Cavernous Angioma, Cavernous Malformation, Central Cervical Cord Syndrome, Central Cord Syndrome, Central Pain Syndrome, Central Pontine Myelinolysis, Cephalic Disorders, Ceramidase Deficiency, Cerebellar Degeneration, Cerebellar Hypoplasia, Cerebral Aneurysms, Cerebral Arteriosclerosis, Cerebral Atrophy, Cerebral Beriberi, Cerebral Cavemous Malformation, Cerebral Gigantism, Cerebral Hypoxia, Cerebral Palsy, Cerebro-Oculo-Facio-Skeletal Syndrome (COFS), Charcot-Marie-Tooth Disease, Charcot-Marie-Tooth syndrome, classical rhizomelic chondrodysplasia punctata (RCDP), Chiari Malformation, Cholesterol Ester Storage Disease, Chorea, Choreoacanthocytosis, Chronic Inflammatory Demyelinating Polyneuropathy (CIDP), Chronic Orthostatic Intolerance, Chronic Pain, Cockayne Syndrome Type II, Coffin Lowry Syndrome, Colpocephaly, Coma, Complex Regional Pain Syndrome, Congenital Facial Diplegia, Congenital Myasthenia, Congenital Myopathy, Congenital Vascular Cavernous Malformations, Corticobasal Degeneration, Cranial Arteritis, Craniosynostosis, Cree encephalitis, Creutzfeldt- Jakob Disease, Cumulative Trauma Disorders, Cushing&#39;s Syndrome, Cytomegalic Inclusion Body Disease, Cytomegalovirus Infection, Dancing Eyes-Dancing Feet Syndrome, Dandy-Walker Syndrome, Dawson Disease, Deafness, De Morsier&#39;s Syndrome, Dejerine-Klumpke Palsy, Dementia, Dementia -Multi -Infarct, Dementia—Semantic, Dementia—Subcortical, Dementia With Lewy Bodies, Dentate Cerebellar Ataxia, Dentatorubral Atrophy, Dermatomyositis, Developmental Dyspraxia, Devic&#39;s Syndrome, Diabetic Neuropathy, Diffuse Sclerosis, Dravet Syndrome, Duchenne muscular dystrophy, Dysautonomia, Dysgraphia, Dyslexia, Dysphagia, Dyspraxia, Dyssynergia Cerebellaris Myoclonica, Dyssynergia Cerebellaris Progressiva, Dystonias, Early Infantile Epileptic Encephalopathy, Empty Sella Syndrome, Encephalitis, Encephalitis Lethargica, Encephaloceles, Encephalopathy, Encephalopathy (familial infantile), Encephalotrigeminal Angiomatosis, Epilepsy, Epileptic Hemiplegia, Erb&#39;s Palsy, Erb-Duchenne and Dejerine-Klumpke Palsies, Essential Tremor, Extrapontine Myelinolysis, Fabry Disease, Fahr&#39;s Syndrome, Fainting, Familial Dysautonomia, Familial Hemangioma, Familial Idiopathic Basal Ganglia Calcification, Familial Periodic Paralyses, Familial Spastic Paralysis, Farber&#39;s Disease, Febrile Seizures, Fibromuscular Dysplasia, Fisher Syndrome, Floppy Infant Syndrome, Foot Drop, Friedreich&#39;s Ataxia, Frontotemporal Dementia, Gaucher Disease, Generalized Gangliosidoses, Gerstmann&#39;s Syndrome, Gerstmann-Straussler-Scheinker Disease, Giant Axonal Neuropathy, Giant Cell Arteritis, Giant Cell Inclusion Disease, glioblastoma, Globoid Cell Leukodystrophy, Glossopharyngeal Neuralgia, Glycogen Storage Disease, Guillain-Barre Syndrome, Hallervorden-Spatz Disease, Head Injury, Headache, Hemicrania Continua, Hemifacial Spasm, Hemiplegia Alterans, Hereditary Neuropathies, Hereditary Spastic Paraplegia, Heredopathia Atactica Polyneuritiformis, Herpes Zoster, Herpes Zoster Oticus, Hirayama Syndrome, Holmes-Adie syndrome, Holoprosencephaly, HTLV-1 Associated Myelopathy, Hughes Syndrome, Huntington&#39;s Disease, Hydranencephaly, Hydrocephalus, Hydrocephalus—Normal Pressure, Hydromyelia, Hypercortisolism, Hypersomnia, Hypertonia, Hypotonia, Hypoxia, Immune-Mediated Encephalomyelitis, Inclusion Body Myositis, Incontinentia Pigmenti, Infantile Hypotonia, Infantile Neuroaxonal Dystrophy, Infantile Phytanic Acid Storage Disease, Infantile Refsum Disease, Infantile Spasms, Inflammatory Myopathies, Iniencephaly, Intestinal Lipodystrophy, Intracranial Cysts, Intracranial Hypertension, Isaacs&#39; Syndrome, Joubert Syndrome, Kearns-Sayre Syndrome, Kennedy&#39;s Disease, Kinsbourne syndrome, Kleine-Levin Syndrome, Klippel-Feil Syndrome, Klippel-Trenaunay Syndrome (KTS), Kliiver-Bucy Syndrome, Korsakoff s Amnesic Syndrome, Krabbe Disease, Kugelberg-Welander Disease, Kuru, Lambert-Eaton Myasthenic Syndrome, Landau-Kleffner Syndrome, Lateral Femoral Cutaneous Nerve Entrapment, Lateral Medullary Syndrome, Learning Disabilities, Leigh&#39;s Disease, Lennox-Gastaut Syndrome, Lesch-Nyhan Syndrome, Leukodystrophy, Levine-Critchley Syndrome, Lewy Body Dementia, Lipid Storage Diseases, Lipoid Proteinosis, Lissencephaly, Locked-In Syndrome, Lou Gehrig&#39;s Disease, Lupus -Neurological Sequelae, Lyme Disease—Neurological Complications, Machado—Joseph Disease, Macrencephaly, Megalencephaly, Melkersson-Rosenthal Syndrome, Meningitis, Meningitis and Encephalitis, Menkes Disease, Meralgia Paresthetica, Metachromatic Leukodystrophy, Microcephaly, Migraine, Miller Fisher Syndrome, Mini Stroke, Mitochondrial Myopathy, Moebius Syndrome, Monomelic Amyotrophy, Motor Neuron Diseases, Moyamoya Disease, Mucolipidoses, Mucopolysaccharidoses, Multi-Infarct Dementia, Multifocal Motor Neuropathy, Multiple Sclerosis, Multiple System Atrophy, Multiple System Atrophy with Orthostatic Hypotension, Muscular Dystrophy, Myasthenia -Congenital, Myasthenia Gravis, Myelinoclastic Diffuse Sclerosis, Myoclonic Encephalopathy of Infants, Myoclonus, Myopathy, Myopathy- Congenital, Myopathy -Thyrotoxic, Myotonia, Myotonia Congenita, Narcolepsy, Neuroacanthocytosis, Neurodegeneration with Brain Iron Accumulation, Neurofibromatosis, Neuroleptic Malignant Syndrome, Neurological Complications of AIDS, Neurological Complications of Lyme Disease, Neurological Consequences of Cytomegalovirus Infection, Neurological Manifestations of Pompe Disease, Neurological Sequelae Of Lupus, Neuromyelitis Optica, Neuromyotonia, Neuronal Ceroid Lipofuscinosis, Neuronal Migration Disorders, Neuropathy- Hereditary, Neurosarcoidosis, Neurosyphilis, Neurotoxicity, Nevus Cavernosus, Niemann-Pick Disease, O′Sullivan-McLeod Syndrome, Occipital Neuralgia, Ohtahara Syndrome, Olivopontocerebellar Atrophy, Opsoclonus Myoclonus, Orthostatic Hypotension, Overuse Syndrome, Pain -Chronic, Pantothenate Kinase-Associated Neurodegeneration, Paraneoplastic Syndromes, Paresthesia, Parkinson&#39;s Disease, Paroxysmal Choreoathetosis, Paroxysmal Hemicrania, Parry -Romberg, Pelizaeus-Merzbacher Disease, Pena Shokeir II Syndrome, Perineural Cysts, Periodic Paralyses, Peripheral Neuropathy, Periventricular Leukomalacia, Persistent Vegetative State, Pervasive Developmental Disorders, Phytanic Acid Storage Disease, Pick&#39;s Disease, Pinched Nerve, Piriformis Syndrome, Pituitary Tumors, Polymyositis, Pompe Disease, Porencephaly, Post-Polio Syndrome, Postherpetic Neuralgia, Postinfectious Encephalomyelitis, Postural Hypotension, Postural Orthostatic Tachycardia Syndrome, Postural Tachycardia Syndrome, Primary Dentatum Atrophy, Primary Lateral Sclerosis, Primary Progressive Aphasia, Prion Diseases, Progressive Hemifacial Atrophy, Progressive Locomotor Ataxia, Progressive Multifocal Leukoencephalopathy, Progressive Sclerosing Poliodystrophy, Progressive Supranuclear Palsy, Prosopagnosia, Pseudo-Torch syndrome, Pseudotoxoplasmosis syndrome, Pseudotumor Cerebri, Psychogenic Movement, Ramsay Hunt Syndrome I, Ramsay Hunt Syndrome II, Rasmussen&#39;s Encephalitis, Reflex Sympathetic Dystrophy Syndrome, Refsum Disease, Refsum Disease—Infantile, Repetitive Motion Disorders, Repetitive Stress Injuries, Restless Legs Syndrome, Retrovirus-Associated Myelopathy, Rett Syndrome, Reye&#39;s Syndrome, Rheumatic Encephalitis, Riley-Day Syndrome, Sacral Nerve Root Cysts, Saint Vitus Dance, Salivary Gland Disease, Sandhoff Disease, Schilder&#39;s Disease, Schizencephaly, Seitelberger Disease, Seizure Disorder, Semantic Dementia, Septo-Optic Dysplasia, Severe Myoclonic Epilepsy of Infancy (SMEI), Shaken Baby Syndrome, Shingles, Shy-Drager Syndrome, Sjogren&#39;s Syndrome, Sleep Apnea, Sleeping Sickness, Sotos Syndrome, Spasticity, Spina Bifida, Spinal Cord Infarction, Spinal Cord Injury, Spinal Cord Tumors, Spinal Muscular Atrophy, Spinocerebellar Atrophy, Spinocerebellar Degeneration, Steele-Richardson-Olszewski Syndrome, Stiff-Person Syndrome, Striatonigral Degeneration, Stroke, Sturge-Weber Syndrome, Subacute Sclerosing Panencephalitis, Subcortical Arteriosclerotic Encephalopathy, Short-lasting, Unilateral, Neuralgiform (SUNCT) Headache, Swallowing Disorders, Sydenham Chorea, Syncope, Syphilitic Spinal Sclerosis, Syringohydromyelia, Syringomyelia, Systemic Lupus Erythematosus, Tabes Dorsalis,Tardive Dyskinesia, Tarlov Cysts, Tay-Sachs Disease, Temporal Arteritis, Tethered Spinal Cord Syndrome, Thomsen&#39;s Myotonia, Thoracic Outlet Syndrome, Thyrotoxic Myopathy, Tic Douloureux, Todd&#39;s Paralysis, Tourette Syndrome, Transient Ischemic Attack, Transmissible Spongiform Encephalopathies, Transverse Myelitis, Traumatic Brain Injury, Tremor, Trigeminal Neuralgia, Tropical Spastic Paraparesis, Troyer Syndrome, Tuberous Sclerosis, Vascular Erectile Tumor, Vasculitis Syndromes of the Central and Peripheral Nervous Systems, Von Economo&#39;s Disease, Von Hippel-Lindau Disease (VHL), Von Recklinghausen&#39;s Disease, Wallenberg&#39;s Syndrome, Werdnig-Hoffman Disease, Wernicke-Korsakoff Syndrome, West Syndrome, Whiplash, Whipple&#39;s Disease, Williams Syndrome, Wilson Disease, Wolman&#39;s Disease, and X-Linked Spinal and Bulbar Muscular Atrophy. In some embodiments, the pharmaceutical formulation comprises a therapeutic nucleic acid encoding a therapeutic gene expression product. In some instances, the therapeutic gene expression product is effective to modulate an activity or an expression of a target gene or gene expression product selected from the group consisting of Sarcoglycan Alpha (SGCA), glutamic acid decarboxylase 65 (GAD65), glutamic acid decarboxylase 67 (GAD67), CLN2, Nerve Growth Factor (NGF), Survival Of Motor Neuron 1, Telomeric (SMN1), Factor X (FIX), Retinoid Isomerohydrolase (RPE65), sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2a), β-Glucocerebrosidase (GCase), Frataxin (FXN), Huntingtin (HTN), methyl-CpG binding protein 2 (MECP2), a peroxisomal biogenesis factor (PEX), progranulin (GRN), an antitubulin agent, copper-zinc superoxide dismutase (SOD1), Glucosylceramidase Beta (GBA), NPC Intracellular Cholesterol Transporter 1 (NPC1), and a NLRP3 inflammasome. In some instances, the therapeutic gene expression product comprises gene editing components. In some instances, the gene editing components are selected from the group consisting of small interfering RNA (siRNA), short hairpin RNA (shRNA), a microRNA (miRNA), artificial site-specific RNA endonuclease (ASRE), zinc finger endonuclease (ZFN), CRISPR/Cas, and transcription factor like effector nuclease (TALEN). 
     Aspects disclosed herein provide methods of manufacturing a recombinant AAV particle from the AAV capsid of the present disclosure, the method comprising: (a) introducing into a cell a nucleic acid comprising: (i) a first nucleic acid sequence encoding a therapeutic gene expression product; (ii) a second nucleic acid sequence encoding a recombinant viral genome comprising a capsid (Cap) gene modified to express the AAV capsid of the present disclosure; and (iii) a third nucleic acid sequence encoding an AAV helper virus genome; and (b) assembling the recombinant AAV particle, the recombinant AAV particle comprising the AAV capsid encapsidating the first nucleic acid. 
     Aspects disclosed herein provide methods of manufacturing comprising: (a) introducing into a cell a nucleic acid comprising: (i) a first nucleic acid sequence encoding a therapeutic gene expression product enclosed by a 5′ and a 3′ inverted terminal repeat (ITR) sequence; (ii) a second nucleic acid sequence encoding a viral genome comprising a 5′ ITR sequence, a Replication (Rep) gene, Capsid (Cap) gene, and a 3′ ITR, wherein the Cap gene encodes the AAV capsid protein described herein; and (iii) a third nucleic acid sequence encoding a first helper virus protein selected from the group consisting of E4orf6, E2a, and VA RNA, and optionally, a second helper virus protein comprising Ela or E1b55k; (b) expressing in the cell the AAV capsid protein described herein; (c) assembling an AAV particle comprising the AAV capsid proteins disclosed herein; and (d) packaging the first nucleic acid sequence in the AAV particle. In some instances, the cell is mammalian. In some instances, the cell is immortalized. In some instances, the immortalized cell is an embryonic stem cell. In some instances, the embryonic stem cell is a human embryonic stem cell. In some instances, the human embryonic stem cell is a human embryonic kidney 293 (HEK-293) cell. In some instances, the Cap gene is derived from the deoxyribose nucleic acid (DNA) provided in any one of SEQ ID NOs: 6-10. In some instances, the nucleic acid sequence comprising the Cap gene is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the DNA sequences provided in U.S. App. Ser. No. 16/582,635, incorporated herein by reference. In some instances, the 5′ ITR and the 3′ ITR are derived from an AAV2 serotype. In some instances, the 5′ ITR and the 3′ ITR are derived from an AAVS serotype. In some instances, the 5′ ITR and the 3′ ITR are derived from an AAV9 serotype. In some instances, the first nucleic acid sequence and the second nucleic acid sequence are in trans. In some instances, the first nucleic acid sequence and the second nucleic acid sequence are in cis. In some instances, the first nucleic acid sequence, the second nucleic acid sequence and the third nucleic acid sequence, are in trans. 
     Aspects disclosed herein provide methods of manufacturing a recombinant AAV particle, the method comprising: (a) providing a recombinant AAV genome comprising: (i) an AAV capsid gene, and (ii) a recognition sequence for a Cre recombinase, wherein the recognition sequence facilitates a recombinase-dependent change that is detectable, and wherein the recombinase recognition sequence comprises two Cre-recognition sites; (iii) transfecting a population of cells expressing the Cre recombinase with the recombinant AAV genome, whereby the Cre recombinase induces a recombination event to generate the recombinase-dependent change in the recombinant AAV genome, and wherein the recombinase-dependent change comprises an inversion of the sequence that is flanked by the Cre-recognition sites; (iv) detecting an increased rate of the recombinase-dependent change a target cell in the population of cells; (v) detecting a decreased rate of the recombinase-dependent change in an off-target cell in the population of cells; and (vi) identifying a recombinant AAV genome generated by the recombinase-dependent change, wherein said identified rAAV genome comprises the inversion, and wherein said identified recombinant AAV genome encodes an AAV capsid particle characterized having an increased specificity for the target cell and a decreased specificity for the off-target cell. In some embodiments, the off-target cell is a hepatocyte. In some embodiments, the target cell is a cell selected from the group consisting of a neuron, a glial cell, a oligodendrocyte, an ependymal cell, an astrocyte, a Schwann cell, a satellite cell, and an enteric glial cell. 
     Aspects disclosed herein provide kits comprising: (a) a first vector comprising the recombinant vector of the present disclosure; (b) a second vector encoding a helper virus protein; and (c) a third vector comprising a therapeutic nucleic acid encoding a therapeutic gene expression product. 
     Aspects disclosed herein provide kits comprising: (a) a first vector comprising a first nucleic acid sequence encoding a viral genome comprising in a 5′ to 3′ direction: (i) a 5′ inverted terminal repeat (ITR) sequence; (ii) a Replication (Rep) gene; (iii) a Capsid (Cap) gene encoding the AAV capsid proteins described herein, and (iv) a 3′ ITR; and (b) optionally, a second vector comprising a second nucleic acid sequence encoding a helper virus protein comprising at least one of E4orf6, E2a, VA RNA, Ela and E1b55k. In some instances, the kit further comprises a cell. In some instances, the cell is mammalian. In some instances, the cell is immortalized. In some instances, the immortalized cell is an embryonic stem cell. In some instances, the embryonic stem cell is a human embryonic stem cell. In some instances, the human embryonic stem cell is a human embryonic kidney 293 (HEK-293) cell. In some instances, the kit further comprises an AAV vector comprising a heterologous nucleic acid encoding a therapeutic gene expression product. In some instances, the AAV vector is an episome. 
     INCORPORATION BY REFERENCE 
     All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which: 
         FIG. 1  shows a multiplexed selection approach to identify capsids with specific and broad tropisms. Steps 1-6 describe the workflow in Round-1 (R1) selection, steps 7-9 describe Round-2 (R2) selection using synthetic pool method, steps la, 2a, and 6a-b show the incorporation of deep sequencing to recover capsids after R1 and R2 selection, and steps 10-11 describe positive and/or negative selection criteria followed by variant characterization. 
         FIG. 2  shows a structural model of the AAV9 capsid (PDB 3UX1) with the insertion site for the 7-mer-i library highlighted in red in the 60-meric (left), trimeric (middle), and monomeric (right) forms. 
         FIG. 3  shows Empirical Cumulative Distribution Frequency (ECDF) of R1 DNA and virus libraries that were recovered by deep sequencing post Gibson assembly and virus production, respectively. 
         FIG. 4  shows distributions of variants recovered from three R1 libraries from Tek-Cre, SNAP25-Cre, and GFAP-Cre brain tissue (n=2 per Cre line), with capsid libraries sorted by decreasing order of the enrichment score. The enrichment scores of the AAV-PHP.V2 variant are mapped as well. 
         FIG. 5  is a schematic of R2 synthetic pool (left) and PCR pool (right) library design. 
         FIG. 6  shows an overlapping bar chart showing the percentage of library overlap between the mentioned libraries and their theoretical composition. 
         FIG. 7  shows histograms of DNA and virus libraries from the two methods, where the variants in a library are binned by their read counts (in log10 scale) and the height of the histogram is proportional to their frequency. 
         FIG. 8  shows distributions of R2 brain libraries from all Cre transgenic lines (n=2 mice per Cre Line, mean is plotted) and both methods, where the libraries are sorted in decreasing order of enrichment score (log10 scale). The total number of positively enriched variants from these libraries are highlighted by dotted straight lines and AAV9&#39;s relative enrichment is mapped on the synthetic pool plot. 
         FIG. 9  shows a comparison of the enrichment scores (log10 scale) of two alternate codon replicates for 8462 variants from the Tek-Cre brain library (n=2 mice, mean is plotted). The broken line separates the high-confidence signal (&gt;0.3) from noise. For the high-confidence signal (below), a linear least-squares regression is determined between the 2 codons and the regression line (best fit). The coefficient of determination R2 is shown. 
         FIG. 10  shows heatmaps representing the magnitude (log2 fold change) of a given AA&#39;s relative enrichment or depletion at each position given statistical significance is reached (boxed if P-value &lt;0.0001, two-sided, two-proportion z-test, p-values corrected for multiple comparisons using Bonferroni correction). R2 DNA normalized to oligopool (top, ˜9000 AA sequences), R2 virus normalized to R2 DNA (middle, n=˜9000 sequences), R2 Tek brain library with enrichment over 0.3 (high-confidence signal) from synthetic pool method normalized to R2 virus (bottom, 154 sequences) (n=2 for brain library, one per mouse. All other libraries, n=1). 
         FIG. 11  shows heatmaps of Cre-independent relative enrichment across organs (n=2 mice per Cre line, mean across 6 samples from 3 Cre lines is plotted) for variants enriched in the brain tissue of at least one Cre-dependent synthetic pool selection (lighter text, n=2 mice per cell-type, mean is plotted) (left). Zoom-in of the most CNS-enriched variants (middle), and of the variants that are characterized in the current study along with spike-in library controls (right) are shown. 
         FIG. 12  shows clustering analysis of variants from synthetic pool brain libraries after enrichment in Tek-Cre (left), GFAP-Cre (middle), and combined SNAP-Cre and Syn-Cre (right) selections. Size of nodes represents relative enrichment in brain. Thickness of edges (connecting lines) representing degree of relatedness. Distinct families (yellow) with the corresponding AA frequency logos (AA size represents prevalence and color encodes AA properties). 
         FIG. 13  shows the 7-mer insertion peptide sequences of AAV-PHP variants between AA positions 588-589 of AAV9 capsid. 
         FIG. 14  shows AAV9 (left) and AAV-PHP.V1 (right) mediated expression using ssAAV:CAG-mNeongreen genome (n=3, 3 weeks of expression in C57BL/6J adult mice with 3×10 11  vg IV dose/mouse). The images were acquired under same microscope settings as that of the sagittal sections of brain (top) with higher magnification image from cortex (bottom). aGLUT1 antibody staining was used in the image to show vasculature. 
         FIG. 15  shows vascular transduction by ssAAV-PHP.V1:CAG-DIO-EYFP in Tek-Cre adult mice (left) (n=2, 4 weeks of expression, 1×10 12  vg IV dose/mouse), and by ssAAV-PHP.V1:Ple261-iCre in Ai14 reporter mice (right) (n=2, 3 weeks of expression, 3×10 11  vg IV dose/mouse). Tissues are stained with aGLUT1. 
         FIG. 16  shows percentage of vasculature stained with aGLUT1 that overlaps with mNeongreen (XFP) expression in cortex. One-way ANOVA non-parametric Kruskal-Wallis test (P-value 0.0036), and follow-up multiple comparisons using uncorrected Dunn&#39;s test (P-value of 0.0070 for AAV9 vs PHP.V1) are reported. **P &lt;0.01 is shown, P &gt;0.05 is not shown; data is mean ±S.E.M, n=3 mice per AAV variant, cells quantified from 2-4 images per mouse per cell-type. 
         FIG. 17  shows percentage of cells stained with each cell-type specific marker (aGLUT1, aS100 for astrocytes, aNeuN for neurons, aOlig2 for oligodendrocyte lineage cells) that overlaps with mNeongreen (XFP) expression in cortex. Kruskal-Wallis test (P-value of 0.0078), and uncorrected Dunn&#39;s test (P-value of 0.0235 for neuron vs vascular cells, and 0.0174 for neuron vs astrocyte, respectively) are reported. *P 0.05 is shown, and P &gt;0.05 is not shown; data is mean ±S.E.M, n=3 mice, cells quantified from 2-4 images per mouse per cell-type. 
         FIG. 18  shows efficiency of vascular transduction (as described in  FIG. 16 ) in Tek-Cre mice (n=2, mean from 3 images per mouse per brain region). 
         FIG. 19  shows efficiency of vascular transduction in Ai14 mice (n=2, a mean from 4 images per mouse per brain region). 
         FIG. 20  shows transduction by AAV-PHP.B4-B6 and Cl variants, as well as B, eB, and AAV9 controls in sagittal brain and liver sections. Across each set of images (column-wise) acquired, microscope settings were matched with AAV-PHP.eB. The white box on the sagittal brain images marks the thalamus and not the precise region of the figures to the right. Vectors are packaged with ssAAV:CAG-2xNLS-EGFP genome (n=3 per group, lx10 11  vg IV dose/adult C57BL/6J mouse, 3 weeks of expression). Tissues are stained with cell-type specific markers: aNeuN for neurons, aS100 for astrocytes and aOlig2 for oligodendrocyte lineage cells. Liver tissues are stained with a DNA stain, DAPI. 
         FIGS. 21-23  show the percentage of aNeuN+( FIG. 21 ), aS 100 +( FIG. 22 ), and aOlig2+( FIG. 23 ) cells with detectable nuclear-localized EGFP in the indicated brain regions (n=3 per group, lx10 11  vg dose). A two-way ANOVA with correction for multiple comparisons using Tukey&#39;s test is reported with adjusted P-values (****P &lt;0.0001, ***P &lt;0.001, **P &lt;0.01, *P &lt;0.05, is shown, and P &gt;0.05 is not shown on the plot; 95% CI, data is mean ±S.E.M. The dataset comprises a mean of 2 images per region per cell-type marker per mouse). 
         FIG. 24  shows the design of the 3-mer-s PHP.B library with combinations of three AA diversification between AA 587-597 of AAV-PHP.B (or corresponding AA 587-590 of AAV9). Shared AA identity with the parent AAV-PHP.B is shown along with unique motifs for AAV-PHP.N and AAV-PHP.eB. 
         FIG. 25  shows distributions of R2 brain and liver libraries (at AA level) by enrichment score (normalized to R2 virus library, with variants sorted in decreasing order of enrichment score). The enrichment of AAV-PHP.eB and AAV-PHP.N across all libraries are mapped on the plot. 
         FIG. 26  shows heatmap represents the magnitude (log2 fold change) of a given AA&#39;s relative enrichment or depletion at each position across the diversified region, only if statistical significance is reached on fold change (boxed if p-value &lt;0.0001, two-sided, two-proportion z-test, p-values corrected for multiple comparisons using Bonferroni correction). Plot includes variants that were highly enriched in brain (&gt;0.5 mean enrichment score, where mean is drawn across Vglut2, Vgat and GFAP, n=1 library per mouse line (sample pooled from 2 mice per line)) and underrepresented in liver (&lt;0.0) (32 AA sequences). 
         FIG. 27  shows clustering analysis of enriched variants from Vgat brain library with node size representing the degree of negative enrichment in liver and the thickness of edges (connecting lines) representing degree of relatedness between nodes. Two distinct families are highlighted in yellow and their corresponding AA frequency logos are shown below (AA size represents prevalence and color encodes AA properties). 
         FIG. 28  shows the percentage of neurons, astrocytes and oligodendrocyte lineage cells with ssAAV-PHP.N:CAG-2xNLS-EGFP in the indicated brain regions (n=3, lx10 11  vg IV dose per adult C57BL/6J mouse, 3 weeks of expression, data is mean±S.E.M, 6-8 images for cortex, thalamus and striatum, and 2 images for ventral midbrain, per mouse per cell-type marker using 20x objective covering the entire regions). A two-way ANOVA with correction for multiple comparisons using Tukey&#39;s test gave adjusted P-values reported as ****P &lt;0.0001, ns for P &gt;0.05, 95% CI. 
         FIG. 29  shows transduction by ssAAV-PHP.N:CAG-NLS-EGFP (n=2, 2×10 11  vg IV dose per adult C57BL/6J mouse, 3 weeks of expression) with NeuN staining (magenta) across three brain areas (cortex, SNc (substantia nigra pars compacta) and thalamus). 
         FIG. 30  shows clustering analysis showing the brain-enriched sequence families of all variants described herein, either identified in prior studies (PHP.B-B3, PHP.eB) or in the current study (PHP.B4-B8, PHP.V1-2, PHP.C1-3). The thickness of edges (connecting lines) representing degree of relatedness between nodes. The AA sequences inserted between 588-589 (of AAV9 capsid) for all the variants discussed are shown below. 
         FIG. 31  shows transduction of AAV9, AAV-PHP.V1 and AAV-PHP.N across three different mouse strains: C57BL/6J, BALB/cJ and FVB/NJ in sagittal brain sections (right), along with a higher magnification image of the thalamus brain region (left). 
         FIG. 32  shows transduction by AAV-PHP.B, AAV-PHP.C1-C3 in C57BL/6J and BALB/cJ mice in sagittal brain sections (right), along with a higher magnification image of the thalamus brain region (left). In  FIGS. 31 and 32 , the white box on the sagittal brain images represents the location of thalamus and not the precise area that is zoomed-in on the figure to the left. The microscope settings of acquired images were matched across all sagittal sections and across all thalamus regions. The insets in AAV-PHP.V1 are zoom-ins with enhanced brightness. The indicated capsids were used to package ssAAV:CAG-mNeongreen (n=2-3 per group, 1×10 11  vg IV dose per 6-8 weeks old adult mouse, 3 weeks of expression. The data reported in  FIGS. 31 and 32  are from one independent trial where all viruses were freshly prepared and titered in the same assay for dosage consistency. AAV-PHP.C2 and AAV-PHP.C3 were further validated in an independent trial for BALB/cJ, n=2 per group). 
         FIG. 33  provides 7-mer and 11-mer variants that were positively enriched in the brain tissue in all cell lines. 
         FIG. 34  provides 7-mer and 11-mer variants enriched in one specific library, but negatively enriched in all other brain and liver libraries. 
         FIG. 35  provides 11-mer variants that were positively enriched in all cre lines in liver tissue. 
         FIG. 36  is a diagram of the genetic switch used in M-CREATE. The Acceptor Vector shows the position of the forward and reverse primers between the Lox sites that are used for selective recovery of capsids from the Cre+cells. The Rep-AAPACap vector shows a deletion of 480 bp in Cap gene in addition to the stop codons that are designed to prevent synthesis of VP1, VP2, and VP3 proteins. AAP protein translation is unaffected by these modifications. 
         FIG. 37  is a schematic of the protocol to selectively recover rAAV genomes from the target population using the Cre-Lox flipping strategy and preparation of the sample for deep sequencing. 
         FIG. 38  illustrates the library coverage for R1 DNA and virus libraries obtained from specific sequencing depths. 
         FIG. 39  shows the percentage of variant overlap within the sampled DNA and virus, or across different Cre lines within tissues, or across tissues from R1 selection. 
         FIG. 40  shows the distributions of AAV capsid read counts for libraries recovered by NGS from brain tissue across different Cre transgenic mice post R1 selection. The dotted line is illustrative only and roughly separates the signal from noise (see Methods for estimation of signal v.s. noise) where signal in this context represents the input for the R2 selection. 
         FIG. 41  shows rAAV genome recovery from tissues using different treatments are shown with total rAAV genome recovery from 0.1 g of liver. 
         FIG. 42  shows the percentage of rAAV genomes recovered per ng of total extracted DNA. 
         FIG. 43  shows the CT value (cycle threshold from qPCR) of rAAV genome extracted by trizol that were treated with Smal restriction enzyme or untreated. 
         FIG. 44  shows CT value of mitochondrial DNA (internal control for smaller genome recovery, fold change =10.79 (2ACT)) recovered from 1 ng of total DNA from liver tissue. In  FIGS. 41-44 , n= 4  mice; 2 from GFAP-Cre line and 2 from Tek-Cre line, each data point is drawn from the mean of three technical replicates, error bar is mean±S.E.M., Mann-Whitney test, two-tailed (exact P-value of 0.0286 (*P &lt;0.05), in  FIGS. 41, 42, and 44, and 0.1143  (n.s., P &gt;0.05, CI 95%) in  FIG. 43 ). The data reported  FIGS. 41, 42, and 44  are from one independent trial, and  FIG. 43 , from three independent trials. 
         FIG. 45  shows the vector yields obtained per 10 ng of capsid DNA library across R1 and R2 vector productions. 
         FIG. 46  shows distributions of the DNA and virus libraries produced by the synthetic pool and PCR pool methods by the standard score of NGS read counts. The variants in virus libraries are sorted by the decreasing order of standard score and their scores from respective DNA libraries are mapped onto them. 
         FIG. 47  shows correlations between the standard scores of read counts for the DNA and virus libraries (n=1 per library) produced by the synthetic pool and PCR pool methods is determined by linear least-squares regression, and the regression line (best fit) and R2 representing the coefficient of determination. 
         FIG. 48  shows distributions of capsid libraries from brain tissue of two mice used in each Cre line selection, as produced by the synthetic pool (left) and PCR pool (right) designs. The distribution of spike-in library introduced in the synthetic pool library design is shown at center. 
         FIG. 49  illustrates correlations of enrichment scores of variants from the brain libraries (n=2 per Cre line, mean is plotted) produced by synthetic pool and PCR pool methods is determined by the same method described with respect to  FIG. 47 . 
         FIG. 50  shows correlation analysis between the enrichment score (log10) of two alternate codon replicates of variants from the GFAP-Cre (left), SNAP-Cre (center), and Syn-Cre (right) brain libraries by linear least-squares regression (n=2 per Cre line, mean is plotted). The dotted line separates the high-confidence signal from noise. High confidence signal (below) is assessed by a linear regression line (best fit) and R2 represents the coefficient of determination. 
         FIG. 51  shows the difference in enrichment score between the two codon replicates of a variant, across different brain libraries, with over 8000 variants recovered in replicates. 
         FIG. 52  shows heatmaps represent the magnitude (log2 fold change) of AA bias in “output” library 1 normalized to “input” library 2 that reach statistical significance (boxed if P-value &lt;0.0001, two-sided, two-proportion z-test, except in R1 DNA normalized to known NNK template where one-proportion z-test was performed, and P-values corrected for multiple comparisons using Bonferroni correction). R1 DNA library normalized to NNK template (top left, ˜9 million sequences), R1 virus normalized to R1 DNA libraries (bottom left, ˜10 million sequences), R2 GFAP library with enrichment score above 1.0 in brain normalized to R2 virus (top right, 20 sequences,) and R2 SNAP library with enrichment score above 1.2 normalized to R2 virus (bottom right, 17 sequences) are shown (n=1 for DNA, virus, and n=2 for brain libraries). 
         FIG. 53  shows clustering analysis of positively enriched variants from Tek, GFAP, and combined neuron brain libraries (SNAP and Syn) by PCR pool design, and by synthetic pool design with spike-in library are shown with size of nodes representing their relative enrichment in brain, and the thickness of edges (connecting lines) representing the extent of shared AA identity between nodes. A distinct family is highlighted in yellow with the corresponding AA frequency logo below (AA size reflects prevalence and color coded based on AA properties). 
         FIG. 54  shows expression of AAV9 (top row) and AAV-PHP.V1 (bottom row) packaging CAG promoter driving mNeonGreen across all organs (n=3, 3×10 11  vg dose per adult C57BL/6J mouse, 3 weeks of expression). 
         FIG. 55  shows expression in cortical astrocytes (S100+) after IV delivery of AAV-PHP.V1 (left) and AAV-PHP.eB (right) capsids carrying the GfABC1D promoter driving expression of nuclear-localized mTurquoise2 reporter (1×10 12  vg dose per adult mouse, 4 weeks of expression). Percentage of cortical S100+cells that overlapped with mTurquoise2 expression is quantified (n=2, each data point is mean from 3 images per mouse). 
         FIG. 56 . shows expression of single-stranded (ss) AAV9, PHP.eB in Ai14-tdTomato reporter adult mouse (n=2-3 per group, 3×10 11  vg dose per adult mouse, 3 weeks of expression) 
         FIG. 57  shows expression of PHP.V1 in Ai14-tdTomato reporter adult mouse (n=2-3 per group, 3×10 11  vg dose per adult mouse, 3 weeks of expression) 
         FIG. 58  shows expression of packaging Ple261 promoter carrying iCre transgene in Ai14-tdTomato reporter adult mouse (n=2-3 per group, 3×10 11  vg dose per adult mouse, 3 weeks of expression) on the left. In the right column,  FIG. 58  shows expression of self-complementary (sc) AAV-PHP.V1 carrying CB6 ubiquitous promoter driving EGFP (above) and CAG promoter driving EGFP (below). Lectin DyLight 594 staining is also shown (n=2-3, 3×10 11  vg dose per adult C57BL/6J mouse, 2 weeks of expression). Experiments in  FIGS. 56, 57 , and the left column of  FIG. 58  are reported from one independent trial from a fresh batch of viruses, and titered in the same assay for dosage consistency. Experiments in  FIG. 58  were validated in two independent trials (n=2 per group). 
         FIG. 59  shows transduction of mouse brain by the AAV-PHP.V2 variant and control AAV9, carrying the CAG promoter that drives the expression of mNeonGreen (n=3, 3×10 11  vg IV dose per C57BL/6J adult mouse, 3 weeks of expression). The sagittal brain images (left) were acquired using the same microscope settings to that of the sagittal brain images in  FIG. 14 . 
       Higher magnification images of AAV-PHP.V2 transduced brain sections stained with aGLUT or aS100 or aOlig2 are shown. 
         FIG. 60  shows Transduction of brain vasculature by AAV-PHP.V2 carrying CAG-DIO-EYFP in Tek-Cre adult mice (left, 1×10 12  vg IV dose per mouse, 4 weeks of expression), and its efficiency (right) is determined by the overlap of aGLUT1 staining with EYFP expression across different brain areas (n=2, mean of 3 images per brain region per mouse). 
         FIG. 61  shows transduction of astrocytes by AAV-PHP.V2 in GFAP-Cre adult mouse (1×10 12  vg IV dose per mouse, 4 weeks of expression). Percentage of cortical S100 + cells that overlapped with EYFP expression is quantified (n=2, mean of 3 images per mouse). 
         FIG. 62  shows transduction levels of liver hepatocytes quantified as the percentage of DAPI + cells that are EGFP + (n=3, vectors packaged with CAG-2xNLS-EGFP, lx10 11  vg IV dose/adult C57BL/6J mouse, 3 weeks of expression, mean±S.E.M, 4 images per mouse per group. One-way ANOVA, non-parametric Kruskal-Wallis test gave an approximate P-value of 0.0088). 
         FIG. 63  shows transduction of brain tissue by AAV-PHP.B4, B7, AAV-PHP.X1 (ARQMDLS), and AAV-PHP.X2 (TNKVGNI) packaging CAG-mNeonGreen genome (n=3, lx10 11  vg IV dose/adult C57BL/6J mouse, 3 weeks of expression), were acquired using the same microscope settings to that of AAV9 and AAV-PHP.V1 sagittal brain images in  FIG. 14 . 
         FIG. 64  shows transduction of the brain by AAV-PHP.B8 using the CAG-mRuby2 genome (n=3, 3×10 11  vg IV dose/ adult C57BL/6J mouse, 3 weeks of expression). 
         FIG. 65  shows Transduction of AAV9 (left), AAV-PHP.X3 (QNVTKGV) (middle) and AAV-PHP.X4 (LNAIKNI) (right) vectors packaging CAG-2xNLS-EGFP (n=2, 1×10 11  vg IV dose/ adult C57BL/6J mouse, 3 weeks of expression). Data in  FIGS. 62-65  is reported from one independent trial. 
         FIGS. 66-67  show distributions of R1 ( FIGS. 66 ) and R 2  ( FIG. 67 ) brain libraries (at AA level, standard score (SS) of RCs sorted in decreasing order of scores). The SS for AAV-PHP.N and AAV-PHP.eB across libraries are mapped on the zoomed-in view of this plot (dotted line box). 
         FIG. 68  shows a heatmap of AA distributions across the diversified region of the positively enriched variants from R2 liver library (top 100 sequences) normalized to the R2 virus (input library). 
         FIG. 69  shows clustering analysis of positively enriched variants from GFAP and Vglut2 brain libraries are shown with size of nodes representing their relative negative enrichment in liver, and the thickness of edges (connecting lines) representing their relative identity between nodes. 
         FIG. 70  shows expression of AAV-PHP.B (top row) and AAV-PHP.N (bottom row) packaged with ssAAV:CAG-mNeonGreen across all organs (n=3, 3×10 11  vg IV dose per adult C57BL/6J mouse, 3 weeks of expression). 
         FIG. 71  shows transduction of mouse brain by the AAV-PHP.N variant, carrying the CAG promoter that drives the expression of mNeonGreen (n=3, 1×10 11  vg IV dose per C57BL/6J adult mouse, 3 weeks of expression). Fluorescence in situ hybridization chain reaction (FITC-HCR) was used to label excitatory neurons with Vglutl and inhibitory neurons with Gadl. Few cells where EGFP expression co-localized with specific cell markers are highlighted by asterisks symbol. 
         FIG. 72  shows transduction of AAV9, AAV-PHP.eB and AAV-PHP.V1 in human brain microvascular endothelial cell culture (HBMEC). The vectors were packaged with ssAAV:CAG-mNeongreen. The mean fluorescence intensity across the groups were quantified (n=3 tissue culture wells of 0.95 cm 2  surface area per group, 3 images per well per group per dose was imaged after three days of expression, doses lx10 8  vg and lx10 10  vg per 0.95 cm 2  surface area). A two-way ANOVA with correction for multiple comparisons using Tukey&#39;s test gave adjusted P-value of 0.0051 for AAV9 vs PHP.V1, 0.0096 for PHP.eB vs PHP.V1, 0.8222 for AAV9 vs PHP.eB for lx10 8  vg, and 0.0052 for AAV9 vs PHP.V1, 0.0049 for PHP.eB vs PHP.V1, 0.9996 for AAV9 vs PHP.eB for 1×10 1 ° vg (**P &lt;0.01, is shown and P &gt;0.05 is not shown on the plot; mean ±S.E.M., 95% CI). 
         FIG. 73  shows the transduction of cortex brain region by AAV-PHP.B, AAV-PHP.C2 and AAV-PHP.C3 across two different mouse strains: C57BL/6J and BALB/cJ. The vectors were packaged with ssAAV:CAG-mNeongreen (n=2-3 per group, lx10 11  vg IV dose/adult mouse, 3 weeks of expression). All images were acquired using the same microscope settings. The data reported in  FIGS. 72 and 73  are from one independent trial where all viruses were freshly prepared and tittered in the same assay for dosage consistency, with additional validation for AAV-PHP.C2 and AAV-PHP.C3 in an independent trial for BALB/cJ. 
         FIG. 74  provides details of a spike-in library of AAV9 and ˜50 additional variants (and their alternative codon duplicates), identified in prior publications (includes well characterized variants like AAV-PHP.B or AAVPHP.eB as well as many variants identified using the previous methodology but uncharacterized in vivo) act as internal selection controls and standards for the relative performance of the new variants. Deverman, B. E. et al. Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain. Nat. Biotechnol. 34,204-209 (2016) and Chan, K. Y. et al. Engineered AAVs for efficient noninvasive gene delivery to the central and peripheral nervous systems. Nat. Neurosci. 20, 1172-1179 (2017); the content of each of which is incorporated herein by reference. The spike-in library was generated as part of the synthetic pool library. 
         FIG. 75  shows enrichment scores for the spike-in library. 
     
    
    
     DETAILED DESCRIPTION 
     While preferred instances of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such instances are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the instances of the disclosure described herein may be employed in practicing the disclosure. It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby. 
     Provided herein are modified adeno-associated (AAV) virus capsid compositions useful for integrating a transgene into a target cell or environment (e.g., a cell-type or tissue) in a subject when they are administered systemically (e.g., intravenous, intranasal) to the subject. The modified AAV capsid proteins of the present disclosure comprise at least one insertion or substitution of an amino acid in a corresponding parental AAV capsid protein that confers a desired tropism such as an increased or decreased specificity as compared to a reference AAV capsid protein, or increased or decreased transgene transduction efficiency as compared to a reference AAV capsid protein. 
     Disclosed herein are AAV capsids engineered with desired tropisms, such as an increased specificity of viral transduction in a target in vivo environment, such as a tissue or cell. In some embodiments, the AAV capsids of the present disclosure are engineered to specifically target the central nervous system (CNS) of a subject. In some embodiments, the AAV capsids of the present disclosure are engineered to specifically target the liver of a subject. The AAV capsids can encapsidate a viral vector with a heterologous nucleic acid encoding, for example, a therapeutic gene expression product. Highly specific transduction of the heterologous nucleic acid in a target in vivo environment (e.g., brain, liver) can be achieved upon systemic delivery to a subject of the AAV capsid of the present disclosure encapsidating a heterologous nucleic acid. The AAV capsids disclosed herein are advantageous for many applications, such as diagnosing and/or treating monogenetic disorders of the brain (e.g., GLUT1-deficiency syndrome, mucoploysaccharidosis type IIIC), producing adoptive cellular therapies, and biomedical research applications. 
     The AAV capsids comprise AAV capsid proteins (e.g., VP1, VP2, and VP3), each with an insertion or a substitution of at least one amino acid at an amino acid in the 588 loop of a parental AAV capsid protein structure (AAV9 VP1 numbering). The 588 loop contains the site of heparan sulfate binding of AAV2 and amenable to peptide display. The only known receptors for AAV9 is N-linked terminal galactose and AAV receptor (AAVR), but many indications point toward there being others. Modifications to AAV9 588 loop are shown herein to confer an increased specificity and transgene transduction in target in vivo environments as compared to a reference AAV in rodent models. In some cases, the parental AAV capsid protein has an AAV serotype 9 (alias AAV9). 
     The most common method of AAV-mediated transgene delivery is by direct injection to the target in vivo environment, which is disadvantageous for many reasons, including risk of injury or death, pain, and higher cost, as compared to less invasive methods. For example, intracranial injection can cause hemorrhaging of the brain. Previous AAV-mediated delivery by intravenous administration avoids a need for a direct injection, but suffers from reduced specificity for the target in vivo environment (e.g., tissue or cell) resulting in off-target transduction events and necessitating a larger viral load to achieve sufficient therapeutic levels in the target in vivo environment. This is especially evident when the AAV must cross the blood brain barrier (BBB). 
     Methods are disclosed comprising systemically administering an AAV capsid of the present disclosure encapsidating a viral vector comprising a transgene (e.g., therapeutic nucleic acid) with an increased specificity, as compared to a reference AAV capsid protein. The AAV capsids of the present disclosure are capable of crossing the BBB, and transducing a transgene in a particular target cell-type (e.g., neuron, endothelial cell) in a subject. Accordingly, the AAV capsid proteins of the present disclosure are suitable for transgene therapy to treat human disease, particularly disease that effects the target in vivo environment. 
     The transgenes contained in a recombinant AAV (rAAV) vector and encapsidated by the AAV capsid proteins of the present disclosure are also provided herein. The transgenes disclosed herein are delivered to a subject for a variety of purposes, such as to treat a disease or condition in the subject. The transgene can be gene editing components that modulate the activity or expression of a target gene or gene expression product. Alternatively, the transgene is a gene encoding a therapeutic gene expression product that is effective to modulate the activity or expression of itself, or another target gene or gene expression product. 
     Provided herein, are methods of identifying the 7-mer or 3-mer peptide insertions comprising multiplexed Cre recombination-based AAV targeted evolution (M-CREATE). The M-CREATE method of the present disclosure supports (1) the calculation of a true enrichment score for each variant by using deep sequencing to correct for biases in viral production prior to selection, (2) reduced propagation of bias in successive rounds of selection through the creation of a post-round 1 synthetic pool library with equal variant representation, (3) the reduction of false positives by including two codon replicates of each selected variant in the pool, and (4) both positive and negative selection criteria by comparing deep sequencing of recovered capsid libraries among multiple targets (cells types or organs). These features allow informed, confident choices on variants worthy of validation and characterization in vivo. 
     Disclosed herein are methods of producing the AAV capsids comprising the AAV capsid proteins and viral vector encoding a therapeutic nucleic acid. The AAV capsid proteins are produced by introducing into a cell (e.g., immortalized stem cell) a first vector encoding the transgene (e.g., containing the therapeutic nucleic acid), a second vector encoding the AAV genome with a AAV capsid protein, and a third vector encoding helper virus proteins, required for assembly of the AAV capsid structure and packaging of the transgene in the AAV capsid. The assembled AAV capsid can be isolated and purified from the cell using suitable methods known in the art. 
     The recombinant AAV vectors comprising a nucleic acid sequence encoding the AAV capsid proteins of the present disclosure as also provided herein. For example, the viral vectors of the present disclosure comprise a nucleic acid sequence comprising the AAV viral Cap (Capsid) encoding VP1, VP2, and VP3, at least one of which is modified to produce the AAV capsid proteins of the present disclosure. The recombinant AAV vector provided can be derived from an AAV serotype (e.g., AAV9). 
     I. COMPOSITIONS 
     Recombinant adeno-associated virus (rAAV) mediated gene delivery leverages the AAV mechanism of viral transduction for nuclear expression of an episomal heterologous nucleic acid (e.g., a transgene, therapeutic nucleic acid). Upon delivery to a host in vivo environment, a rAAV will (1) bind or attach to cellular surface receptors on the target cell, (2) endocytose, (3) traffic to the nucleus, (4) uncoat the virus to release the encapsidated heterologous nucleic acid , (5) convert of the heterologous nucleic acid from single-stranded to double-stranded DNA as a template for transcription in the nucleus, and (6) transcribe of the episomal heterologous nucleic acid in the nucleus of the host cell (“transduction”). rAAVs engineered to have an increased specificity (binding to cellular surface receptors on the target cell) and transduction efficiency (transcription of the episomal heterologous nucleic acid in the host cell) are desirable for gene therapy applications. 
     A rAAV comprises an AAV capsid that can be engineered to encapsidate a heterologous nucleic acid (e.g., therapeutic nucleic acid, gene editing machinery). The AAV capsid is made up of three AAV capsid protein monomers, VP1, VP2, and VP3. Sixty copies of these three VP proteins interact in a 1:1:10 ratio to form the viral capsid ( FIG. 2 ). VP1 covers the whole of VP2 protein in addition to a ˜137 amino acid N-terminal region (VP1u), VP2 covers the whole of VP3 in addition to ˜65 amino acid N-terminal region (VP1/2 common region). The three capsid proteins share a conserved amino acid sequence of VP3, which in some cases is the region beginning at amino acid position 138 (e.g., AA139-736). 
     The AAV VP3 structure contains highly conserved regions that are common to all serotypes, a core eight-stranded β-barrel motif (f3B-(3I) and a small a-helix (aA). The loop regions inserted between the β-strands consist of the distinctive HI loop between β-strands H and I, the DE loop between β-strands D and E, and nine variable regions (VRs), which form the top of the loops. These VRs, such as the AA588 loop, are found on the capsid surface and can be associated with specific functional roles in the AAV life cycle including receptor binding, transduction and antigenic specificity. 
     Disclosed herein are AAV capsids comprising AAV capsid proteins with a substitution at the 588 loop that confer a desired tropism characterized by a higher specificity for transduction in specific cell-types, including for e.g., brain cell types (e.g., brain endothelial cells, neurons, astrocytes) and liver cell types. In particular, the AAV capsid proteins disclosed herein enable rAAV-mediated transduction of a heterologous nucleic acid (e.g., transgene) in the brain or the liver of a subject. The AAV capsids of the present disclosure, or the AAV capsid proteins, may be formulated as a pharmaceutical composition. In addition, the AAV capsids or the AAV capsid proteins can be isolated and purified to be used for a variety of applications. 
     A. Adeno-Associated Virus (AAV) Capsid Proteins 
     Disclosed herein are recombinant AAV (rAAV) capsids comprise AAV capsid proteins that are engineered with a modified capsid protein (e.g., VP1, VP2, VP3). In some embodiments, the rAAV capsid proteins of the present disclosure are generated using the methods disclosed herein (e.g., M-CREATE). In some embodiments, the AAV capsid proteins are used in the methods of delivering a therapeutic nucleic acid (e.g., a transgene) to a subject. In some instances, the rAAV capsid proteins have desired AAV tropisms rendering them particularly suitable for certain therapeutic applications, e.g., the treatment of a disease or disorder in a subject such as those disclosed herein. 
     The rAAV capsid proteins are engineered for optimized entry into and through the blood brain barrier (BBB) of a subject upon systemic administration of the rAAV to the subject, such as those provided in Tables 2-3, and  FIG. 33 . Prior methods of AAV-mediated delivery of a therapeutic transgene to the brain required intracranial injection. Intracranial injection is an invasive procedure that causes a subject discomfort, and in some cases, pain. For example, intracranial injection can cause hemorrhaging of the brain. Additionally, intracranial delivery has limited spread and is highly heterogeneous. The rAAV capsid proteins provided in Tables 2-4, and  FIG. 33  are engineered to have tropisms that eliminate the need for intracranial injection, while also achieving widespread and efficient transduction of an encapsidated transgene. In particular, the tropisms comprise at least one of an increased specificity and efficiency (e.g., of viral transduction) in the central nervous system (CNS) of a subject, as compared to a reference AAV. 
     The engineered AAV capsid proteins described herein have, in some cases, an insertion of an amino acid that is heterologous to the parental AAV capsid protein at the amino acid position in the 588 loop. In some embodiments, the amino acid is not endogenous to the parental AAV capsid protein at the amino acid position of the insertion. The amino acid may be a naturally occurring amino acid in the same or equivalent amino acid position as the insertion of the substitution in a different AAV capsid protein. 
     Aspects provided herein provide amino acid insertions comprising seven amino acid polymer (7-mer) inserted at AA588 589, and may additionally include a substitution of one or two amino acids at amino acid positions flanking the 7-mer sequence (e.g., AA587-588 and/or AA589-590) to produce an eleven amino acid polymer (11-mer) at the 588 loop of a parental AAV capsid protein. The 7-mers described herein were advantageously generated using polymerase chain reaction (PCR) with degenerate primers, where each of the seven amino acids is encoded by a deoxyribose nucleic acid (DNA) sequence N-N-K. “N” is any of the four DNA nucleotides and K is guanine (G) or thymine (T). This method of generating random 7-mer amino acid sequences enables 1.28 billion possible combinations at the protein level. Since the 7-mers developed are random, some amino acids in the 7-mer may be naturally occurring in the AAC capsid protein at that amino acid position, while other amino acids may differ. 
     Recombinant AAVs (rAAVs) were generated, each with a unique 7-mer or 11-mer at the 588 loop and each encapsidating a reporter gene that, when administered systemically in multiple transgenic animals, enabled the selective amplification and recovery of sequences that effectively transduced the reporter gene in a target in vivo environment of the transgenic animal. 7-mers and 11-mers that were found to be positively enriched in the target in vivo environment (e.g, central nervous system, liver) are provided herein. “Enrichment” is the prevalence of a given 7-mer or an 11-mer in the tissue of the in vivo environment compared to its prevalence in the viral library that was administered to the transgenic animal. An enrichment score above 0 indicates a positive enrichment. An enrichment score below 0 indicates a negative enrichment. A subset of the rAAVs with desired enrichment profiles were tested individually in vivo to determine exact systemic expression (e.g., specificity and transduction efficiency). rAAVs from this subset exhibiting a desired tropism comprising increased specificity of viral transduction, and in some cases, transduction efficiency are considered to be uniquely suited for targeted rAAV-mediated transgene delivery useful for a wide variety of purposes (e.g., therapeutic, diagnostic, scientific discovery). 
     The rAAV particles with the 7-mers or 11-mers described herein have an increased transduction efficiency in a target in vivo environment (e.g., tissue or cell type). In some instances, the increased transduction efficiency comprises a 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21-fold, 22-fold, 23-fold, 24-fold, 25-fold, 26-fold, 27-fold, 28-fold, 29-fold, 30-fold, 31-fold, 32-fold, 33-fold, 34-fold, 35-fold, 36-fold, 37-fold, 38-fold, 39-fold, 40-fold, 41-fold, 42-fold, 43-fold, 44-fold, 45-fold, 46-fold, 47-fold, 48-fold, 49-fold, 50-fold, 75-fold, or 100-fold increase, or more, relative to a reference AAV. In some instances, the increased transduction efficiency is at least 30-fold. In some instances, the increased transduction efficiency is at least 40-fold. In some instances, the increased transduction efficiency is at least 50-fold. In some instances, the increased transduction efficiency is at least 60-fold. In some instances, the increased transduction efficiency is at least 80-fold. In some instances, the increased transduction efficiency is at least 90-fold. In some instances, the increased transduction efficiency is at least 100-fold. 
     The rAAV particles with the 7-mers or 11-mers described herein have an increased specificity in a target in vivo environment (e.g., tissue or cell type), as compared to a reference AAV. Detecting whether a rAAV possesses more or less specificity for a target in vivo environment than a reference AAV, includes measuring a level of gene expression product (e.g., RNA or protein) expressed from the heterologous nucleic acid encapsidated by the rAAV in a tissue sample obtained from the target in vivo environment in a subject; and comparing the measured level to a control level (such as, for e.g., the gene expression product expressed from a heterologous nucleic acid encapsidated by a reference AAV (e.g., AAV9)). Suitable methods for measuring expression of a gene expression product luciferase reporter assay and quantitative polymerase chain reaction (qPCR). 
     The increased specificity is correlated with an increased enrichment in the target in vivo environment, which in some cases is represented with an enrichment score provided herein in  FIGS. 33-35 . As a non-limiting example, AAV-PHP.V2 (TTLKPFL), which is shown herein to be positively enriched in the brain (enrichment score of ˜2.51) also exhibited an increase in reporter gene expression (e.g., measured by fluorescence reporter assay) in the brain (-60% of cortical brain vasculature cells and ˜60% cortical astrocytes transduced with the reporter gene) as compared to a reference AAV9 (˜0%). Without being bound by a particular theory, the inventors of the present disclosure would expect to see this correlation for all rAAVs disclosed herein, and further, would expect that a more significant the enrichment score (whether negative or positive) would correlate with a more significant specificity to the in vivo environment(s) as indicated by a measured level of the gene expression product in the in vivo environment(s). 
     Transduction efficiency, as disclosed herein, may be measured by at least one of (1) a number of cells in a target in vivo or off-target in vivo environment expressing the heterologous nucleic acid encapsidated by the modified AAV capsid proteins disclosed herein, and (2) a quantity of expression of the heterologous nucleic acid in a single cell. Specificity for a target in vivo environment may be inferred when a presence, or an increase in a level, of rAAV-mediated transduction in a target in vivo environment is observed, as compared to a reference AAV. A lack of, or reduced, specificity to an off-target in vivo environment may be inferred when an absence, or a decrease in a level, of rAAV-mediated transduction in the off-target in vivo environment is observed, as compared to a reference AAV. 
     The reference AAV may have a serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAVS, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or variant thereof. For example, the reference AAV can have a serotype selected from the group consisting of AAV-PHP.B, AAV-PHP.eB, and AAV-PHP.S. 
     The rAAV capsid proteins of the present disclosure comprise an insertion of an amino acid in an amino acid sequence of an AAV capsid protein. The AAV capsid, from which an engineered AAV capsid protein of the present disclosure is produced, is referred to as a “parental” AAV capsid. In some cases, the parental AAV has a serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12. The complete genome of AAV-1 is provided in GenBank Accession No. NC 002077; the complete genome of AAV-2 is provided in GenBank Accession No. NC 001401 and Srivastava et al., J. Virol., 45: 555-564 (1983); the complete genome of AAV-3 is provided in GenBank Accession No. NC 1829; the complete genome of AAV-4 is provided in GenBank Accession No. NC 001829; the AAV-5 genome is provided in GenBank Accession No. AF085716; the complete genome of AAV-6 is provided in GenBank Accession No. NC 00 1862; at least portions of AAV-7 and AAV-8 genomes are provided in GenBank Accession Nos. AX753246 and AX753249, respectively; the AAV -9 genome is provided in Gao et al., J. Virol., 78: 6381-6388 (2004); the AAV-10 genome is provided in Mol. Ther., 13(1): 67-76 (2006); the AAV-11 genome is provided in Virology, 330(2): 375-383 (2004); portions of the AAV-12 genome are provided in Genbank Accession No. DQ813647; portions of the AAV-13 genome are provided in Genbank Accession No. EU285562. 
     In some cases, the parental AAV is derived from an AAV with a serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12. The AAV capsid protein that is “derived” from another may be a variant AAV capsid protein. A variant may include, for example, a heterologous amino acid in an amino acid sequence of the AAV capsid protein. The heterologous amino acid may be non-naturally occurring in the AAV capsid protein. The heterologous amino acid may be naturally occurring in a different AAV capsid protein. In some instances, the parental AAV capsid is described in U.S. Pat. App. Ser. Nos. 6 2 / 7 36,904; 16/582,635; 62/832,812; and 62/832,826; the content of each of which is incorporated herein. For example, the parental AAV capsid may be modified at the 455 loop of the AAV capsid protein (e.g., substitutions of 7-mer at AA452-458, AAV9 VP1 numbering). 
     In some instances, the parental AAV is AAV9. In some instances, the amino acid sequence of the AAV9 capsid protein comprises SEQ ID NO: 1. The amino acid sequence of AAV9 VP1 capsid protein (&gt;trIQ6JC401Q6JC40 9VIRU Capsid protein VP1 OS=Adeno-associated virus 9 OX=235455 GN=cap PE=1 SV=1) is provided in SEQ ID NO: 1 (MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPG NGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGG NLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRL NFGQTGDTESVPDPQPIGEPPAAPS GVGSLTMAS GGGAPVADNNEGADGVGSSS GNWH CDS QWLGDRVITTSTRTWALPTYNNHLYKQISNSTS GGSSNDNAYFGYSTPWGYFDFN RFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFT DSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGS QAVGRSSFYCLEYFPS QML RTGNNFQFSYEFENVPFHSSYAHS QSLDRLMNPLIDQYLYYLSKTINGS GQNQQTLKFSV AGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGP AMASHKEGEDRFFPLS GSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVA TNHQSAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGF GMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPE IQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL). In some instances, the parental AAV capsid protein sequence is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous to SEQ ID NO: 1. 
     Disclosed herein are insertions of an amino acid (or amino acid sequence) in an amino acid sequence of an AAV capsid protein at an amino acid position between amino acid 588 and amino acid 589. As used herein, “AA588_589” indicates that the insertion of the amino acid (or amino acid sequence) is immediately after an amino acid (AA) at position 588 and immediately before an AA at position 589 within an amino acid sequence of a parental AAV VP capsid protein (VP1 numbering). Amino acids 587-591 include a motif comprising “AQAQA” as set forth in SEQ ID NO: 1. Exemplary AAV capsid protein sequences are provided in Table 1. For example, QAVRTSL is inserted at AA588_589 in an AAV9 capsid amino acid sequence, and is provided SEQ ID NO: 3. In another example, TLAVPFK is inserted at AA588_589 in an AAV9 capsid amino acid sequence, and is provided in SEQ ID NO: 2. It is envisioned that the 7-mer insertions disclosed herein ( FIGS. 33-35 , Tables 2-4) may be inserted at AA588_589 in an amino acid sequence of a parental AAV9 capsid protein, a variant thereof, or equivalent amino acid position a parental AAV of a different serotype (e.g., AAV1, AAV2, AAV3, and the like). 
     The 11-mers described herein may, in some cases, comprise a 7-mer insertion at AA588 589, and substitutions of one or more amino acids at amino acid positions AA587-590. In some cases, the amino acids 587-590 are substituted with an amino acid that is not endogenous to the parental AAV capsid protein at that position. In some cases, AA587 is substituted with D (e.g., A587D). In some cases, AA587 is substituted with an A (e.g., Q587A). In some cases, AA587 is substituted with an S (e.g., Q587S). In some cases, AA587 is substituted with a G (e.g., Q587G). In some cases, AA588 is substituted with a G (e.g., Q588G). In some cases, AA589 is substituted with an N (e.g., A589N). In some cases, AA590 is substituted with a P (e.g., A590). In a non-limiting example SEQ ID NO: 4 (PHP-AAV.eB) comprises an insertion at AA588_AA589 of TLAVPFK, and substitutions A587D and Q588G. In another non-limiting example, SEQ ID NO: 5 (PHP-AAV.N) comprises an insertion at AA588_AA589 of TTLKPFS, and substitutions A587D, Q588G, A589N, and Q590P. It is envisioned that any 7-mer insertion disclosed herein in addition to a substitution with any amino acid at amino acid positions 587-590 may comprise an 11-mer. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Exemplary AAV Capsid Protein Sequences 
               
            
           
           
               
               
               
            
               
                 SEQ 
                   
                   
               
               
                 ID NO: 
                 Identifier 
                 Sequence 
               
               
                   
               
               
                 2 
                 AAV- 
                 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDN 
               
               
                   
                 PHP.B 
                 ARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQ 
               
               
                   
                   
                 LKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKK 
               
               
                   
                   
                 RLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQP 
               
               
                   
                   
                 AKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGA 
               
               
                   
                   
                 PVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPT 
               
               
                   
                   
                 YNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFS 
               
               
                   
                   
                 PRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIAN 
               
               
                   
                   
                 NLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGY 
               
               
                   
                   
                 LTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPF 
               
               
                   
                   
                 HSSYAHSQSLDRLMNPLIDQYLYYLSRTINGSGQNQQTLKFSV 
               
               
                   
                   
                 AGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGAS 
               
               
                   
                   
                 SWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTG 
               
               
                   
                   
                 RDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQ TLA   
               
               
                   
                   
                   VPFK AQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHT 
               
               
                   
                   
                 DGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLN 
               
               
                   
                   
                 SFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNV 
               
               
                   
                   
                 EFAVNTEGVYSEPRPIGTRYLTRNL 
               
               
                   
               
               
                 3 
                 AAV- 
                 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDN 
               
               
                   
                 PHP.S 
                 ARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQ 
               
               
                   
                   
                 LKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKK 
               
               
                   
                   
                 RLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQP 
               
               
                   
                   
                 AKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGA 
               
               
                   
                   
                 PVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPT 
               
               
                   
                   
                 YNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFS 
               
               
                   
                   
                 PRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIAN 
               
               
                   
                   
                 NLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGY 
               
               
                   
                   
                 LTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPF 
               
               
                   
                   
                 HSSYAHSQSLDRLMNPLIDQYLYYLSRTINGSGQNQQTLKFSV 
               
               
                   
                   
                 AGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGAS 
               
               
                   
                   
                 SWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTG 
               
               
                   
                   
                 RDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQ QAV   
               
               
                   
                   
                   RTSL AQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHT 
               
               
                   
                   
                 DGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLN 
               
               
                   
                   
                 SFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNV 
               
               
                   
                   
                 EFAVNTEGVYSEPRPIGTRYLTRNL 
               
               
                   
               
               
                 4 
                 AAV- 
                 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDN 
               
               
                   
                 PHP.eB 
                 ARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQ 
               
               
                   
                   
                 LKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKK 
               
               
                   
                   
                 RLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQP 
               
               
                   
                   
                 AKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGA 
               
               
                   
                   
                 PVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPT 
               
               
                   
                   
                 YNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFS 
               
               
                   
                   
                 PRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIAN 
               
               
                   
                   
                 NLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGY 
               
               
                   
                   
                 LTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPF 
               
               
                   
                   
                 HSSYAHSQSLDRLMNPLIDQYLYYLSRTINGSGQNQQTLKFSV 
               
               
                   
                   
                 AGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGAS 
               
               
                   
                   
                 SWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTG 
               
               
                   
                   
                 RDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQS DGTLA   
               
               
                   
                   
                   VPFKAQ AQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHT 
               
               
                   
                   
                 DGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLN 
               
               
                   
                   
                 SFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNV 
               
               
                   
                   
                 EFAVNTEGVYSEPRPIGTRYLTRNL 
               
               
                   
               
               
                 5 
                 AAV- 
                 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDN 
               
               
                   
                 PHP.N 
                 ARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQ 
               
               
                   
                   
                 LKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKK 
               
               
                   
                   
                 RLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQP 
               
               
                   
                   
                 AKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGA 
               
               
                   
                   
                 PVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPT 
               
               
                   
                   
                 YNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFS 
               
               
                   
                   
                 PRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIAN 
               
               
                   
                   
                 NLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGY 
               
               
                   
                   
                 LTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPF 
               
               
                   
                   
                 HSSYAHSQSLDRLMNPLIDQYLYYLSRTINGSGQNQQTLKFSV 
               
               
                   
                   
                 AGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGAS 
               
               
                   
                   
                 SWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTG 
               
               
                   
                   
                 RDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQS AQTLA   
               
               
                   
                   
                   VPFSNP AQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHT 
               
               
                   
                   
                 DGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLN 
               
               
                   
                   
                 SFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNV 
               
               
                   
                   
                 EFAVNTEGVYSEPRPIGTRYLTRNL 
               
               
                   
               
            
           
         
       
     
     The rAAV capsid proteins described herein may be isolated and purified. The AAV may be isolated and purified by methods standard in the art such as by column chromatography or cesium chloride gradients. Methods for purifying AAV from helper virus are known in the art and may include methods disclosed in, for example, Clark et al., Hum. Gene Ther., 10(6): 1031-1039 (1999); Schenpp and Clark, Methods Mol. Med., 69: 427-443 (2002); U.S. Patent No. 6,566,118 and WO 98/09657. 
     The rAAV capsid protein can be conjugated to a nanoparticle, a second molecule, or a viral capsid protein. In some cases, the nanoparticle or viral capsid protein would encapsidate the therapeutic nucleic acid described herein. In some instances, the second molecule is a therapeutic agent, e.g., a small molecule, antibody, antigen-binding fragment, peptide, or protein, such as those described herein. In some instances, the second molecule is a detectable moiety. For example, the modified AAV capsid protein conjugated to a detectable moiety may be used for in vitro, ex vivo, or in vivo biomedical research applications, the detectable moiety used to visualize the modified capsid protein. The modified AAV capsid protein conjugated to a detectable moiety may also be used for diagnostic purposes. 
     AAV Capsid Proteins Targeting the Central Nervous System 
     Disclosed herein are AAV capsid proteins with a substitution or an insertion of at least one amino acid at an amino acid position described above in a parental AAV capsid protein that confers an increased specificity for a central nervous system (CNS) or peripheral nervous system (PNS) in a subject, even when delivered systemically. One of the many advantages of the AAV capsid proteins described herein is their ability to target the CNS and penetrate the blood brain barrier (BBB). 
     The in vivo environment can be a cell. The cell can be a cell-type selected from the group consisting of a central nervous system (CNS) cell and a peripheral nervous system (PNS) cell. Non-limiting examples of CNS cells include a neuron and a glial cell. Glial cells can be selected from the group consisting of an oligodendrocyte, an ependymal cell, and an astrocyte. Non-limiting examples of a PNS cell includes a neuron or a glial cell. The glial cell can be selected from the group consisting of a Schwann cell, a satellite cell, and an enteric glial cell. 
     The in vivo environment can be a tissue. The tissue can be the brain, or the spinal cord. The tissue can be a region of an organ, example, the cerebrum, the cerebellum, the brainstem, the cortex, the striatum, the thalamus, the lateral ventricles, the putamen, the hypothalamus, the medulla, the pons, the hippocampus, the amygdala, the motor cortex, or a combination thereof. 
     Disclosed herein are AAV capsid proteins with at least one amino acid insertion or substitution in a parental AAV capsid protein. The insertion or substitution can be of at least one, two, three, four, five, six, seven, eight, nine, ten, or eleven amino acids, or more. In some instances, the amino acids are contiguous. In some instances, the amino acids are not contiguous. 
     In some instances, the insertion is of at least one amino acid is provided in any one of the sequences provided in any one of Tables 2-3, or  FIG. 6 . In some instances, the insertion is of at least two amino acids provided in any one of the sequences provided in any one of Tables 2-3, or  FIG. 6 . In some instances, the insertion is of at least three amino acids provided in any one of the sequences provided in any one of Tables 2-3, or  FIG. 6 . In some instances, the insertion is of at least four amino acids provided in any one of the sequences provided in any one of Tables 2-3, or  FIG. 6 . In some instances, the insertion is of at least five amino acids provided in any one of Tables 2-3, or  FIG. 6 . In some instances, the insertion is of at least six amino acids provided in any one of Tables 2-3, or  FIG. 6 . In some instances, the insertion is of at least seven amino acids provided in any one of Tables 2-3, or  FIG. 6 . 
     Disclosed herein are AAV capsid proteins with an insertion of at least one amino acid X1, wherein X1 is selected from the group consisting of A, E, D, G, R, S and T. In some instances, the insertion further comprises two amino acids, wherein X2 is selected from the group consisting of A, G, I, L, M, N, Q, R, T, and Y. In some instances, the insertion further comprises three amino acids, wherein X3 is selected from the group consisting of E, K, L, T, and Q. In some instances, the insertion further comprises at least four amino acids, wherein X1 is selected from the group consisting of A, E, D, G, R, S and T, X2 is selected from the group consisting of A, G, I, L, M, N, Q, R, T, and Y, X3 is selected from the group consisting of E, K, L, T, and Q, and X4 is selected from the group consisting of G, I, K, L, M, R, T, and V. In some instances, the insertion further comprises five amino acids wherein X5 is selected from the group consisting of A, D, G, P, L, Q, and V. In some instances, the insertion further comprises at least six amino acids, wherein X6 is selected from the group consisting of F, K, L, N, P, Q, S, and V. In some instances, the insertion further comprises at least seven amino acids, wherein X7 is selected from the group consisting of I, K, L, P, S, and V. 
     In some embodiments, Xl, X2, X3, X4, X5, X6, and X7 are contiguous (X1-X2-X3-X4-X5-X6-X7). In some embodiments, any two of Xl, X2, X3, X4, X5, X6, and X7 are contiguous. In some embodiments, any three of Xl, X2, X3, X4, X5, X6, and X7 are contiguous. In some embodiments, any four of Xl, X2, X3, X4, X5, X6, and X7 are contiguous. In some embodiments, any five of Xl, X2, X3, X4, X5, X6, and X7 are contiguous. In some embodiments, any six of Xl, X2, X3, X4, X5, X6, and X7 are contiguous. In some embodiments, any seven of Xl, X2, X3, X4, X5, X6, and X7 are contiguous. In some embodiments, Xl, X2, X3, X4, X5, X6, and X7 are not contiguous. In some embodiments, the insertion is not TLAVPFK. 
     The 7-mers disclosed herein, in some cases share a common motif. A 7-mer (X1-X2-X3-X4-X5-X6-X7) may in some cases advantageously have a T in position Xl, an L in position X2, a P in positive X5, an F in position X6, and a K or L in position X7. In some embodiments, the 7-mer comprises T-L-X3-X4-P-F-K, wherein X3 and X4 are any amino acid. In some embodiments, the 7-mer comprises T-L-X3-X4-P-F-L, wherein X3 and X4 are any amino acid. In some cases, X3 is not an A. In some cases, X3 is A, S, Q, or E or L. In some cases, X4 is not a V. In some cases, X4 is R, K, V, or a Q. 
     In some cases, the 7-mer may be X1 L A V PF K, wherein X1 is any amino acid other than T, S, or N; X1-X2-A-V-P-F-K, wherein X1 is any amino acid other than T, S, or N, and X2 is any amino acid other than L or V; or Xl-X2-X3-V-P-F-K, wherein X1 is any amino acid other than T, S, or N, X2 is any amino acid other than L or V, and X3 is any amino acid other than A, S, Q, P, or T; or Xl-X2-X3-X4-P-F-K, wherein X1 is any amino acid other than T, S, or N, X2 is any amino acid other than L or V, X3 is any amino acid other than A, S, Q, P, or T, and X4 is any amino acid other than V, T, Q, N, L, or M. In some instances, the 7-mer is T-L-A-X4-P-F-K, wherein X is any amino acid other than V. In some instances, the 7-mer is T-L-A-X4-P-F-K, wherein X is any amino acid other than, T, Q, N, L, or M. 
     In some cases, the 7-mer (X1 X2 X3 X4 X5 X6 X7) comprises TALKPFL. In some instances, the 7-mer comprises TTLKPFL. In some instances, the 7-mer comprises TLQIPFK. In some instances, the 7-mer comprises TMQKPFI. In some instances, the 7-mer comprises SIERPFK. In some instances, the 7-mer comprises RYQGDSV. 
     In some instances, the AAV capsid protein comprises an insertion of at least or about three, four, five, six, or seven amino acids of an amino acid sequence T-X2 L K P F L at an amino acid position 588_589 in a parental AAV9 capsid protein (SEQ ID NO: 1), wherein X2 is A or T. In some cases, the AAV capsid protein has an increased specificity for viral transduction in brain vascular cells (GLUT1+), as compared to a reference AAV (e.g., AAV9). In some cases, the AAV capsid protein has increased specificity for viral tranduction in astrocytes, as compared to a reference AAV (e.g., AAV9). 
     In some instances, the AAV capsid protein comprises an insertion of at least or about three, four, five, six, or seven amino acids of an amino acid sequence T-X2-Q-X4-P-F-X7 at an amino acid position 588_589 in a parental AAV9 capsid protein (SEQ ID NO: 1), wherein X2 is L or M; X4 is I, K, or L; X7 is K or I. In some cases, the AAV capsid protein has increased specificity for viral transduction in neurons and astrocytes, as compared to a reference AAV (e.g., AAV9). In some instances, the amino acid sequence is TLQIPFK. In some instances, the amino acid sequence is TMQKPFI. In some instances, the amino acid sequence is TLQLPFK. 
     In some instances, the AAV capsid protein comprises an insertion of at least or about three, four, five, six, or seven amino acids of an amino acid sequence S-I E R P F K at an amino acid position 588_589 in a parental AAV9 capsid protein (SEQ ID NO: 1). In some cases, the AAV capsid protein has increased specificity for viral transduction in neurons and astrocytes, as compared to a reference AAV (e.g., AAV9). 
     In some instances, the AAV capsid protein comprises an insertion of at least or about three, four, five, six, or seven amino acids of an amino acid sequence R-Y-Q-G-D-S-V at an amino acid position 588_589 in a parental AAV9 capsid protein (SEQ ID NO: 1). In some cases, the AAV capsid protein has increased specificity for viral transduction in astrocytes, as compared to a reference AAV (e.g., AAV9). 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 List of 7-mer targeting peptides that can target 
               
               
                 the CNS with greater efficiency and specificity 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                   
                   
                 Brain- 
               
               
                 SEQ 
                   
                 SEQ 
                 7 mer 
                 mean- 
               
               
                 ID 
                 Sequence (7 mer Amino 
                 ID 
                 Amino 
                 enrich 
               
               
                 NO 
                 acid) 
                 NO 
                 acid 
                 (log10) 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 10 
                 ACGTTGCAGATTCCTTTTAAG 
                 435 
                 TLQIPFK 
                 2.730563 
               
               
                   
               
               
                 11 
                 ACCGCCCTCAAACCCTTCCTC 
                 436 
                 TALKPFL 
                 2.536397 
               
               
                   
               
               
                 12 
                 ACCACCCTCAAACCCTTCCTC 
                 437 
                 TTLKPFL 
                 2.513839 
               
               
                   
               
               
                 13 
                 AGCATCGAAAGACCCTTCAAA 
                 438 
                 SIERPFK 
                 2.357132 
               
               
                   
               
               
                 14 
                 ACCCAAAACAGACCCTTCCTC 
                 439 
                 TQNRPFL 
                 2.22812 
               
               
                   
               
               
                 15 
                 ACCATGCAAAAACCCTTCATC 
                 440 
                 TMQKPFI 
                 2.165359 
               
               
                   
               
               
                 16 
                 ACTAGTACGCGGCCGTTTTTG 
                 441 
                 TSTRPFL 
                 2.14357 
               
               
                   
               
               
                 17 
                 GGCACCTTCGTCCCCCCCACC 
                 442 
                 GTFVPPT 
                 2.110151 
               
               
                   
               
               
                 18 
                 TGGTCGACTAATGCGGGTTAT 
                 443 
                 WSTNAGY 
                 1.979177 
               
               
                   
               
               
                 19 
                 ACCCTCGAAAGACCCTTCACC 
                 444 
                 TLERPFT 
                 1.892846 
               
               
                   
               
               
                 20 
                 ACTGCTGCTAAGCCGTTTCTG 
                 445 
                 TAAKPFL 
                 1.863541 
               
               
                   
               
               
                 21 
                 ATTAGGATTGGTTATTCGCAG 
                 446 
                 IRIGYSQ 
                 1.835644 
               
               
                   
               
               
                 22 
                 GAGCGTGTGGGTTTTGCTCAG 
                 447 
                 ERVGFAQ 
                 1.73803 
               
               
                   
               
               
                 23 
                 GCCGACCTCCTCAACTACAGA 
                 448 
                 ADLLNYR 
                 1.599571 
               
               
                   
               
               
                 24 
                 CTCGTCGCCGGCTTCAGCCAA 
                 449 
                 LVAGFSQ 
                 1.591793 
               
               
                   
               
               
                 25 
                 TCGTCTCTGAAGCCTTTTCTG 
                 450 
                 SSLKPFL 
                 1.574365 
               
               
                   
               
               
                 26 
                 ACGCATTCTAGGCCGTTTATT 
                 451 
                 THSRPFI 
                 1.566468 
               
               
                   
               
               
                 27 
                 CCTTTTCCTGGTTATTCTGCG 
                 452 
                 PFPGYSA 
                 1.555967 
               
               
                   
               
               
                 28 
                 ATTATTGTTGGGTATAGTCAG 
                 453 
                 IIVGYSQ 
                 1.515858 
               
               
                   
               
               
                 29 
                 AGGTATCAGGGTGATTCTGTT 
                 454 
                 RYQGDSV 
                 1.486197 
               
               
                   
               
               
                 30 
                 AGGTATGCTGGGGATTCTATT 
                 455 
                 RYAGDSI 
                 1.456352 
               
               
                   
               
               
                 31 
                 AAGACGATTGGGGAGCATTGG 
                 456 
                 KTIGEHW 
                 1.44094 
               
               
                   
               
               
                 32 
                 TGGATGACGCATGGTTCTGCG 
                 457 
                 WMTHGSA 
                 1.432726 
               
               
                   
               
               
                 33 
                 GCTGCTTCGAGGCCGTTTCTT 
                 458 
                 AASRPFL 
                 1.3791 
               
               
                   
               
               
                 34 
                 ACTACTTCTAGGCCGTTTCTT 
                 459 
                 TTSRPFL 
                 1.353519 
               
               
                   
               
               
                 35 
                 GCTCGTGTGGGTTTGGCTCAG 
                 460 
                 ARVGLAQ 
                 1.345499 
               
               
                   
               
               
                 36 
                 GGGACTAGTCCGAATCTTGCG 
                 461 
                 GTSPNLA 
                 1.306429 
               
               
                   
               
               
                 37 
                 GTGCGTACGGGGTATAGTTCT 
                 462 
                 VRTGYSS 
                 1.302979 
               
               
                   
               
               
                 38 
                 ATTCGGGTGGGGTATAGTTCT 
                 463 
                 IRVGYSS 
                 1.286795 
               
               
                   
               
               
                 39 
                 ACTACTTTGCGTCCGTTTATT 
                 464 
                 TTLRPFI 
                 1.284391 
               
               
                   
               
               
                 40 
                 GGTATTACGTCTCTGTTTAAG 
                 465 
                 GITSLFK 
                 1.251837 
               
               
                   
               
               
                 41 
                 ACCACCCAAACCCCCTTCAGA 
                 466 
                 TTQTPFR 
                 1.192121 
               
               
                   
               
               
                 42 
                 ACTGTGTCGAGGCCGTTTTTG 
                 467 
                 TVSRPFL 
                 1.171604 
               
               
                   
               
               
                 43 
                 TATGGGACGAATCCGTTTCGT 
                 468 
                 YGTNPFR 
                 1.13664 
               
               
                   
               
               
                 44 
                 CCCACCATGGTCGACAGCAGC 
                 469 
                 PTMVDSS 
                 1.099202 
               
               
                   
               
               
                 45 
                 AATAAGGCTAGTTCTCAGAAT 
                 470 
                 NKASSQN 
                 1.084711 
               
               
                   
               
               
                 46 
                 ACTTTTCGTAGTTCGAATGAT 
                 471 
                 TFRSSND 
                 1.032118 
               
               
                   
               
               
                 47 
                 CTGCCGGATCGTTATCCTAAT 
                 472 
                 LPDRYPN 
                 1.019382 
               
               
                   
               
               
                 48 
                 CAGAATGTTACGAAGGGTGTT 
                 473 
                 QNVTKGV 
                 1.000865 
               
               
                   
               
               
                 49 
                 GCTCAGTCTCCGATTCTTCGG 
                 474 
                 AQSPILR 
                 0.979922 
               
               
                   
               
               
                 50 
                 GTGGCTACTGGTTATTCTTCG 
                 475 
                 VATGYSS 
                 0.978116 
               
               
                   
               
               
                 51 
                 TTGAATTCGAATCGGCCTAAT 
                 476 
                 LNSNRPN 
                 0.9672 
               
               
                   
               
               
                 52 
                 ACGATTCGTAATAATACGATT 
                 477 
                 TIRNNTI 
                 0.959641 
               
               
                   
               
               
                 53 
                 GATCTTAGTCGTATTTTTAAG 
                 478 
                 DLSRIFK 
                 0.950386 
               
               
                   
               
               
                 54 
                 CTCAGCAACAGCGCCAGCCAA 
                 479 
                 LSNSASQ 
                 0.941212 
               
               
                   
               
               
                 55 
                 CTCGAAAACATGGTCATGAGC 
                 480 
                 LENMVMS 
                 0.937552 
               
               
                   
               
               
                 56 
                 TTGACTCAGCAGACGGATGTT 
                 481 
                 LTQQTDV 
                 0.936974 
               
               
                   
               
               
                 57 
                 GCCGGCCTCGCCCACGCCACC 
                 482 
                 AGLAHAT 
                 0.936046 
               
               
                   
               
               
                 58 
                 AATGCTCATACTGCGCTGGAT 
                 483 
                 NAHTALD 
                 0.933184 
               
               
                   
               
               
                 59 
                 TGGCCGATTCTTCGGGCTGAT 
                 484 
                 WPILRAD 
                 0.931545 
               
               
                   
               
               
                 60 
                 GCGCTGGCTGGTCTGTCTCTG 
                 485 
                 ALAGLSL 
                 0.930454 
               
               
                   
               
               
                 61 
                 CGTATTGATATGCCTTTTAAG 
                 486 
                 RIDMPFK 
                 0.920205 
               
               
                   
               
               
                 62 
                 ACGATGTCGTTGAGTAGTAGT 
                 487 
                 TMSLSSS 
                 0.919718 
               
               
                   
               
               
                 63 
                 CAAAACGTCGCCAAAAACCTC 
                 488 
                 QNVAKNL 
                 0.916602 
               
               
                   
               
               
                 64 
                 TTCGACAACCCCAACAACGTC 
                 489 
                 FDNPNNV 
                 0.915503 
               
               
                   
               
               
                 65 
                 GTCCTCATGAGAGAAAGCCTC 
                 490 
                 VLMRESL 
                 0.914386 
               
               
                   
               
               
                 66 
                 CACCACGACACCGGCAGCGCC 
                 491 
                 HHDTGSA 
                 0.912951 
               
               
                   
               
               
                 67 
                 GATGCGCCGAATAGTGTGGAT 
                 492 
                 DAPNSVD 
                 0.901999 
               
               
                   
               
               
                 68 
                 ACTGCGAGTAGGGATGCTAAG 
                 493 
                 TASRDAK 
                 0.889552 
               
               
                   
               
               
                 69 
                 ACTAATTATACGAAGCAGATT 
                 494 
                 TNYTKQI 
                 0.889024 
               
               
                   
               
               
                 70 
                 CCTCATAATGCTTCTGTGTTG 
                 495 
                 PHNASVL 
                 0.864707 
               
               
                   
               
               
                 71 
                 ACCAACAACACCAGAAACATC 
                 496 
                 TNNTRNI 
                 0.862873 
               
               
                   
               
               
                 72 
                 ATGGTTCCTAATCATTCTGGT 
                 497 
                 MVPNHSG 
                 0.859604 
               
               
                   
               
               
                 73 
                 CCGGTGCCGCAGTCTGAGCAT 
                 498 
                 PVPQSEH 
                 0.852761 
               
               
                   
               
               
                 74 
                 CTCTACCACGGCGGCAGCACC 
                 499 
                 LYHGGST 
                 0.851158 
               
               
                   
               
               
                 75 
                 ATGATTCATACTAGGGAGACG 
                 500 
                 MIHTRET 
                 0.850116 
               
               
                   
               
               
                 76 
                 ACTAATAATACGAAGCCTCTT 
                 501 
                 TNNTKPL 
                 0.849557 
               
               
                   
               
               
                 77 
                 CTCAACACCACCAGAAACACC 
                 502 
                 LNTTRNT 
                 0.839304 
               
               
                   
               
               
                 78 
                 GCTTTGCATAATTCTCAGAAT 
                 503 
                 ALHNSQN 
                 0.827469 
               
               
                   
               
               
                 79 
                 GTGAATAGTACGAGGAATGTT 
                 504 
                 VNSTRNV 
                 0.826565 
               
               
                   
               
               
                 80 
                 GGTGTTGGGGTTGTTACGAAG 
                 505 
                 GVGVVTK 
                 0.820556 
               
               
                   
               
               
                 81 
                 GTTTTGTCGAAGCAGTTGGCT 
                 506 
                 VLSKQLA 
                 0.816495 
               
               
                   
               
               
                 82 
                 TCTAAGTCTCTGCAGCCGTTG 
                 507 
                 SKSLQPL 
                 0.815502 
               
               
                   
               
               
                 83 
                 TCGAATTCTACGCGGTTGGTT 
                 508 
                 SNSTRLV 
                 0.815119 
               
               
                   
               
               
                 84 
                 CTCAACCAAACCAAACAAATC 
                 509 
                 LNQTKQI 
                 0.811665 
               
               
                   
               
               
                 85 
                 AACAACAGCGTCAGACAACTC 
                 510 
                 NNSVRQL 
                 0.806765 
               
               
                   
               
               
                 86 
                 ATGTCGGGGTATTCGCATACT 
                 511 
                 MSGYSHT 
                 0.804194 
               
               
                   
               
               
                 87 
                 GTCAAAGTCCCCATCATCACC 
                 512 
                 VKVPIIT 
                 0.799412 
               
               
                   
               
               
                 88 
                 GGGAATAATACTCGGGCGGTG 
                 513 
                 GNNTRAV 
                 0.797634 
               
               
                   
               
               
                 89 
                 CAGAATCAGATTAAGAATATT 
                 514 
                 QNQIKNI 
                 0.797303 
               
               
                   
               
               
                 90 
                 ACCAACATGACCAAACCCCTC 
                 515 
                 TNMTKPL 
                 0.791064 
               
               
                   
               
               
                 91 
                 AACAACAGCACCAGACTCAGC 
                 516 
                 NNSTRLS 
                 0.790429 
               
               
                   
               
               
                 92 
                 AGCATGCTCACCAGCATCGTC 
                 517 
                 SMLTSIV 
                 0.790252 
               
               
                   
               
               
                 93 
                 CTCAACGCCACCGCCAGCAGA 
                 518 
                 LNATASR 
                 0.785269 
               
               
                   
               
               
                 94 
                 AATACTGGGGTGCAGGTTAGG 
                 519 
                 NTGVQVR 
                 0.785026 
               
               
                   
               
               
                 95 
                 ACCAACACCGTCAGACCCATC 
                 520 
                 TNTVRPI 
                 0.780569 
               
               
                   
               
               
                 96 
                 CAGTCTACGCCGGGGGCTACT 
                 521 
                 QSTPGAT 
                 0.779842 
               
               
                   
               
               
                 97 
                 GGTCATTCTGAGAATTCTCGT 
                 522 
                 GHSENSR 
                 0.778724 
               
               
                   
               
               
                 98 
                 CTCCCCACCCAAACCCTCAGA 
                 523 
                 LPTQTLR 
                 0.773756 
               
               
                   
               
               
                 99 
                 AAGAATGCTCCGATGCCTGAT 
                 524 
                 KNAPMPD 
                 0.772397 
               
               
                   
               
               
                 100 
                 AGCAACAGCACCAAACCCACC 
                 525 
                 SNSTKPT 
                 0.768053 
               
               
                   
               
               
                 101 
                 CCTATTATTACTATGCCTGCT 
                 526 
                 PIITMPA 
                 0.767318 
               
               
                   
               
               
                 102 
                 CCCCTCAAAGGCATCGGCAGC 
                 527 
                 PLKGIGS 
                 0.767253 
               
               
                   
               
               
                 103 
                 GTCCTCAGCGTCAACCACCTC 
                 528 
                 VLSVNHL 
                 0.765014 
               
               
                   
               
               
                 104 
                 AGTGTGGCTGCTGTGCATATG 
                 529 
                 SVAAVHM 
                 0.764013 
               
               
                   
               
               
                 105 
                 AGCAACAGCATCAGACTCGTC 
                 530 
                 SNSIRLV 
                 0.763114 
               
               
                   
               
               
                 106 
                 CATAATGATACTCGTCCTCTT 
                 531 
                 HNDTRPL 
                 0.759962 
               
               
                   
               
               
                 107 
                 CCTCCGGTGAGGTCTACGGCT 
                 532 
                 PPVRSTA 
                 0.755777 
               
               
                   
               
               
                 108 
                 ATGAACACCGTCAGAAACCTC 
                 533 
                 MNTVRNL 
                 0.754463 
               
               
                   
               
               
                 109 
                 TTGACGGATGGTCATCGGATT 
                 534 
                 LTDGHRI 
                 0.754429 
               
               
                   
               
               
                 110 
                 ACGAATGCGGTGAAGCTGACT 
                 535 
                 TNAVKLT 
                 0.752766 
               
               
                   
               
               
                 111 
                 CGTGTGGATGCGGTTTTGAGT 
                 536 
                 RVDAVLS 
                 0.750458 
               
               
                   
               
               
                 112 
                 ACCAACGCCGTCAAAAGCACC 
                 537 
                 TNAVKST 
                 0.749271 
               
               
                   
               
               
                 113 
                 ATGCTCAACACCACCGCCAGA 
                 538 
                 MLNTTAR 
                 0.747921 
               
               
                   
               
               
                 114 
                 GGCAACAACACCAGACCCACC 
                 539 
                 GNNTRPT 
                 0.747523 
               
               
                   
               
               
                 115 
                 CAGTCTGGGGGTAGTGCGTTG 
                 540 
                 QSGGSAL 
                 0.744332 
               
               
                   
               
               
                 116 
                 GTGATTGCTTTGTCTACTATG 
                 541 
                 VIALSTM 
                 0.743901 
               
               
                   
               
               
                 117 
                 CTGAATACGATTCGGAATGTG 
                 542 
                 LNTIRNV 
                 0.743811 
               
               
                   
               
               
                 118 
                 AGCAACGCCACCAGAAGCGTC 
                 543 
                 SNATRSV 
                 0.740235 
               
               
                   
               
               
                 119 
                 GGCAACGGCACCAAATTCATC 
                 544 
                 GNGTKFI 
                 0.736944 
               
               
                   
               
               
                 120 
                 AGCAACACCATCAGACCCGTC 
                 545 
                 SNTIRPV 
                 0.736197 
               
               
                   
               
               
                 121 
                 AGCAACAGCACCAGAGCCATC 
                 546 
                 SNSTRAI 
                 0.734716 
               
               
                   
               
               
                 122 
                 TTTCATTTGACTGATAGGGGT 
                 547 
                 FHLTDRG 
                 0.733831 
               
               
                   
               
               
                 123 
                 CCGATTAGGTCGGATACTGTG 
                 548 
                 PIRSDTV 
                 0.731286 
               
               
                   
               
               
                 124 
                 GCCAACCAAATCCACAACACC 
                 549 
                 ANQIHNT 
                 0.730071 
               
               
                   
               
               
                 125 
                 TCGAATACGGTTCGGAATACT 
                 550 
                 SNTVRNT 
                 0.727807 
               
               
                   
               
               
                 126 
                 ACCAGCCTCGGCTACAGCAGC 
                 551 
                 TSLGYSS 
                 0.727167 
               
               
                   
               
               
                 127 
                 ATCAGCGGCGTCCAAACCAGA 
                 552 
                 ISGVQTR 
                 0.724309 
               
               
                   
               
               
                 128 
                 TCGAATGCTGTTAGGCAGACT 
                 553 
                 SNAVRQT 
                 0.72361 
               
               
                   
               
               
                 129 
                 TCTCTTTCTGAGCTTAGGATT 
                 554 
                 SLSELRI 
                 0.723061 
               
               
                   
               
               
                 130 
                 AGTACTGAGAAGAGGGATGAG 
                 555 
                 STEKRDE 
                 0.721349 
               
               
                   
               
               
                 131 
                 AACCTCGTCGGCACCCTCATC 
                 556 
                 NLVGTLI 
                 0.72103 
               
               
                   
               
               
                 132 
                 GGCAACAACATCAGACTCACC 
                 557 
                 GNNIRLT 
                 0.719643 
               
               
                   
               
               
                 133 
                 GCCGTCCCCACCGGCCTCAGA 
                 558 
                 AVPTGLR 
                 0.717951 
               
               
                   
               
               
                 134 
                 GGTGATAATAGTCTGACTAGG 
                 559 
                 GDNSLTR 
                 0.717624 
               
               
                   
               
               
                 135 
                 CACGCCGACAACAGACTCAAA 
                 560 
                 HADNRLK 
                 0.714815 
               
               
                   
               
               
                 136 
                 AGCAACGCCACCAGAAACTTC 
                 561 
                 SNATRNF 
                 0.714052 
               
               
                   
               
               
                 137 
                 GGGAATACGACTAGGGGGCTG 
                 562 
                 GNTTRGL 
                 0.713945 
               
               
                   
               
               
                 138 
                 CTCAACCTCACCGCCACCAAC 
                 563 
                 LNLTATN 
                 0.71358 
               
               
                   
               
               
                 139 
                 AGCGGCGTCAGCACCACCGAC 
                 564 
                 SGVSTTD 
                 0.711249 
               
               
                   
               
               
                 140 
                 TATGCTAAGACGTTGGCTATG 
                 565 
                 YAKTLAM 
                 0.710703 
               
               
                   
               
               
                 141 
                 ATGAACCACACCAAACCCACC 
                 566 
                 MNHTKPT 
                 0.710702 
               
               
                   
               
               
                 142 
                 GCTAATGCTACGAGGAATTCT 
                 567 
                 ANATRNS 
                 0.707423 
               
               
                   
               
               
                 143 
                 CAAAACCAAACCAAAATCACC 
                 568 
                 QNQTKIT 
                 0.707235 
               
               
                   
               
               
                 144 
                 TCTAATGCTATTAAGGCTACG 
                 569 
                 SNAIKAT 
                 0.70699 
               
               
                   
               
               
                 145 
                 TATCCTGATAATTATGGTAAG 
                 570 
                 YPDNYGK 
                 0.706343 
               
               
                   
               
               
                 146 
                 GGCAACACCACCATCAGAAGC 
                 571 
                 GNTTIRS 
                 0.705947 
               
               
                   
               
               
                 147 
                 CTGGGTCTTACGGGTGCGGTT 
                 572 
                 LGLTGAV 
                 0.705628 
               
               
                   
               
               
                 148 
                 GCGAATAGTACGCGTATTCTG 
                 573 
                 ANSTRIL 
                 0.704068 
               
               
                   
               
               
                 149 
                 GATAATGCGATTCGTGCGCAG 
                 574 
                 DNAIRAQ 
                 0.703945 
               
               
                   
               
               
                 150 
                 ACGAATTATACTAAGCTTATG 
                 575 
                 TNYTKLM 
                 0.702916 
               
               
                   
               
               
                 151 
                 CATAATAGTCCTAGTAGTTAT 
                 576 
                 HNSPSSY 
                 0.70154 
               
               
                   
               
               
                 152 
                 GAGAATATGGTTCGGTCTGTG 
                 577 
                 ENMVRSV 
                 0.700139 
               
               
                   
               
               
                 153 
                 ACCAACAACATCAAAAGCTAC 
                 578 
                 TNNIKSY 
                 0.698201 
               
               
                   
               
               
                 154 
                 ACTAATTCGACGAGGCCTGTG 
                 579 
                 TNSTRPV 
                 0.696679 
               
               
                   
               
               
                 155 
                 CATACGGCTCCGCCTCATCCT 
                 580 
                 HTAPPHP 
                 0.695156 
               
               
                   
               
               
                 156 
                 GCCCTCAGCCAAAACACCAGC 
                 581 
                 ALSQNTS 
                 0.692869 
               
               
                   
               
               
                 157 
                 CCGAATGTGACTAAGAATGCT 
                 582 
                 PNVTKNA 
                 0.692038 
               
               
                   
               
               
                 158 
                 CTTGATACGCTGACGGGTTAT 
                 583 
                 LDTLTGY 
                 0.690142 
               
               
                   
               
               
                 159 
                 CCTAATAGTGTGCGTTCTGTT 
                 584 
                 PNSVRSV 
                 0.689082 
               
               
                   
               
               
                 160 
                 ACCAGCAGCCACCTCCCCCAC 
                 585 
                 TSSHLPH 
                 0.688469 
               
               
                   
               
               
                 161 
                 GTTCTTCATGGTGGTAATGAT 
                 586 
                 VLHGGND 
                 0.687991 
               
               
                   
               
               
                 162 
                 AGCAACTACGTCAAAAGCGCC 
                 587 
                 SNYVKSA 
                 0.687276 
               
               
                   
               
               
                 163 
                 GACAACAACAGCACCAGATGG 
                 588 
                 DNNSTRW 
                 0.687057 
               
               
                   
               
               
                 164 
                 ACTGTGATGAAGGGTTTTACT 
                 589 
                 TVMKGFT 
                 0.687036 
               
               
                   
               
               
                 165 
                 AGCCACACCCTCAGCACCCTC 
                 590 
                 SHTLSTL 
                 0.686592 
               
               
                   
               
               
                 166 
                 CCTGGTCCTCAGCAGGCGAAG 
                 591 
                 PGPQQAK 
                 0.685365 
               
               
                   
               
               
                 167 
                 ACTTTTGGGACGTCTAAGTTG 
                 592 
                 TFGTSKL 
                 0.684153 
               
               
                   
               
               
                 168 
                 AGCAACGCCGTCACCAACAGA 
                 593 
                 SNAVTNR 
                 0.683838 
               
               
                   
               
               
                 169 
                 TTGTCTATGTCGACGGTGCCT 
                 594 
                 LSMSTVP 
                 0.682136 
               
               
                   
               
               
                 170 
                 AGGCGTTATGATGGTAGGGAG 
                 595 
                 RRYDGRE 
                 0.681722 
               
               
                   
               
               
                 171 
                 ACCAACAGCACCAAAAACATC 
                 596 
                 TNSTKNI 
                 0.678439 
               
               
                   
               
               
                 172 
                 ATTAATGCTCAGTGGTCTGCG 
                 597 
                 INAQWSA 
                 0.672085 
               
               
                   
               
               
                 173 
                 GCCCAAACCAGCAACGACCCC 
                 598 
                 AQTSNDP 
                 0.669325 
               
               
                   
               
               
                 174 
                 ACGAATGAGACTAGGATGGTG 
                 599 
                 TNETRMV 
                 0.669307 
               
               
                   
               
               
                 175 
                 GCCAACGCCACCAGAGGCGTC 
                 600 
                 ANATRGV 
                 0.667153 
               
               
                   
               
               
                 176 
                 GCGGATGTGAATGCTTCGGGT 
                 601 
                 ADVNASG 
                 0.666574 
               
               
                   
               
               
                 177 
                 CTTAGTGAGCGGAATTATGTG 
                 602 
                 LSERNYV 
                 0.665675 
               
               
                   
               
               
                 178 
                 AGTGGTGAGCTTGCGCGGGCG 
                 603 
                 SGELARA 
                 0.663381 
               
               
                   
               
               
                 179 
                 GGGAATACTGCTAAGAATATT 
                 604 
                 GNTAKNI 
                 0.662988 
               
               
                   
               
               
                 180 
                 GACAACGCCGTCAGACCCCTC 
                 605 
                 DNAVRPL 
                 0.662435 
               
               
                   
               
               
                 181 
                 ATGATGCTCAGCGGCCTCAAC 
                 606 
                 MMLSGLN 
                 0.660837 
               
               
                   
               
               
                 182 
                 CCCAACACCATCAGAAACGTC 
                 607 
                 PNTIRNV 
                 0.660528 
               
               
                   
               
               
                 183 
                 GAGAATTTGACGCGTGGGGTG 
                 608 
                 ENLTRGV 
                 0.659884 
               
               
                   
               
               
                 184 
                 TTTAGTGCTCGGAGTACGGGG 
                 609 
                 FSARSTG 
                 0.659095 
               
               
                   
               
               
                 185 
                 GAAAACGCCACCAGAACCTAC 
                 610 
                 ENATRTY 
                 0.657305 
               
               
                   
               
               
                 186 
                 GGCAACAGCACCAGAATGAAC 
                 611 
                 GNSTRMN 
                 0.656999 
               
               
                   
               
               
                 187 
                 GGGAATTCTACTAAGTCTCCT 
                 612 
                 GNSTKSP 
                 0.656908 
               
               
                   
               
               
                 188 
                 ATGCAAACCACCCTCCACCTC 
                 613 
                 MQTTLHL 
                 0.656525 
               
               
                   
               
               
                 189 
                 AACCCCACCCTCATCACCCTC 
                 614 
                 NPTLITL 
                 0.655924 
               
               
                   
               
               
                 190 
                 CTTATTTCTGGTCATGCGCAG 
                 615 
                 LISGHAQ 
                 0.655121 
               
               
                   
               
               
                 191 
                 TTCAACCAAGTCAGAAGCCTC 
                 616 
                 FNQVRSL 
                 0.65493 
               
               
                   
               
               
                 192 
                 ATGAACATCCTCAGAGAAGTC 
                 617 
                 MNILREV 
                 0.654097 
               
               
                   
               
               
                 193 
                 AGTGCTATGATGCGTGGTGTT 
                 618 
                 SAMMRGV 
                 0.653566 
               
               
                   
               
               
                 194 
                 CCCATGCTCAGCAGCCCCAGC 
                 619 
                 PMLSSPS 
                 0.652269 
               
               
                   
               
               
                 195 
                 CAGAATAATACGCTTAGGTCG 
                 620 
                 QNNTLRS 
                 0.651653 
               
               
                   
               
               
                 196 
                 AGGCATTCGGATCCGGTGGAG 
                 621 
                 RHSDPVE 
                 0.65144 
               
               
                   
               
               
                 197 
                 GTGAATACGACGTTTTCTACG 
                 622 
                 VNTTFST 
                 0.65142 
               
               
                   
               
               
                 198 
                 ATCAACAGCACCAGAGGCATC 
                 623 
                 INSTRGI 
                 0.650374 
               
               
                   
               
               
                 199 
                 ACCAACGCCGTCAGAGACCTC 
                 624 
                 TNAVRDL 
                 0.649865 
               
               
                   
               
               
                 200 
                 TCTAATAGTACTAGGCTGTCG 
                 625 
                 SNSTRLS 
                 0.649567 
               
               
                   
               
               
                 201 
                 ACGTCTATTGCTGGGTCGTTT 
                 626 
                 TSIAGSF 
                 0.647793 
               
               
                   
               
               
                 202 
                 CTCGGCAGCACCTACCCCAGA 
                 627 
                 LGSTYPR 
                 0.644533 
               
               
                   
               
               
                 203 
                 GCCAACGTCACCACCCAAAGA 
                 628 
                 ANVTTQR 
                 0.643299 
               
               
                   
               
               
                 204 
                 AGCAACAGCGTCAGACTCAGC 
                 629 
                 SNSVRLS 
                 0.641635 
               
               
                   
               
               
                 205 
                 GCTACGAATGAGCCTGATCGG 
                 630 
                 ATNEPDR 
                 0.641008 
               
               
                   
               
               
                 206 
                 AGAATGAACCCCGAACAAAGC 
                 631 
                 RMNPEQS 
                 0.640866 
               
               
                   
               
               
                 207 
                 TCGGTTAGTGGTTCGGCTAAT 
                 632 
                 SVSGSAN 
                 0.640362 
               
               
                   
               
               
                 208 
                 CCGAGTAATAGTACTACGCGT 
                 633 
                 PSNSTTR 
                 0.639664 
               
               
                   
               
               
                 209 
                 CTTTTTCATGGTACGCATGAG 
                 634 
                 LFHGTHE 
                 0.639176 
               
               
                   
               
               
                 210 
                 CCCCAACTCGGCAGCACCAAA 
                 635 
                 PQLGSTK 
                 0.638022 
               
               
                   
               
               
                 211 
                 CCCATCAGCGCCAACAGACTC 
                 636 
                 PISANRL 
                 0.637814 
               
               
                   
               
               
                 212 
                 AATTCGGGTAATCTTTCTATG 
                 637 
                 NSGNLSM 
                 0.637206 
               
               
                   
               
               
                 213 
                 AAGGTTGAGGCTATTGGGATG 
                 638 
                 KVEAIGM 
                 0.63585 
               
               
                   
               
               
                 214 
                 AAGACTTTGCTTAATAGTGTT 
                 639 
                 KTLLNSV 
                 0.635567 
               
               
                   
               
               
                 215 
                 AATGCTATGGATCTTAAGGTG 
                 640 
                 NAMDLKV 
                 0.633592 
               
               
                   
               
               
                 216 
                 ATGATGGGTAATGGGTCTTCG 
                 641 
                 MMGNGSS 
                 0.632846 
               
               
                   
               
               
                 217 
                 AGCAACAGCACCAAAGCCCTC 
                 642 
                 SNSTKAL 
                 0.630646 
               
               
                   
               
               
                 218 
                 TCTAATAGTACGCGGGGTACG 
                 643 
                 SNSTRGT 
                 0.630359 
               
               
                   
               
               
                 219 
                 AGCCTCAGCAGCCTCCACGTC 
                 644 
                 SLSSLHV 
                 0.629137 
               
               
                   
               
               
                 220 
                 ACCCTCAGCGGCGCCCTCACC 
                 645 
                 TLSGALT 
                 0.628275 
               
               
                   
               
               
                 221 
                 CCGAATACGGTGAGGAATAAT 
                 646 
                 PNTVRNN 
                 0.627943 
               
               
                   
               
               
                 222 
                 AATAATAATGTGAAGATGACT 
                 647 
                 NNNVKMT 
                 0.627302 
               
               
                   
               
               
                 223 
                 TCTGTTGCTAAGCCTTTTATG 
                 648 
                 SVAKPFM 
                 0.627075 
               
               
                   
               
               
                 224 
                 ACGCAGACGACTCGGCTTTCG 
                 649 
                 TQTTRLS 
                 0.625657 
               
               
                   
               
               
                 225 
                 AATAATGCGACTCGGGGTGGG 
                 650 
                 NNATRGG 
                 0.622765 
               
               
                   
               
               
                 226 
                 ACCAACACCACCGCCAGAATC 
                 651 
                 TNTTARI 
                 0.621665 
               
               
                   
               
               
                 227 
                 GAGAATAGTATTCGGACTATT 
                 652 
                 ENSIRTI 
                 0.619021 
               
               
                   
               
               
                 228 
                 ATTAGGGAGACTTCTGGGAAG 
                 653 
                 IRETSGK 
                 0.618732 
               
               
                   
               
               
                 229 
                 AGCATCTACTACCACACCGAA 
                 654 
                 SIYYHTE 
                 0.618441 
               
               
                   
               
               
                 230 
                 GTCCTCGACAACAGCGGCCAA 
                 655 
                 VLDNSGQ 
                 0.618183 
               
               
                   
               
               
                 231 
                 AGCCACAACATGACCATCAGA 
                 656 
                 SHNMTIR 
                 0.614114 
               
               
                   
               
               
                 232 
                 ACCATCCCCAGCGCCGGCAAA 
                 657 
                 TIPSAGK 
                 0.612816 
               
               
                   
               
               
                 233 
                 CCTAGGCATACTTTGAGTCAG 
                 658 
                 PRHTLSQ 
                 0.60799 
               
               
                   
               
               
                 234 
                 TCGCTTAATGTGCAGGATGTG 
                 659 
                 SLNVQDV 
                 0.607203 
               
               
                   
               
               
                 235 
                 TATCAGTCTCTGGCTAATCCG 
                 660 
                 YQSLANP 
                 0.607005 
               
               
                   
               
               
                 236 
                 ATTCTGAATATGTCTACGGAT 
                 661 
                 ILNMSTD 
                 0.606834 
               
               
                   
               
               
                 237 
                 GTCCTCCACCTCGGCCACAGC 
                 662 
                 VLHLGHS 
                 0.605932 
               
               
                   
               
               
                 238 
                 GGTGTTACTCAGACTCCGCGT 
                 663 
                 GVTQTPR 
                 0.6059 
               
               
                   
               
               
                 239 
                 ACCAACGACACCAGAAGAATC 
                 664 
                 TNDTRRI 
                 0.604587 
               
               
                   
               
               
                 240 
                 CAAACCGACCACACCAGCAGA 
                 665 
                 QTDHTSR 
                 0.604031 
               
               
                   
               
               
                 241 
                 AAGAATGAGATTAAGAATGTG 
                 666 
                 KNEIKNV 
                 0.603838 
               
               
                   
               
               
                 242 
                 TGGACTGGTAATGAGAGGCTT 
                 667 
                 WTGNERL 
                 0.603412 
               
               
                   
               
               
                 243 
                 GGTAAGAGTGATGCTATGCGG 
                 668 
                 GKSDAMR 
                 0.603322 
               
               
                   
               
               
                 244 
                 GGGAATGCGACTCGTACTTAT 
                 669 
                 GNATRTY 
                 0.602016 
               
               
                   
               
               
                 245 
                 GCCAGCAACCTCGGCCTCCCC 
                 670 
                 ASNLGLP 
                 0.600647 
               
               
                   
               
               
                 246 
                 AGCAGCCTCAGCGGCAGCCCC 
                 671 
                 SSLSGSP 
                 0.599424 
               
               
                   
               
               
                 247 
                 GCCGCCGTCAACCAAGGCGTC 
                 672 
                 AAVNQGV 
                 0.599283 
               
               
                   
               
               
                 248 
                 GACCAAAACCAACCCAGAGAA 
                 673 
                 DQNQPRE 
                 0.59872 
               
               
                   
               
               
                 249 
                 CTCACCCAAGACAAACAAGCC 
                 674 
                 LTQDKQA 
                 0.59843 
               
               
                   
               
               
                 250 
                 TTCGGCAGCAACGAACACAAC 
                 675 
                 FGSNEHN 
                 0.59773 
               
               
                   
               
               
                 251 
                 CTGAATCAGCCGAATGTGCGG 
                 676 
                 LNQPNVR 
                 0.59703 
               
               
                   
               
               
                 252 
                 ATGAATACTACGCGTAATCTG 
                 677 
                 MNTTRNL 
                 0.596819 
               
               
                   
               
               
                 253 
                 ACTAATTCTACTAGGACGAGT 
                 678 
                 TNSTRTS 
                 0.596817 
               
               
                   
               
               
                 254 
                 TTTGAGCTTTCGCATGTGCCT 
                 679 
                 FELSHVP 
                 0.595373 
               
               
                   
               
               
                 255 
                 GAAGGCCTCGGCAACGCCGCC 
                 680 
                 EGLGNAA 
                 0.59339 
               
               
                   
               
               
                 256 
                 CTCACCGTCACCCTCAACCAC 
                 681 
                 LTVTLNH 
                 0.593104 
               
               
                   
               
               
                 257 
                 TTTGGGAATGCGATTCAGTCT 
                 682 
                 FGNAIQS 
                 0.593063 
               
               
                   
               
               
                 258 
                 CCGGGTGGGGGTTTGACTCCG 
                 683 
                 PGGGLTP 
                 0.591748 
               
               
                   
               
               
                 259 
                 CCCAACATGACCAGAAGCCTC 
                 684 
                 PNMTRSL 
                 0.591633 
               
               
                   
               
               
                 260 
                 AGTCAGCTGCATAGGTTGCAG 
                 685 
                 SQLHRLQ 
                 0.588997 
               
               
                   
               
               
                 261 
                 AATGATCCTGTGCTGACTGTG 
                 686 
                 NDPVLTV 
                 0.587622 
               
               
                   
               
               
                 262 
                 GAAAACAGCGTCAGAACCACC 
                 687 
                 ENSVRTT 
                 0.586872 
               
               
                   
               
               
                 263 
                 GATCGTGTGACTAATCCGAAG 
                 688 
                 DRVTNPK 
                 0.585898 
               
               
                   
               
               
                 264 
                 CTCAACACCGGCCAAATGAGA 
                 689 
                 LNTGQMR 
                 0.584482 
               
               
                   
               
               
                 265 
                 ATTAGTCCTTCTCAGGTGAAG 
                 690 
                 ISPSQVK 
                 0.583565 
               
               
                   
               
               
                 266 
                 TACAGCGCCGACAAAACCACC 
                 691 
                 YSADKTT 
                 0.583168 
               
               
                   
               
               
                 267 
                 ATTGATAGTGCGGGGATGGCG 
                 692 
                 IDSAGMA 
                 0.582726 
               
               
                   
               
               
                 268 
                 AGCAACAACATCAAACTCAAC 
                 693 
                 SNNIKLN 
                 0.582501 
               
               
                   
               
               
                 269 
                 CTTGAGACGCAGCCTAGGACT 
                 694 
                 LETQPRT 
                 0.582054 
               
               
                   
               
               
                 270 
                 AATTTGCATAGTAATGTGCTG 
                 695 
                 NLHSNVL 
                 0.581653 
               
               
                   
               
               
                 271 
                 ATGGTCAAAGACACCCAACTC 
                 696 
                 MVKDTQL 
                 0.581409 
               
               
                   
               
               
                 272 
                 CTCATGCACCTCACCAACCCC 
                 697 
                 LMHLTNP 
                 0.579725 
               
               
                   
               
               
                 273 
                 GTCGCCAGCCCCGACAAAAGA 
                 698 
                 VASPDKR 
                 0.579259 
               
               
                   
               
               
                 274 
                 GTCGTCGCCGTCCTCACCAGC 
                 699 
                 VVAVLTS 
                 0.578391 
               
               
                   
               
               
                 275 
                 AACATCATGGTCAACGTCCCC 
                 700 
                 NIMVNVP 
                 0.578008 
               
               
                   
               
               
                 276 
                 CCGTATAGTGGGCAGCGGACT 
                 701 
                 PYSGQRT 
                 0.577882 
               
               
                   
               
               
                 277 
                 CCTCTGCTTAAGACGAATACT 
                 702 
                 PLLKTNT 
                 0.575523 
               
               
                   
               
               
                 278 
                 GGGAATACTACGGTGCGTGGG 
                 703 
                 GNTTVRG 
                 0.574402 
               
               
                   
               
               
                 279 
                 ACCAACAAAGTCAGAGACGAC 
                 704 
                 TNKVRDD 
                 0.573889 
               
               
                   
               
               
                 280 
                 CCCAGCACCGCCCTCCTCGTC 
                 705 
                 PSTALLV 
                 0.573034 
               
               
                   
               
               
                 281 
                 ATCCAAAGCATCACCCTCAAA 
                 706 
                 IQSITLK 
                 0.572783 
               
               
                   
               
               
                 282 
                 CCCCTCCACGCCAACATGAGC 
                 707 
                 PLHANMS 
                 0.570881 
               
               
                   
               
               
                 283 
                 ACCGCCCACGCCGAAGCCAGA 
                 708 
                 TAHAEAR 
                 0.570572 
               
               
                   
               
               
                 284 
                 ACGAATACGGATTCGTATCGT 
                 709 
                 TNTDSYR 
                 0.569365 
               
               
                   
               
               
                 285 
                 ACCCACGTCAGCCTCGACAGA 
                 710 
                 THVSLDR 
                 0.569228 
               
               
                   
               
               
                 286 
                 TTTAGTACGAAGGATCATGTT 
                 711 
                 FSTKDHV 
                 0.568654 
               
               
                   
               
               
                 287 
                 GACAACACCCAAACCGCCCCC 
                 712 
                 DNTQTAP 
                 0.568553 
               
               
                   
               
               
                 288 
                 AGCAACAGAATCATCAGCGGC 
                 713 
                 SNRIISG 
                 0.568551 
               
               
                   
               
               
                 289 
                 ACTAATTCTGTTAGGAATAAT 
                 714 
                 TNSVRNN 
                 0.568237 
               
               
                   
               
               
                 290 
                 AGCAACGAAGTCAGAAACATG 
                 715 
                 SNEVRNM 
                 0.567847 
               
               
                   
               
               
                 291 
                 GAACTCTACAAACCCAGCAGA 
                 716 
                 ELYKPSR 
                 0.567258 
               
               
                   
               
               
                 292 
                 TGGATTACGGGGGGTGCGAGT 
                 717 
                 WITGGAS 
                 0.567159 
               
               
                   
               
               
                 293 
                 AACAACAGCGTCAGACCCACC 
                 718 
                 NNSVRPT 
                 0.566945 
               
               
                   
               
               
                 294 
                 CACACCGCCGTCCTCAGAACC 
                 719 
                 HTAVLRT 
                 0.565911 
               
               
                   
               
               
                 295 
                 AATACTTTGTCTCTTGCTCCT 
                 720 
                 NTLSLAP 
                 0.564955 
               
               
                   
               
               
                 296 
                 CCCAACGCCAGCGTCAACAGC 
                 721 
                 PNASVNS 
                 0.564885 
               
               
                   
               
               
                 297 
                 GTTACTGGTGGGAATACTTAT 
                 722 
                 VTGGNTY 
                 0.564099 
               
               
                   
               
               
                 298 
                 GTCAACGAAAACCTCATCGAA 
                 723 
                 VNENLIE 
                 0.563902 
               
               
                   
               
               
                 299 
                 GTTTTGACGACTCATTCGAAT 
                 724 
                 VLTTHSN 
                 0.563552 
               
               
                   
               
               
                 300 
                 CACACCCAACTCCCCCTCACC 
                 725 
                 HTQLPLT 
                 0.562802 
               
               
                   
               
               
                 301 
                 CGGCAGTCTCTTGAGGCGTTG 
                 726 
                 RQSLEAL 
                 0.562715 
               
               
                   
               
               
                 302 
                 CCCGACGTCACCGAAGGCAGA 
                 727 
                 PDVTEGR 
                 0.560929 
               
               
                   
               
               
                 303 
                 CCGACTAATATTATGCTTGAT 
                 728 
                 PTNIMLD 
                 0.560313 
               
               
                   
               
               
                 304 
                 CCCAACATGAGCGCCATGATC 
                 729 
                 PNMSAMI 
                 0.559451 
               
               
                   
               
               
                 305 
                 ATTATGAATGGTCAGGCTCTG 
                 730 
                 IMNGQAL 
                 0.558677 
               
               
                   
               
               
                 306 
                 AATAATAATTCGACGCGTTTT 
                 731 
                 NNNSTRF 
                 0.558656 
               
               
                   
               
               
                 307 
                 GCCAACGTCACCAGAAGCACC 
                 732 
                 ANVTRST 
                 0.558653 
               
               
                   
               
               
                 308 
                 CAGAATCTTGGTCTTAATGTG 
                 733 
                 QNLGLNV 
                 0.558202 
               
               
                   
               
               
                 309 
                 CAAGCCCAAATGAGCAGCGCC 
                 734 
                 QAQMSSA 
                 0.557238 
               
               
                   
               
               
                 310 
                 CTCACCATGGCCACCAACGTC 
                 735 
                 LTMATNV 
                 0.556779 
               
               
                   
               
               
                 311 
                 GATCGTGATTCTGTGCAGAAT 
                 736 
                 DRDSVQN 
                 0.55654 
               
               
                   
               
               
                 312 
                 GTCAACCCCACCAACAACCTC 
                 737 
                 VNPTNNL 
                 0.555076 
               
               
                   
               
               
                 313 
                 CCGTCTTTTACGACTATGAGT 
                 738 
                 PSFTTMS 
                 0.554811 
               
               
                   
               
               
                 314 
                 AGGGAGACGCCTATTCCTAAG 
                 739 
                 RETPIPK 
                 0.553803 
               
               
                   
               
               
                 315 
                 AGCCTCAACTTCACCACCGCC 
                 740 
                 SLNFTTA 
                 0.553055 
               
               
                   
               
               
                 316 
                 GGTATGAGTGGGGTTGCTCAG 
                 741 
                 GMSGVAQ 
                 0.552736 
               
               
                   
               
               
                 317 
                 AAGGTTGATGCGGCTCAGAGT 
                 742 
                 KVDAAQS 
                 0.552569 
               
               
                   
               
               
                 318 
                 CATATTACGACTAATGTGTCT 
                 743 
                 HITTNVS 
                 0.552479 
               
               
                   
               
               
                 319 
                 GAGCGGGAGTCGGCTCGTCTT 
                 744 
                 ERESARL 
                 0.551876 
               
               
                   
               
               
                 320 
                 GATAATAGGACTCAGAGGACG 
                 745 
                 DNRTQRT 
                 0.551636 
               
               
                   
               
               
                 321 
                 TCTGCGCCTAATGTGACTATT 
                 746 
                 SAPNVTI 
                 0.551278 
               
               
                   
               
               
                 322 
                 GAAAACGGCACCAGAAACACC 
                 747 
                 ENGTRNT 
                 0.55012 
               
               
                   
               
               
                 323 
                 GGCACCGCCGTCTTCACCGCC 
                 748 
                 GTAVFTA 
                 0.549912 
               
               
                   
               
               
                 324 
                 GCCAACAACATGAGCCAAACC 
                 749 
                 ANNMSQT 
                 0.549519 
               
               
                   
               
               
                 325 
                 GGTAATCATACTTATAATCTG 
                 750 
                 GNHTYNL 
                 0.549264 
               
               
                   
               
               
                 326 
                 CTGAGTCCTTCGAATTCTAAT 
                 751 
                 LSPSNSN 
                 0.549019 
               
               
                   
               
               
                 327 
                 ATGGGCGCCGACCACAGAACC 
                 752 
                 MGADHRT 
                 0.548749 
               
               
                   
               
               
                 328 
                 AGTAATTCGACGCGTGGTTCT 
                 753 
                 SNSTRGS 
                 0.548705 
               
               
                   
               
               
                 329 
                 AATACTTCTGGTGTGCCTAAG 
                 754 
                 NTSGVPK 
                 0.548579 
               
               
                   
               
               
                 330 
                 ATCAACACCGCCGTCGTCAGC 
                 755 
                 INTAVVS 
                 0.54846 
               
               
                   
               
               
                 331 
                 ATGGGGGTTCAGGCTTATGTG 
                 756 
                 MGVQAYV 
                 0.54845 
               
               
                   
               
               
                 332 
                 GGGAATTTTGTTAAGCCGAAT 
                 757 
                 GNFVKPN 
                 0.547942 
               
               
                   
               
               
                 333 
                 GTGAATTCGGTGAGGATGATT 
                 758 
                 VNSVRMI 
                 0.547171 
               
               
                   
               
               
                 334 
                 CACGACAGCGCCAACAGCAGA 
                 759 
                 HDSANSR 
                 0.54671 
               
               
                   
               
               
                 335 
                 CTCGGCGTCAGAGACGACAGC 
                 760 
                 LGVRDDS 
                 0.545708 
               
               
                   
               
               
                 336 
                 AGCAACGCCGTCAGAGCCAAC 
                 761 
                 SNAVRAN 
                 0.545674 
               
               
                   
               
               
                 337 
                 CCCGTCCTCGCCGCCACCATG 
                 762 
                 PVLAATM 
                 0.545458 
               
               
                   
               
               
                 338 
                 AATAAGCCTGTGTCTGGTAAT 
                 763 
                 NKPVSGN 
                 0.544076 
               
               
                   
               
               
                 339 
                 GTCGGCCTCGCCAGCCACACC 
                 764 
                 VGLASHT 
                 0.541637 
               
               
                   
               
               
                 340 
                 GGCAGCAACACCAACATGAAA 
                 765 
                 GSNTNMK 
                 0.540634 
               
               
                   
               
               
                 341 
                 GTTGCTACTACTGTGCATAAT 
                 766 
                 VATTVHN 
                 0.540191 
               
               
                   
               
               
                 342 
                 AACACCCTCGACAGCAGAGTC 
                 767 
                 NTLDSRV 
                 0.539923 
               
               
                   
               
               
                 343 
                 AGGGAGGCTAATTTGCAGGCG 
                 768 
                 REANLQA 
                 0.539717 
               
               
                   
               
               
                 344 
                 GTCGACGCCCTCAGCCACATG 
                 769 
                 VDALSHM 
                 0.539694 
               
               
                   
               
               
                 345 
                 CTTCGTCTTGCTGGTCTTGCT 
                 770 
                 LRLAGLA 
                 0.539525 
               
               
                   
               
               
                 346 
                 ATTACGGTGACTAATTATTCG 
                 771 
                 ITVTNYS 
                 0.539447 
               
               
                   
               
               
                 347 
                 TGGGACAGCGGCAGCGGCGAA 
                 772 
                 WDSGSGE 
                 0.539198 
               
               
                   
               
               
                 348 
                 TTCGCCAGCGAAACCGTCGCC 
                 773 
                 FASETVA 
                 0.537906 
               
               
                   
               
               
                 349 
                 CAGAATGTTACGGGGACGAGG 
                 774 
                 QNVTGTR 
                 0.537867 
               
               
                   
               
               
                 350 
                 CTTAATGGGTTGAATGTGTCT 
                 775 
                 LNGLNVS 
                 0.537505 
               
               
                   
               
               
                 351 
                 AATATGCTTAAGCAGAGTGAG 
                 776 
                 NMLKQSE 
                 0.537354 
               
               
                   
               
               
                 352 
                 TTTAATGAGGTTCCGAAGGCG 
                 777 
                 FNEVPKA 
                 0.537144 
               
               
                   
               
               
                 353 
                 CTCGGCGACATCACCGGCTTC 
                 778 
                 LGDITGF 
                 0.537047 
               
               
                   
               
               
                 354 
                 ATTTCGGCTTCTCATTCTCGT 
                 779 
                 ISASHSR 
                 0.536983 
               
               
                   
               
               
                 355 
                 AGTCAGACGCAGATTGCTCTT 
                 780 
                 SQTQIAL 
                 0.53684 
               
               
                   
               
               
                 356 
                 AATATGGCTACTCAGATGAAG 
                 781 
                 NMATQMK 
                 0.536747 
               
               
                   
               
               
                 357 
                 GTCAACCACAACATCAGACTC 
                 782 
                 VNHNIRL 
                 0.53652 
               
               
                   
               
               
                 358 
                 AACGCCCTCCAAGTCCCCGTC 
                 783 
                 NALQVPV 
                 0.536474 
               
               
                   
               
               
                 359 
                 AATTCGACGGGTATTGATACG 
                 784 
                 NSTGIDT 
                 0.53526 
               
               
                   
               
               
                 360 
                 GTTGTTGCTGGGCATCTTAAT 
                 785 
                 VVAGHLN 
                 0.534827 
               
               
                   
               
               
                 361 
                 CGGAATGATGCGATTCTGAAT 
                 786 
                 RNDAILN 
                 0.53456 
               
               
                   
               
               
                 362 
                 AATCGGGTTGATTCGCGGGCT 
                 787 
                 NRVDSRA 
                 0.53408 
               
               
                   
               
               
                 363 
                 TCGACGCATTCTACTTATGTG 
                 788 
                 STHSTYV 
                 0.533753 
               
               
                   
               
               
                 364 
                 AGTGGGCATGGGACTCTGCGG 
                 789 
                 SGHGTLR 
                 0.532267 
               
               
                   
               
               
                 365 
                 CCCAACAGCACCACCCTCAGC 
                 790 
                 PNSTTLS 
                 0.531723 
               
               
                   
               
               
                 366 
                 GACACCAAACCCACCAACACC 
                 791 
                 DTKPTNT 
                 0.531589 
               
               
                   
               
               
                 367 
                 AACAGAGACACCATCAACAAC 
                 792 
                 NRDTINN 
                 0.531212 
               
               
                   
               
               
                 368 
                 CCCAGAACCCTCAGCGACGGC 
                 793 
                 PRTLSDG 
                 0.530962 
               
               
                   
               
               
                 369 
                 ACCAAAGACGTCACCACCAGC 
                 794 
                 TKDVTTS 
                 0.53023 
               
               
                   
               
               
                 370 
                 CTGGGTAATCCTACGCCTTCT 
                 795 
                 LGNPTPS 
                 0.529624 
               
               
                   
               
               
                 371 
                 TATCAGGCGATGTATAGGGAT 
                 796 
                 YQAMYRD 
                 0.528592 
               
               
                   
               
               
                 372 
                 AGCGGCGTCCAAGAATTCGAC 
                 797 
                 SGVQEFD 
                 0.528493 
               
               
                   
               
               
                 373 
                 TCTAATCATCTGTCGACGGTT 
                 798 
                 SNHLSTV 
                 0.528046 
               
               
                   
               
               
                 374 
                 CACGACGAAAGAGCCAACATG 
                 799 
                 HDERANM 
                 0.526932 
               
               
                   
               
               
                 375 
                 ACGAATAGTACTCGGTCGCCT 
                 800 
                 TNSTRSP 
                 0.526707 
               
               
                   
               
               
                 376 
                 CCGACGGATAAGTCTTATCCG 
                 801 
                 PTDKSYP 
                 0.525687 
               
               
                   
               
               
                 377 
                 ATGACCGAACAAAGAACCGCC 
                 802 
                 MTEQRTA 
                 0.524729 
               
               
                   
               
               
                 378 
                 ACCAACGCCACCCACAGCAAA 
                 803 
                 TNATHSK 
                 0.523113 
               
               
                   
               
               
                 379 
                 AATCGGGGTACTTTTAGTGCG 
                 804 
                 NRGTFSA 
                 0.522986 
               
               
                   
               
               
                 380 
                 AACACCACCAAATTCAACACC 
                 805 
                 NTTKFNT 
                 0.522899 
               
               
                   
               
               
                 381 
                 GGGATTCCGCCTTTGACTAAG 
                 806 
                 GIPPLTK 
                 0.522659 
               
               
                   
               
               
                 382 
                 ACCGCCAACAGCACCCAAAGA 
                 807 
                 TANSTQR 
                 0.522626 
               
               
                   
               
               
                 383 
                 ACTCAGGGTCCGGGTCCGAAG 
                 808 
                 TQGPGPK 
                 0.522239 
               
               
                   
               
               
                 384 
                 ATGAATACTACGCGGAATTAT 
                 809 
                 MNTTRNY 
                 0.522193 
               
               
                   
               
               
                 385 
                 CTCAGAGAAGGCACCTTCATG 
                 810 
                 LREGTFM 
                 0.521554 
               
               
                   
               
               
                 386 
                 AACATCAGCAGCACCGACAGA 
                 811 
                 NISSTDR 
                 0.521154 
               
               
                   
               
               
                 387 
                 TCGGTTAGTCGGCCGTTTCAG 
                 812 
                 SVSRPFQ 
                 0.520365 
               
               
                   
               
               
                 388 
                 CCTATGTCTGCTAATGGGAAG 
                 813 
                 PMSANGK 
                 0.518792 
               
               
                   
               
               
                 389 
                 GAAGTCAGAGGCAACACCTTC 
                 814 
                 EVRGNTF 
                 0.518514 
               
               
                   
               
               
                 390 
                 TCGCAGAAGGGTCTGTCTGTG 
                 815 
                 SQKGLSV 
                 0.518477 
               
               
                   
               
               
                 391 
                 ATTGCTGAGAATCTGAGTGCT 
                 816 
                 IAENLSA 
                 0.517935 
               
               
                   
               
               
                 392 
                 TTCAGCAACATGAACCTCAAA 
                 817 
                 FSNMNLK 
                 0.517734 
               
               
                   
               
               
                 393 
                 CCTCGTGAGCGGACTTATACT 
                 818 
                 PRERTYT 
                 0.516835 
               
               
                   
               
               
                 394 
                 TGGTCGCATAATCCGGATTCT 
                 819 
                 WSHNPDS 
                 0.516248 
               
               
                   
               
               
                 395 
                 GGTCTTCAGACTCTGATTACG 
                 820 
                 GLQTLIT 
                 0.516123 
               
               
                   
               
               
                 396 
                 AGCAACAGAACCAAAGACATC 
                 821 
                 SNRTKDI 
                 0.515515 
               
               
                   
               
               
                 397 
                 GTGACTGAGACGATTCTTAAG 
                 822 
                 VTETILK 
                 0.515459 
               
               
                   
               
               
                 398 
                 TATGTTACGCGTAGTGGTCTG 
                 823 
                 YVTRSGL 
                 0.515319 
               
               
                   
               
               
                 399 
                 ATGAACGCCAACATCCTCGTC 
                 824 
                 MNANILV 
                 0.514679 
               
               
                   
               
               
                 400 
                 ACTACGAATCGGGTGGGTACG 
                 825 
                 TTNRVGT 
                 0.51427 
               
               
                   
               
               
                 401 
                 GTCCTCAAAAGCCACCTCCAA 
                 826 
                 VLKSHLQ 
                 0.51406 
               
               
                   
               
               
                 402 
                 AGCGACGGCCTCAGAACCCAA 
                 827 
                 SDGLRTQ 
                 0.514048 
               
               
                   
               
               
                 403 
                 CTCAACAGCGTCAAACCCAAC 
                 828 
                 LNSVKPN 
                 0.513525 
               
               
                   
               
               
                 404 
                 AGCCAAGCCATCGCCCCCAAA 
                 829 
                 SQAIAPK 
                 0.51343 
               
               
                   
               
               
                 405 
                 TCTAATAATGTTCGTGCTCCG 
                 830 
                 SNNVRAP 
                 0.513257 
               
               
                   
               
               
                 406 
                 ATCAGCCACCTCAGCGAAAGC 
                 831 
                 ISHLSES 
                 0.511503 
               
               
                   
               
               
                 407 
                 CTCCTCCCCGGCATGAGATTC 
                 832 
                 LLPGMRF 
                 0.5114 
               
               
                   
               
               
                 408 
                 CTTCTTGCGTCGGCTACGAAG 
                 833 
                 LLASATK 
                 0.510718 
               
               
                   
               
               
                 409 
                 AACCACAACGGCATGGTCAGC 
                 834 
                 NHNGMVS 
                 0.510709 
               
               
                   
               
               
                 410 
                 CACAGCGGCGAACTCAACAAC 
                 835 
                 HSGELNN 
                 0.510488 
               
               
                   
               
               
                 411 
                 CTTTCTGCGCCTAAGGATACT 
                 836 
                 LSAPKDT 
                 0.509967 
               
               
                   
               
               
                 412 
                 ATTGGGACTCATGTGGGGACG 
                 837 
                 IGTHVGT 
                 0.509748 
               
               
                   
               
               
                 413 
                 GCCGTCGCCCTCGCCAGCGCC 
                 838 
                 AVALASA 
                 0.509614 
               
               
                   
               
               
                 414 
                 ATTACTCTTCAGTCTAATGCT 
                 839 
                 ITLQSNA 
                 0.509556 
               
               
                   
               
               
                 415 
                 GTCGACCACAGCCTCACCAGC 
                 840 
                 VDHSLTS 
                 0.509193 
               
               
                   
               
               
                 416 
                 GGCAACGAAGCCACCAGCCTC 
                 841 
                 GNEATSL 
                 0.508729 
               
               
                   
               
               
                 417 
                 CCCGTCAGCAGCACCACCCTC 
                 842 
                 PVSSTTL 
                 0.507682 
               
               
                   
               
               
                 418 
                 CCCTACCAAACCAGCAGCGCC 
                 843 
                 PYQTSSA 
                 0.507351 
               
               
                   
               
               
                 419 
                 ACCAGCGTCGTCAGCGCCTTC 
                 844 
                 TSVVSAF 
                 0.507058 
               
               
                   
               
               
                 420 
                 AACGGCGTCCCCGACAACAAC 
                 845 
                 NGVPDNN 
                 0.50693 
               
               
                   
               
               
                 421 
                 AGCACCCAAACCATCGGCTTC 
                 846 
                 STQTIGF 
                 0.505905 
               
               
                   
               
               
                 422 
                 ATTCGGAGTTCTGATCTTGCG 
                 847 
                 IRSSDLA 
                 0.505273 
               
               
                   
               
               
                 423 
                 AACACCCAAAGCGCCAAATAC 
                 848 
                 NTQSAKY 
                 0.504002 
               
               
                   
               
               
                 424 
                 AGTCTTGAGTCGTCTAGGCAG 
                 849 
                 SLESSRQ 
                 0.503575 
               
               
                   
               
               
                 425 
                 CTCAACCTCGCCAGCGGCAGA 
                 850 
                 LNLASGR 
                 0.503463 
               
               
                   
               
               
                 426 
                 ATGCAGGAGCGTCGGGAGTAT 
                 851 
                 MQERREY 
                 0.503055 
               
               
                   
               
               
                 427 
                 TGGCTCCTCAAAGACGGCTAC 
                 852 
                 WLLKDGY 
                 0.502893 
               
               
                   
               
               
                 428 
                 GGCAGATACCAAACCACCAGC 
                 853 
                 GRYQTTS 
                 0.502776 
               
               
                   
               
               
                 429 
                 AATACGACGAAGTTTCCGTTT 
                 854 
                 NTTKFPF 
                 0.502408 
               
               
                   
               
               
                 430 
                 ATGAGCAACGGCATCAGCAGA 
                 855 
                 MSNGISR 
                 0.501897 
               
               
                   
               
               
                 431 
                 CCGATTCGGGATCCGGAGAAG 
                 856 
                 PIRDPEK 
                 0.501479 
               
               
                   
               
               
                 432 
                 AGTACGCTGAGTCCGCATGTT 
                 857 
                 STLSPHV 
                 0.501315 
               
               
                   
               
               
                 433 
                 TTCATGCCCATCAGCAGCGCC 
                 858 
                 FMPISSA 
                 0.50124 
               
               
                   
               
               
                 434 
                 AGAAGCGACACCCAAAGCAGC 
                 859 
                 RSDTQSS 
                 0.500462 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 11-mer targeting peptides that can target the CNS 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                   
                   
                 Mean 
               
               
                 SEQ 
                   
                 SEQ 
                   
                 enrichment 
               
               
                 ID 
                   
                 ID 
                 11-mer Amino 
                 score (log 10 
               
               
                 NO 
                 11-mer DNA Sequence 
                 NO 
                 Acid Sequence 
                 scale) 
               
               
                   
               
               
                 860 
                 GCCCAAACCACCCTCAAACCCTTCAGCAACC 
                 864 
                 AQTTLKPFSN 
                 1.101507461 
               
               
                   
                 CC 
                   
                 P 
                   
               
               
                   
               
               
                 861 
                 GACGGCACCACCCTCAAACCCTTCAGCAACC 
                 865 
                 DGTTLKPFSN 
                 0.172425316 
               
               
                   
                 CC 
                   
                 P 
                   
               
               
                   
               
               
                 862 
                 GACGGCACCGCCCTCAAACCCTTCCTCGCCC 
                 866 
                 DGTALKPFLA 
                 0.647416071 
               
               
                   
                 AA 
                   
                 Q 
                   
               
               
                   
               
               
                 863 
                 GATGGGACGACTCTTAAGCCGTTTCTGGCAC 
                 867 
                 DGTTLKPFLA 
                 sequence 
               
               
                   
                 AG 
                   
                 Q 
                 predicted based 
               
               
                   
                   
                   
                   
                 on PHP.eB 
               
               
                   
                   
                   
                   
                 sequence 
               
               
                   
               
            
           
         
       
     
     AAV Capsid Proteins Targeting the Liver 
     Disclosed herein are AAV capsid proteins with a substitution or an insertion of at least one amino acid at an amino acid position described above in a parental AAV capsid protein that confers an increased specificity for the liver. In some instances, the insertion comprises at least one amino acid provided in any one of the sequences provided in Table 4 and/or  FIG. 35 . In some instances, the insertion comprises at least two amino acids provided in any one of the sequences provided in Table 4 and/or  FIG. 35 . In some instances, the insertion comprises at least three amino acids provided in any one of the sequences provided in Table 4 and/or  FIG. 35 . In some instances, the insertion comprises at least four amino acids provided in any one of the sequences provided in Table 4 and/or  FIG. 35 . In some instances, the insertion comprises at least five amino acids provided in Table 4 and/or  FIG. 35 . In some instances, the insertion comprises at least six amino acids provided in Table 4 and/or  FIG. 35 . In some instances, the insertion comprises at least seven amino acids provided in Table 4 and/or  FIG. 35 . In some instances, the amino acids are contiguous. In some instances, the amino acids are not contiguous. In some instances, the insertion is at an amino acid position 588_589 in a parental AAV capsid protein. In some instances, the parental capsid protein is AAV9 capsid protein, provided in SEQ ID NO: 1. 
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 List of 7-mer targeting peptides that target the liver 
               
            
           
           
               
               
               
               
               
            
               
                 SEQ ID 
                   
                 SEQ ID 
                 7 mer amino 
                 liver-mean- 
               
               
                 NO 
                 DNA sequence for 7 mer amino acid 
                 NO 
                 acid 
                 enrich (log10) 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 868 
                 GCCGAATACAACACCGGCGTC 
                 950 
                 AEYNTGV 
                 0.585999 
               
               
                   
               
               
                 869 
                 GCTAATGCGATGGGTGATCAT 
                 951 
                 ANAMGDH 
                 0.611245 
               
               
                   
               
               
                 870 
                 GAAAGCATCCAACAACTCAGC 
                 952 
                 ESIQQLS 
                 0.525712 
               
               
                   
               
               
                 871 
                 TTTAAGGTTAGTATTCAGCAG 
                 953 
                 FKVSIQQ 
                 0.591275 
               
               
                   
               
               
                 872 
                 TTTCTTCTTACTTCGGATCCG 
                 954 
                 FLLTSDP 
                 0.515975 
               
               
                   
               
               
                 873 
                 TTTCAGCAGGATACTTATCTG 
                 955 
                 FQQDTYL 
                 0.548808 
               
               
                   
               
               
                 874 
                 TTTTCTGGTAGTGCTAGGGTT 
                 956 
                 FSGSARV 
                 0.509852 
               
               
                   
               
               
                 875 
                 TTCAGCGTCACCAGACAAGCC 
                 957 
                 FSVTRQA 
                 0.718621 
               
               
                   
               
               
                 876 
                 GGGGCTGCTGGTAAGAGTTTG 
                 958 
                 GAAGKSL 
                 0.571307 
               
               
                   
               
               
                 877 
                 GGTCATATTTCGGCGCTTGCG 
                 959 
                 GHISALA 
                 0.552655 
               
               
                   
               
               
                 878 
                 GGGCTGAAGGTGTCTGGGTCT 
                 960 
                 GLKVSGS 
                 0.777335 
               
               
                   
               
               
                 879 
                 GGTCTTCAGTCGCCGAGGTCT 
                 961 
                 GLQSPRS 
                 0.572095 
               
               
                   
               
               
                 880 
                 GGGAGGACGGTGCAGGATAGG 
                 962 
                 GRTVQDR 
                 0.528631 
               
               
                   
               
               
                 881 
                 GGCACCAAAACCCACAGCCTC 
                 963 
                 GTKTHSL 
                 0.518459 
               
               
                   
               
               
                 882 
                 GGCACCACCAGAAGCCCCACC 
                 964 
                 GTTRSPT 
                 0.560705 
               
               
                   
               
               
                 883 
                 CACCCCAGCCTCAGACAAAGC 
                 965 
                 HPSLRQS 
                 0.508478 
               
               
                   
               
               
                 884 
                 CATTCTGCGAAGACTATTGCG 
                 966 
                 HSAKTIA 
                 0.579949 
               
               
                   
               
               
                 885 
                 CACAGCGGCCCCACCAGCAGC 
                 967 
                 HSGPTSS 
                 0.505295 
               
               
                   
               
               
                 886 
                 ATCAACAAAGACAACAACCAA 
                 968 
                 INKDNNQ 
                 0.531458 
               
               
                   
               
               
                 887 
                 ATCAACACCGCCAGCTACAAA 
                 969 
                 INTASYK 
                 0.542125 
               
               
                   
               
               
                 888 
                 AAAGCCATCAGCACCGCCAAC 
                 970 
                 KAISTAN 
                 0.616958 
               
               
                   
               
               
                 889 
                 AAAGCCAACAGCCAAATCACC 
                 971 
                 KANSQIT 
                 0.519425 
               
               
                   
               
               
                 890 
                 AAAGCCTACAGCGTCCAAGTC 
                 972 
                 KAYSVQV 
                 1.15776 
               
               
                   
               
               
                 891 
                 AAACTCAGCACCCTCCACCAA 
                 973 
                 KLSTLHQ 
                 0.568791 
               
               
                   
               
               
                 892 
                 AAGCGGGCGCCTGCTGATTCT 
                 974 
                 KRAPADS 
                 0.611303 
               
               
                   
               
               
                 893 
                 AAAACCAACAACACCGTCCTC 
                 975 
                 KTNNTVL 
                 0.504377 
               
               
                   
               
               
                 894 
                 AAAACCCAACTCGGCCAAATG 
                 976 
                 KTQLGQM 
                 0.519843 
               
               
                   
               
               
                 895 
                 CTTGGTTCTGCGCTTACGAGG 
                 977 
                 LGSALTR 
                 0.563314 
               
               
                   
               
               
                 896 
                 CTCATCAGCACCACCCACAGA 
                 978 
                 LISTTHR 
                 0.502083 
               
               
                   
               
               
                 897 
                 CTTAAGGTTGCGTCTGGTTTG 
                 979 
                 LKVASGL 
                 0.540598 
               
               
                   
               
               
                 898 
                 CTTCTTGCGACTGGGCTTAAG 
                 980 
                 LLATGLK 
                 0.545453 
               
               
                   
               
               
                 899 
                 CTTCCGGGTGGTGCGAGGTTG 
                 981 
                 LPGGARL 
                 0.564086 
               
               
                   
               
               
                 900 
                 CTTAGGGGTTCTGCTCAGGTG 
                 982 
                 LRGSAQV 
                 0.648123 
               
               
                   
               
               
                 901 
                 TTGTCTGCTCAGCTTCCGCGG 
                 983 
                 LSAQLPR 
                 0.503052 
               
               
                   
               
               
                 902 
                 CTTTCTACTGCGTTGACTGTG 
                 984 
                 LSTALTV 
                 0.591029 
               
               
                   
               
               
                 903 
                 CTCACCAGAGTCGGCACCGTC 
                 985 
                 LTRVGTV 
                 0.520831 
               
               
                   
               
               
                 904 
                 ATGGCCAACACCAGAAGCGCC 
                 986 
                 MANTRSA 
                 0.525721 
               
               
                   
               
               
                 905 
                 ATGATCAGAGGCACCAGCAGC 
                 987 
                 MIRGTSS 
                 0.512891 
               
               
                   
               
               
                 906 
                 ATGCTCAGCGGCCAAGCCAGA 
                 988 
                 MLSGQAR 
                 0.668364 
               
               
                   
               
               
                 907 
                 ATGAGAGGCCAAGGCGTCCAC 
                 989 
                 MRGQGVH 
                 0.567657 
               
               
                   
               
               
                 908 
                 AACAACAACCAAAAAGTCTAC 
                 990 
                 NNNQKVY 
                 0.610002 
               
               
                   
               
               
                 909 
                 AACAACAGCGGCCACATCAGC 
                 991 
                 NNSGHIS 
                 0.518228 
               
               
                   
               
               
                 910 
                 AACACCCTCATCAACGTCAGC 
                 992 
                 NTLINVS 
                 0.607212 
               
               
                   
               
               
                 911 
                 AATACTTCTGGTGTGCCTAAG 
                 993 
                 NTSGVPK 
                 0.684638 
               
               
                   
               
               
                 912 
                 AATGTGCCGACTTCGCCGCGG 
                 994 
                 NVPTSPR 
                 0.520245 
               
               
                   
               
               
                 913 
                 CCCGCCAGAATCCCCGGCAGC 
                 995 
                 PARIPGS 
                 0.50309 
               
               
                   
               
               
                 914 
                 CCCCTCAGAAACATCAGCGCC 
                 996 
                 PLRNISA 
                 0.50497 
               
               
                   
               
               
                 915 
                 CCGTTGAGTGGGGGTGTTCGT 
                 997 
                 PLSGGVR 
                 0.598554 
               
               
                   
               
               
                 916 
                 CCCAACAGAGCCGCCAACAAC 
                 998 
                 PNRAANN 
                 0.508143 
               
               
                   
               
               
                 917 
                 CCGCGTCATGCGACTCAGTCG 
                 999 
                 PRHATQS 
                 0.711312 
               
               
                   
               
               
                 918 
                 CCGCGGCCGGTGTCGAATGGG 
                 1000 
                 PRPVSNG 
                 0.7305 
               
               
                   
               
               
                 919 
                 CCTTCTGGTTCTGCGCGGAGT 
                 1001 
                 PSGSARS 
                 0.82811 
               
               
                   
               
               
                 920 
                 CCCAGCCACGCCAGAGCCAGC 
                 1002 
                 PSHARAS 
                 0.520336 
               
               
                   
               
               
                 921 
                 CCCGTCGGCACCAGAACCAGC 
                 1003 
                 PVGTRTS 
                 0.560759 
               
               
                   
               
               
                 922 
                 AGAGACACCGTCAGCAGATAC 
                 1004 
                 RDTVSRY 
                 0.535765 
               
               
                   
               
               
                 923 
                 AGAATCAGCACCCTCGGCCTC 
                 1005 
                 RISTLGL 
                 0.595844 
               
               
                   
               
               
                 924 
                 AGGCTTGATAGGACTGGTTTG 
                 1006 
                 RLDRTGL 
                 0.588542 
               
               
                   
               
               
                 925 
                 AGACTCACCAACAGCAACCAA 
                 1007 
                 RLTNSNQ 
                 0.516161 
               
               
                   
               
               
                 926 
                 CGTCAGCATCAGCTTCCTATG 
                 1008 
                 RQHQLPM 
                 0.505918 
               
               
                   
               
               
                 927 
                 AGAACCGCCAACGCCCTCGGC 
                 1009 
                 RTANALG 
                 0.802247 
               
               
                   
               
               
                 928 
                 CGGACGGATGTGCGGACGAAT 
                 1010 
                 RTDVRTN 
                 0.682169 
               
               
                   
               
               
                 929 
                 CGGACTACGGCTAATTCGCTT 
                 1011 
                 RTTANSL 
                 0.509233 
               
               
                   
               
               
                 930 
                 AGCGGCGGCACCAGAGAAGGC 
                 1012 
                 SGGTREG 
                 0.501512 
               
               
                   
               
               
                 931 
                 AGCATCAGAGCCCCCGTCAGC 
                 1013 
                 SIRAPVS 
                 0.572426 
               
               
                   
               
               
                 932 
                 TCGATTTCTCCTCCTCGTACG 
                 1014 
                 SISPPRT 
                 0.550578 
               
               
                   
               
               
                 933 
                 TCGAAGGTTACGCCTCCTCTG 
                 1015 
                 SKVTPPL 
                 0.687491 
               
               
                   
               
               
                 934 
                 AGCAGCAGACTCAGCGTCGGC 
                 1016 
                 SSRLSVG 
                 0.637971 
               
               
                   
               
               
                 935 
                 AGCACCAGAAACGTCGTCGGC 
                 1017 
                 STRNVVG 
                 0.532567 
               
               
                   
               
               
                 936 
                 TCGGTTCCTCCGAATAGGAAT 
                 1018 
                 SVPPNRN 
                 0.508166 
               
               
                   
               
               
                 937 
                 ACGGCTGCGCATGTTAGTTAT 
                 1019 
                 TAAHVSY 
                 0.520336 
               
               
                   
               
               
                 938 
                 ACCAGAAGCGAAGTCATGAAA 
                 1020 
                 TRSEVMK 
                 0.683884 
               
               
                   
               
               
                 939 
                 GTGGCGGGTCTGACTGTTCAG 
                 1021 
                 VAGLTVQ 
                 0.581695 
               
               
                   
               
               
                 940 
                 GTTGGTTCTAGTAATACGTAT 
                 1022 
                 VGSSNTY 
                 0.502229 
               
               
                   
               
               
                 941 
                 GTTAAGACTGATCGGGTTCTG 
                 1023 
                 VKTDRVL 
                 0.51183 
               
               
                   
               
               
                 942 
                 GTCAACAGCCACGAAAGAGCC 
                 1024 
                 VNSHERA 
                 0.573043 
               
               
                   
               
               
                 943 
                 GTCCAAGTCGCCATGAGAGCC 
                 1025 
                 VQVAMRA 
                 0.520831 
               
               
                   
               
               
                 944 
                 GTCAGCATCAGCGTCATGGGC 
                 1026 
                 VSISVMG 
                 0.510987 
               
               
                   
               
               
                 945 
                 GTGACGGGGGTTCGGATTGGG 
                 1027 
                 VTGVRIG 
                 0.75699 
               
               
                   
               
               
                 946 
                 TGGTCTGATCCTAGTGATCTG 
                 1028 
                 WSDPSDL 
                 0.59828 
               
               
                   
               
               
                 947 
                 TACCTCACCAGCGGCGGCTAC 
                 1029 
                 YLTSGGY 
                 0.538789 
               
               
                   
               
               
                 948 
                 TACCCCCACATGACCCACGAC 
                 1030 
                 YPHMTHD 
                 0.655794 
               
               
                   
               
               
                 949 
                 TATGTTAATAGTGCTCCGAAG 
                 1031 
                 YVNSAPK 
                 0.551681 
               
               
                   
               
            
           
         
       
     
     The AAV capsids and AAV capsid proteins disclosed herein, in some embodiments, are isolated. In some instances, the AAV capsids and AAV capsid proteins disclosed herein are isolated and purified. In addition, the AAV capsids and AAV capsid proteins disclosed herein, either isolated and purified, or not, may be formulated into a pharmaceutical formulation, which in some cases, further comprises a pharmaceutically acceptable carrier. 
     B. Heterologous Nucleic Acids 
     Disclosed herein are therapeutic nucleic acids useful for the treatment or prevention of a disease or condition, or symptom of the disease or condition, disclosed herein. In some embodiments, the therapeutic nucleic acids encode a therapeutic gene expression product. Non-limiting examples of gene expression products include proteins, polypeptides, peptides, enzymes, antibodies, antigen binding fragments, nucleic acid (RNA, DNA, antisense oligonucleotide, siRNA, and the like), and gene editing components, for use in the treatment, prophylaxis, and/or amelioration of the disease or disorder, or symptoms of the disease or disorder. In some instances, the therapeutic nucleic acids are placed in an organism, cell, tissue or organ of a subject by way of a rAAV, such as those disclosed herein. 
     Disclosed herein are rAAVs, each comprising a viral vector (e.g., a single stranded DNA molecule (ssDNA)). In some instances, the viral vector comprises two inverted terminal repeat (ITR) sequences that are about 145 bases each, flanking a transgene. In some embodiments, the transgene comprises a therapeutic nucleic acid, and in some cases, a promoter in cis with the therapeutic nucleic acid in an open reading frame (ORF). The promoter is capable of initiating transcription of therapeutic nucleic acid in the nucleus of the target cell. The ITR sequences can be from any AAV serotype. Non-limiting examples of AAV serotypes include AV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12. In some cases, an ITR is from AAV2. In some cases, an ITR is from AAV9. 
     Disclosed herein are transgenes that can comprise any number of nucleotides. In some cases, a transgene can comprise less than about 100 nucleotides. In some cases, a transgene can comprise at least about 100 nucleotides. In some cases, a transgene can comprise at least about 200 nucleotides. In some cases, a transgene can comprise at least about 300 nucleotides. In some cases, a transgene can comprise at least about 400 nucleotides. In some cases, a transgene can comprise at least about 500 nucleotides. In some cases, a transgene can comprise at least about 1000 nucleotides. In some cases, a transgene can comprise at least about 5000 nucleotides. In some cases, a transgene can comprise at least about 10,000 nucleotides. In some cases, a transgene can comprise at least about 20,000 nucleotides. In some cases, a transgene can comprise at least about 30,000 nucleotides. In some cases, a transgene can comprise at least about 40,000 nucleotides. In some cases, a transgene can comprise at least about 50,000 nucleotides. In some cases, a transgene can comprise between about 500 and about 5000 nucleotides. In some cases, a transgene can comprise between about 5000 and about 10,000 nucleotides. In any of the cases disclosed herein, the transgene can comprise DNA, RNA, or a hybrid of DNA and RNA. In some cases, the transgene can be single stranded. In some cases, the transgene can be double stranded. 
     Disclosed herein are transgenes useful for modulating the expression or activity of a target gene or gene expression product thereof. In some instances, the transgene is encapsidated by an rAAV capsid protein of an rAAV particle described herein. In some instances, the rAAV particle is delivered to a subject to treat a disease or condition disclosed herein in the subject. In some instances, the delivery is systemic (e.g., intravenous, intranasal). 
     The transgenes disclosed herein are useful for expressing an endogenous gene at a level similar to that of a healthy or normal individual. This is particularly useful in the treatment of a disease or condition related to the underexpression, or lack of expression, of a gene expression product. In some embodiments, the transgenes disclosed herein are useful for overexpressing an endogenous gene, such that an expression level of the endogenous gene is above the expression level of a healthy or normal individual. Additionally, transgenes can be used to express exogenous genes (e.g., active agent such as an antibody, peptide, nucleic acid, or gene editing components). In some embodiments, the therapeutic gene expression product is capable of altering, enhancing, increasing, or inducing the activity of one or more endogenous biological processes in the cell. In some embodiments, the transgenes disclosed herein are useful for reducing expressing an endogenous gene, example, a dominant negative gene. In some embodiments, the therapeutic gene expression product is capable of altering, inhibiting, reducing, preventing, eliminating, or impairing the activity of one or more endogenous biological processes in the cell. In some aspects, the increase of gene expression refers to an increase by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%. In one aspect, the protein product of the targeted gene may be increased by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%. In some aspects, the decrease of gene expression refers to an increase by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%. In one aspect, the protein product of the targeted gene may be decreased by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%. 
     When endogenous sequences (endogenous or part of a transgene) are expressed with a transgene, the endogenous sequences can be full-length sequences (wild-type or mutant) or partial sequences. The endogenous sequences can be functional. Non-limiting examples of the function of these full length or partial sequences include increasing the serum half-life of the polypeptide expressed by a transgene (e.g., therapeutic gene) and/or acting as a carrier. 
     A transgene can be inserted into an endogenous gene such that all, some or none of the endogenous gene is expressed. For example, a transgene as described herein can be inserted into an endogenous locus such that some (N-terminal and/or C-terminal to a transgene) or none of the endogenous sequences are expressed, for example as a fusion with a transgene. In other cases, a transgene (e.g., with or without additional coding sequences of the endogenous gene) is integrated into any endogenous locus, for example a safe-harbor locus. For example, a Frataxin (FXN) transgene can be inserted into an endogenous FXN gene. A transgene can be inserted into any gene, e.g., the genes as described herein. 
     At least one advantage of the present disclosure is that virtually any therapeutic nucleic acid may be used to express any therapeutic gene expression product. In some instances, the therapeutic gene expression product is a therapeutic protein or a peptide (e.g., antibody, antigen-binding fragment, peptide, or protein). In one embodiment the protein encoded by the therapeutic nucleic acid is between 50-5000 amino acids in length. In some embodiments the protein encoded is between 50-2000 amino acids in length. In some embodiments the protein encoded is between 50-1000 amino acids in length. In some embodiments the protein encoded is between 50-1500 amino acids in length. In some embodiments the protein encoded is between 50-800 amino acids in length. In some embodiments the protein encoded is between 50-600 amino acids in length. In some embodiments the protein encoded is between 50-400 amino acids in length. In some embodiments the protein encoded is between 50-200 amino acids in length. In some embodiments the protein encoded is between 50-100 amino acids in length. In some embodiments the peptide encoded is between 4-50 amino acids in length. In some embodiments, the protein encoded is a tetrapeptide, a pentapeptide, a hexapeptide, a heptapeptide, an octapeptide, a nonapeptide, or a decapeptide. In some embodiments, the protein encoded comprises a peptide of 2-30 amino acids, such as for example 5-30, 10-30, 2-25, 5-25, 10-25, or 10-20 amino acids. In some embodiments, the protein encoded comprises a peptide of at least 11, 12, 13, 14, 15, 17, 20, 25 or 30 amino acids, or a peptide that is no longer than 50 amino acids, e.g. no longer than 35, 30, 25, 20, 17, 15, 14, 13, 12, 11 or 10 amino acids. 
     Non-limiting examples of therapeutic protein or peptides include an adrenergic agonist, an anti-apoptosis factor, an apoptosis inhibitor, a cytokine receptor, a cytokine, a cytotoxin, an erythropoietic agent, a glutamic acid decarboxylase, a glycoprotein, a growth factor, a growth factor receptor, a hormone, a hormone receptor, an interferon, an interleukin, an interleukin receptor, a kinase, a kinase inhibitor, a nerve growth factor, a netrin, a neuroactive peptide, a neuroactive peptide receptor, a neurogenic factor, a neurogenic factor receptor, a neuropilin, a neurotrophic factor, a neurotrophin, a neurotrophin receptor, an N-methyl-D-aspartate antagonist, a plexin, a protease, a protease inhibitor, a protein decarboxylase, a protein kinase, a protein kinsase inhibitor, a proteolytic protein, a proteolytic protein inhibitor, a semaphoring, a semaphorin receptor, a serotonin transport protein, a serotonin uptake inhibitor, a serotonin receptor, a serpin, a serpin receptor, and a tumor suppressor. In certain embodiments, the therapeutic protein or peptide is selected from the group consisting of brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), macrophage colony-stimulating factor (CSF), epidermal growth factor (EGF), fibroblast growth factor (FGF), gonadotropin, interferon-gamma (IFN), insulin-like growth factor 1 (IFG-1), nerve growth factor (NGF), platelet-derived growth factor (PDGF), pigment epithelium-derived factor (PEDF), transforming growth factor (TGF), transforming growth factor-beta (TGF-B), tumor necrosis factor (TNF), vascular endothelial growth factor (VEGF), prolactin, somatotropin, X-linked inhibitor of apoptosis protein 1 (XIAP1), interleukin 1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-10, viral IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, and IL-18. 
     A therapeutic gene expression product can comprise gene editing components. Non-limiting examples of gene editing components include those required for CRISPR/Cas, artificial site-specific RNA endonuclease (ASRE), zinc finger endonuclease (ZFN), and transcription factor like effector nuclease (TALEN). In a non-limiting example, a subject having Huntington&#39;s disease is identified. The subject is then systemically administered a first amount of a rAAV encapsidating a viral vector encoding ZFN engineered to represses the transcription of the Huntingtin (HTT) gene. In some instances, the route of administration is intravenous. The rAAV will include a modified AAV capsid protein that includes an amino acid sequence provided in any one of Tables 2-3, or  FIG. 33 , so as to allow proper targeting of the ZFN to the nervous system, while retargeting off-target organs, such as the liver. If needed, the subject is administered a second or third dose of the rAAV, until a therapeutically effective amount of the ZFN is expressed in the subject&#39;s nervous system. In another non-limiting example, a subject with cystic fibrosis is identified. The subject is then systemically administered a first amount of a rAAV encapsidating a viral vector encoding ZFN engineered to represses the transcription of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. In some instances, the route of administration is intranasal (e.g., intranasal spray). The rAAV will include a modified AAV capsid protein that includes an amino acid sequence provided in  FIG. 33  or Tables 2-3, so as to allow proper targeting of the ZFN to the lung. If needed, the subject is administered a second or third dose of the rAAV, until a therapeutically effective amount of the ZFN is expressed in the subject&#39;s lung. 
     A therapeutic nucleic acid can comprise a non-protein coding gene e.g., sequences encoding antisense RNAs, RNAi, shRNAs and micro RNAs (miRNAs), miRNA sponges or decoys, recombinase delivery for conditional gene deletion, conditional (recombinase-dependent) expression, includes those required for the gene editing components described herein. The non-protein coding gene may also encode a tRNA, rRNA, tmRNA, piRNA, double stranded RNA, snRNA, snoRNA, and/or long non-coding RNA (IncRNA). In some cases, the non-protein coding gene can modulate the expression or the activity of a target gene or gene expression product. For example, the RNAs described herein may be used to inhibit gene expression in a target cell, for example, a cell in the central nervous system (CNS) or peripheral organ (e.g., lung). In some cases, inhibition of gene expression refers to an inhibition by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%. In some cases, the protein product of the targeted gene may be inhibited by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%. The gene can be either a wild type gene or a gene with at least one mutation. The targeted protein may be either a wild type protein or a protein with at least one mutation. 
     A therapeutic nucleic acid can modulate the expression or activity of a gene or gene expression product expressed from the gene that is implicated in disease or disorder of the brain. For example, the therapeutic nucleic acid, in some cases is a gene or a modified version of the gene described herein. In another example, the therapeutic nucleic acid comprises an effector gene expression product such as a gene editing component specific to target a gene therein. Non-limited examples of genes include Sarcoglycan Alpha (SGCA), glutamic acid decarboxylase 65 (GAD65), glutamic acid decarboxylase 67 (GAD67), CLN2 gene, Nerve Growth Factor (NGF), glial cell derived neurotrophic factor(GDNF), Neurturin, Survival Of Motor Neuron 1, Telomeric (SMN1), β-Glucocerebrosidase (GCase), Frataxin (FXN), Huntingtin (HTN), methyl-CpG binding protein 2 (MECP2), peroxisomal biogenesis factor (PEX), progranulin (GRN), an antitubulin agent, copper-zinc superoxide dismutase (SOD1), Glucosylceramidase Beta (GBA), NPC Intracellular Cholesterol Transporter 1 (NPC1), and NPS3. In some embodiments, the peroxisomal biogenesis factor (PEX) is selected from the group consisting of PEX1, PEX2, PEX3, PEX4, PEX5, PEX6, PEX7, PEX10, PEX11(3, PEX12, PEX13, PEX14, PEX16, PEX19, and PEX26. In some instances, the gene or gene expression product is inhibited. In some instances, the gene or gene expression product is enhanced. 
     A therapeutic nucleic acid modulates expression or activity of a gene or gene expression product expressed from the gene that is implicated in disease or disorder of a particular organ (e.g., lung, heart, liver, muscle, eye). Non-limited examples of genes include Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), Factor X (FIX), RPE65, Retinoid Isomerohydrolase (RPE65), Sarcoglycan Alpha (SGCA), and sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2a). In some embodiments, the therapeutic gene expression product is of human, murine, avian, porcine, bovine, ovine, feline, canine, equine, epine, caprine, lupine or primate origin. In some instances, the gene or gene expression product is inhibited. In some instances, the gene or gene expression product is enhanced. 
     C. AAV Vectors 
     Disclosed herein are adeno-associated virus (AAV) vectors comprising genetic information. AAV vectors described herein are useful for the assembly of a rAAV and viral packaging of a heterologous nucleic acid. In addition, an AAV vector may encode a transgene comprising the heterologous nucleic acid. In some instances, the AAV vector is from an AAV serotypes selected from the group consisting of AV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12. In some instances, the AAV vector is selected from a modified AAV serotype selected from the group consisting of AAV-PHP.B, AAV-PHP.eB, and AAV-PHP.S. 
     An AAV vector can comprise a transgene, which in some cases encodes a heterologous gene expression product (e.g., therapeutic gene expression product, recombinant capsid protein, and the like). The transgene is in cis with two inverted terminal repeats (ITRs) flanking the transgene. The transgene may comprise a therapeutic nucleic acid encoding a therapeutic gene expression product. Due to the limited packaging capacity of the rAAV (˜2.5 kB), in some cases, a longer transgene may be split between two AAV vectors, the first with 3′ splice donor and the second with a 5′ splice acceptor. Upon co-infection of a cell, concatemers form, which are spliced together to express a full-length transgene. 
     A transgene is generally inserted so that its expression is driven by the endogenous promoter at the integration site, namely the promoter that drives expression of the endogenous gene into which a transgene is inserted. In some instances, a transgene comprises a promoter and/or enhancer, for example a constitutive promoter or an inducible or tissue/cell specific promoter. As a non-limiting example, the promoter may be CMV promoter, a CMV-(3-Actin-intron-(3-Globin hybrid promoter (CAG), CBA promoter, FRDA or FXN promoter, UBC promoter, GUSB promoter, NSE promoter, Synapsin promoter, MeCP2 promoter, GFAP promoter, H1 promoter, U6 promoter, NFL promoter, NFH promoter, SCN8A promoter, or PGK promoter. As a non-limiting example, promoters can be tissue-specific expression elements include, but are not limited to, human elongation factor 1a-subunit (EF1a), immediate-early cytomegalovirus (CMV), chicken β-actin (CBA) and its derivative CAG, the (3 glucuronidase (GUSB), and ubiquitin C (UBC). The transgene may include a tissue-specific expression elements for neurons such as, but not limited to, neuron-specific enolase (NSE), platelet-derived growth factor (PDGF), platelet-derived growth factor B-chain (PDGF-(3), the synapsin (Syn), the methyl-CpG binding protein 2 (MeCP2), Ca2+/calmodulin-dependent protein kinase II (CaMKII), metabotropic glutamate receptor 2 (mGluR2), NFL, NFH, np32, PPE, Enk and EAAT2 promoters. The transgene may comprise a tissue-specific expression element for astrocytes such as, but not limited to, the glial fibrillary acidic protein (GFAP) and EAAT2 promoters. The transgene may comprise tissue-specific expression elements for oligodendrocytes such as, but not limited to, the myelin basic protein (MBP) promoter. 
     In some embodiments, the promoter is less than 1 kb. The promoter may have a length of 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800 or more than 800. The promoter may have a length between 200-300, 200-400, 200-500, 200-600, 200-700, 200-800, 300-400, 300-500, 300-600, 300-700, 300-800, 400-500, 400-600, 400-700, 400-800, 500-600, 500-700, 500-800, 600-700, 600-800 or 700-800. The promoter may provide expression of the therapeutic gene expression product for a period of time in targeted tissues such as, but not limited to, the central nervous system and peripheral organs (e.g., lung). Expression of the therapeutic gene expression product may be for a period of 1 hour, 2, hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 3 weeks, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 21 years, 22 years, 23 years, 24 years, 25 years, 26 years, 27 years, 28 years, 29 years, 30 years, 31 years, 32 years, 33 years, 34 years, 35 years, 36 years, 37 years, 38 years, 39 years, 40 years, 41 years, 42 years, 43 years, 44 years, 45 years, 46 years, 47 years, 48 years, 49 years, 50 years, 55 years, 60 years, 65 years, or more than 65 years. Expression of the payload may be for 1-5 hours, 1-12 hours, 1-2 days, 1-5 days, 1-2 weeks, 1-3 weeks, 1-4 weeks, 1-2 months, 1-4 months, 1-6 months, 2-6 months, 3-6 months, 3-9 months, 4-8 months, 6-12 months, 1-2 years, 1-5 years, 2-5 years, 3-6 years, 3-8 years, 4-8 years or 5-10 years or 10-15 years, or 15-20 years, or 20-25 years, or 25-30 years, or 30-35 years, or 35-40 years, or 40-45 years, or 45-50 years, or 50-55 years, or 55-60 years, or 60-65 years. 
     An AAV vector can comprise a genome of a helper virus. Helper virus proteins are required for the assembly of a recombinant AAV (rAAV), and packaging of a transgene containing a heterologous nucleic acid into the rAAV. The helper virus genes are adenovirus genes E4, E2a and VA, that when expressed in the cell, assist with AAV replication. In some embodiments, an AAV vector comprises E2. In some embodiments, an AAV vector comprises E4. In some embodiments, an AAV vector comprises VA. In some instances, the AAV vector comprises one of helper virus proteins, or any combination. 
     An AAV vector can comprise a viral genome comprising a nucleic acid encoding the recombinant AAV (rAAV) capsid protein described herein. The viral genome can comprise a 
     Replication (Rep) gene encoding a Rep protein, and Capsid (Cap) gene encoding an AAP protein in the first open reading frame (ORF1) or a Cap protein in the second open reading frame (ORF2). The Rep protein is selected from the group consisting of Rep78, Rep68, Rep52, and Rep40. In some instances, the Cap gene is modified encoding a modified AAV capsid protein described herein. A wild-type Cap gene encodes three proteins, VP1, VP2, and VP3. In some cases, VP1 is modified. In some cases, VP2 is modified. In some cases, VP3 is modified. In some cases, all three VP1-VP3 are modified. The AAV vector can comprise nucleic acids encoding wild-type Rep78, Rep68, Rep52, Rep40 and AAP proteins. 
     Disclosed herein are AAV vectors comprising any one of SEQ ID NOS: 10-434, 860-863, 868-949, 1068-5661, 14841-14880, and 14961-15053 which are the DNA sequences encoding modified portions of AAV capsid proteins of the present disclosure. In some instances, the AAV vector comprises a nucleic acid sequences provided in any one of SEQ ID NOS: 10-434 and 868-949, encoding 7-mer modified AAV capsid protein portions. The AAV vector of the present disclosure can comprise the VP1 Cap gene comprises any one of SEQ ID NOS: 6-9 provided in Table 5. An AAV vector can comprise 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of any one of SEQ ID NOS: 6-9. 
     In some instances, the AAV9 VP1 gene provided in SEQ ID NO: 6, which is provided in Table 5, is modified to include any one of SEQ ID NOS: 10-434, 860-863, 868-949, 1068-5661, 14841-14880, and 14961-15053. In some instances, the AAV-PHP.eB VP1 (SEQ ID NO: 9), which is also provided in Table 5 is modified to include any one of SEQ ID NOS: 10-434, 860-863, 868-949, 1068-5661, 14841-14880, and 14961-15053. The AAV vector described herein may be used to produce a variant AAV capsid by the methods described herein. 
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 VP1 Capsid Protein Nucleic Acid Sequences 
               
            
           
           
               
               
               
            
               
                 SEQ 
                   
                   
               
               
                 ID NO: 
                 Identifier 
                 Sequence 
               
               
                   
               
               
                 6 
                 AAV9 
                 ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAA 
               
               
                   
                 &gt;AY530579.1 
                 CCTTAGTGAAGGAATTCGCGAGTGGTGGGCTTTGAAACCTG 
               
               
                   
                 Adeno- 
                 GAGCCCCTCAACCCAAGGCAAATCAACAACATCAAGACAA 
               
               
                   
                 associated 
                 CGCTCGAGGTCTTGTGCTTCCGGGTTACAAATACCTTGGACC 
               
               
                   
                 virus 9 
                 CGGCAACGGACTCGACAAGGGGGAGCCGGTCAACGCAGCA 
               
               
                   
                 isolate 
                 GACGCGGCGGCCCTCGAGCACGACAAGGCCTACGACCAGC 
               
               
                   
                 hu.14 capsid 
                 AGCTCAAGGCCGGAGACAACCCGTACCTCAAGTACAACCAC 
               
               
                   
                 protein VP1 
                 GCCGACGCCGAGTTCCAGGAGCGGCTCAAAGAAGATACGTC 
               
               
                   
                 (cap) gene, 
                 TTTTGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAAA 
               
               
                   
                 complete 
                 AGAGGCTTCTTGAACCTCTTGGTCTGGTTGAGGAAGCGGCT 
               
               
                   
                 cds 
                 AAGACGGCTCCTGGAAAGAAGAGGCCTGTAGAGCAGTCTCC 
               
               
                   
                   
                 TCAGGAACCGGACTCCTCCGCGGGTATTGGCAAATCGGGTG 
               
               
                   
                   
                 CACAGCCCGCTAAAAAGAGACTCAATTTCGGTCAGACTGGC 
               
               
                   
                   
                 GACACAGAGTCAGTCCCAGACCCTCAACCAATCGGAGAACC 
               
               
                   
                   
                 TCCCGCAGCCCCCTCAGGTGTGGGATCTCTTACAATGGCTTC 
               
               
                   
                   
                 AGGTGGTGGCGCACCAGTGGCAGACAATAACGAAGGTGCC 
               
               
                   
                   
                 GATGGAGTGGGTAGTTCCTCGGGAAATTGGCATTGCGATTC 
               
               
                   
                   
                 CCAATGGCTGGGGGACAGAGTCATCACCACCAGCACCCGAA 
               
               
                   
                   
                 CCTGGGCCCTGCCCACCTACAACAATCACCTCTACAAGCAA 
               
               
                   
                   
                 ATCTCCAACAGCACATCTGGAGGATCTTCAAATGACAACGC 
               
               
                   
                   
                 CTACTTCGGCTACAGCACCCCCTGGGGGTATTTTGACTTCAA 
               
               
                   
                   
                 CAGATTCCACTGCCACTTCTCACCACGTGACTGGCAGCGAC 
               
               
                   
                   
                 TCATCAACAACAACTGGGGATTCCGGCCTAAGCGACTCAAC 
               
               
                   
                   
                 TTCAAGCTCTTCAACATTCAGGTCAAAGAGGTTACGGACAA 
               
               
                   
                   
                 CAATGGAGTCAAGACCATCGCCAATAACCTTACCAGCACGG 
               
               
                   
                   
                 TCCAGGTCTTCACGGACTCAGACTATCAGCTCCCGTACGTGC 
               
               
                   
                   
                 TCGGGTCGGCTCACGAGGGCTGCCTCCCGCCGTTCCCAGCG 
               
               
                   
                   
                 GACGTTTTCATGATTCCTCAGTACGGGTATCTGACGCTTAAT 
               
               
                   
                   
                 GATGGAAGCCAGGCCGTGGGTCGTTCGTCCTTTTACTGCCT 
               
               
                   
                   
                 GGAATATTTCCCGTCGCAAATGCTAAGAACGGGTAACAACT 
               
               
                   
                   
                 TCCAGTTCAGCTACGAGTTTGAGAACGTACCTTTCCATAGCA 
               
               
                   
                   
                 GCTACGCTCACAGCCAAAGCCTGGACCGACTAATGAATCCA 
               
               
                   
                   
                 CTCATCGACCAATACTTGTACTATCTCTCAAAGACTATTAAC 
               
               
                   
                   
                 GGTTCTGGACAGAATCAACAAACGCTAAAATTCAGTGTGGC 
               
               
                   
                   
                 CGGACCCAGCAACATGGCTGTCCAGGGAAGAAACTACATAC 
               
               
                   
                   
                 CTGGACCCAGCTACCGACAACAACGTGTCTCAACCACTGTG 
               
               
                   
                   
                 ACTCAAAACAACAACAGCGAATTTGCTTGGCCTGGAGCTTC 
               
               
                   
                   
                 TTCTTGGGCTCTCAATGGACGTAATAGCTTGATGAATCCTGG 
               
               
                   
                   
                 ACCTGCTATGGCCAGCCACAAAGAAGGAGAGGACCGTTTCT 
               
               
                   
                   
                 TTCCTTTGTCTGGATCTTTAATTTTTGGCAAACAAGGAACTG 
               
               
                   
                   
                 GAAGAGACAACGTGGATGCGGACAAAGTCATGATAACCAA 
               
               
                   
                   
                 CGAAGAAGAAATTAAAACTACTAACCCGGTAGCAACGGAG 
               
               
                   
                   
                 TCCTATGGACAAGTGGCCACAAACCACCAGAGTGCCCAAGC 
               
               
                   
                   
                 ACAGGCGCAGACCGGCTGGGTTCAAAACCAAGGAATACTTC 
               
               
                   
                   
                 CGGGTATGGTTTGGCAGGACAGAGATGTGTACCTGCAAGGA 
               
               
                   
                   
                 CCCATTTGGGCCAAAATTCCTCACACGGACGGCAACTTTCA 
               
               
                   
                   
                 CCCTTCTCCGCTGATGGGAGGGTTTGGAATGAAGCACCCGC 
               
               
                   
                   
                 CTCCTCAGATCCTCATCAAAAACACACCTGTACCTGCGGAT 
               
               
                   
                   
                 CCTCCAACGGCCTTCAACAAGGACAAGCTGAACTCTTTCAT 
               
               
                   
                   
                 CACCCAGTATTCTACTGGCCAAGTCAGCGTGGAGATCGAGT 
               
               
                   
                   
                 GGGAGCTGCAGAAGGAAAACAGCAAGCGCTGGAACCCGGA 
               
               
                   
                   
                 GATCCAGTACACTTCCAACTATTACAAGTCTAATAATGTTGA 
               
               
                   
                   
                 ATTTGCTGTTAATACTGAAGGTGTATATAGTGAACCCCGCCC 
               
               
                   
                   
                 CATTGGCACCAGATACCTGACTCGTAATCTGTAA 
               
               
                   
               
               
                 7 
                 AAV- 
                 ATGGCTGCCG ATGGTTATCT TCCAGATTGG CTCGAGGACA 
               
               
                   
                 PHP.B 
                 ACCTTAGTGAAGGAATTCGC GAGTGGTGGG CTTTGAAACC 
               
               
                   
                   
                 TGGAGCCCCT CAACCCAAGGCAAATCAACA ACATCAAGAC 
               
               
                   
                   
                 AACGCTCGAG GTCTTGTGCT TCCGGGTTACAAATACCTTG 
               
               
                   
                   
                 GACCCGGCAA CGGACTCGAC AAGGGGGAGC GGTCAACGC 
               
               
                   
                   
                 AGCAGACGCG GCGGCCCTCG AGCACGACAA GCCTACGAC 
               
               
                   
                   
                 CAGCAGCTCAAGGCCGGAGA CAACCCGTAC CTCAAGTACA 
               
               
                   
                   
                 ACCACGCCGA CGCCGAGTTCCAGGAGCGGC TCAAAGAAGA 
               
               
                   
                   
                 TACGTCTTTT GGGGGCAACC TCGGGCGAGCAGTCTTCCAG 
               
               
                   
                   
                 GCCAAAAAGA GGCTTCTTGA ACCTCTTGGT CTGGTTGAGG 
               
               
                   
                   
                 AAGCGGCTAA GACGGCTCCT GGAAAGAAGA 
               
               
                   
                   
                 GGCCTGTAGA GCAGTCTCCTCAGGAACCGG ACTCCTCCGC 
               
               
                   
                   
                 GGGTATTGGC AAATCGGGTG CACAGCCCGCTAAAAAGAGA 
               
               
                   
                   
                 CTCAATTTCG GTCAGACTGG CGACACAGAG 
               
               
                   
                   
                 TCAGTCCCAGACCCTCAACC AATCGGAGAA CCTCCCGCAG 
               
               
                   
                   
                 CCCCCTCAGG TGTGGGATCT CTTACAATGG CTTCAGGTGG 
               
               
                   
                   
                 TGGCGCACCA GTGGCAGACA ATAACGAAGGTGCCGATGGA 
               
               
                   
                   
                 GTGGGTAGTT CCTCGGGAAA TTGGCATTGC 
               
               
                   
                   
                 GATTCCCAATGGCTGGGGGA CAGAGTCATC ACCACCAGCA 
               
               
                   
                   
                 CCCGAACCTG GGCCCTGCCCACCTACAACA ATCACCTCTA 
               
               
                   
                   
                 CAAGCAAATC TCCAACAGCA CATCTGGAGG 
               
               
                   
                   
                 ATCTTCAAAT GACAACGCCT ACTTCGGCTA CAGCACCCCC 
               
               
                   
                   
                 TGGGGGTATTTTGACTTCAA CAGATTCCAC TGCCACTTCT 
               
               
                   
                   
                 CACCACGTGA CTGGCAGCGACTCATCAACA ACAACTGGGG 
               
               
                   
                   
                 ATTCCGGCCT AAGCGACTCA ACTTCAAGCTCTTCAACATT 
               
               
                   
                   
                 CAGGTCAAAG AGGTTACGGA CAACAATGGA 
               
               
                   
                   
                 GTCAAGACCA TCGCCAATAA CCTTACCAGC ACGGTCCAGG 
               
               
                   
                   
                 TCTTCACGGA CTCAGACTATCAGCTCCCGT ACGTGCTCGG 
               
               
                   
                   
                 GTCGGCTCAC GAGGGCTGCC TCCCGCCGTTCCCAGCGGAC 
               
               
                   
                   
                 GTTTTCATGA TTCCTCAGTA CGGGTATCTG 
               
               
                   
                   
                 ACGCTTAATGATGGAAGCCA GGCCGTGGGT CGTTCGTCCT 
               
               
                   
                   
                 TTTACTGCCT GGAATATTTC CCGTCGCAAA TGCTAAGAAC 
               
               
                   
                   
                 GGGTAACAAC TTCCAGTTCA GCTACGAGTTTGAGAACGTA 
               
               
                   
                   
                 CCTTTCCATA GCAGCTACGC TCACAGCCAA 
               
               
                   
                   
                 AGCCTGGACCGACTAATGAA TCCACTCATC GACCAATACT 
               
               
                   
                   
                 TGTACTATCT CTCTAGAACT ATTAACGGTT CTGGACAGAA 
               
               
                   
                   
                 TCAACAAACG CTAAAATTCA GTGTGGCCGG 
               
               
                   
                   
                 ACCCAGCAAC ATGGCTGTCC AGGGAAGAAA CTACATACCT 
               
               
                   
                   
                 GGACCCAGCTACCGACAACA ACGTGTCTCA ACCACTGTGA 
               
               
                   
                   
                 CTCAAAACAA CAACAGCGAATTTGCTTGGC CTGGAGCTTC 
               
               
                   
                   
                 TTCTTGGGCT CTCAATGGAC GTAATAGCTT GATGAATCCT 
               
               
                   
                   
                 GGACCTGCTA TGGCCAGCCA CAAAGAAGGA 
               
               
                   
                   
                 GAGGACCGTTTCTTTCCTTT GTCTGGATCT TTAATTTTTG 
               
               
                   
                   
                 GCAAACAAGG TACCGGCAGAGACAACGTGG 
               
               
                   
                   
                 ATGCGGACAA AGTCATGATA ACCAACGAAG 
               
               
                   
                   
                 AAGAAATTAAAACTACTAAC CCGGTAGCAA CGGAGTCCTA 
               
               
                   
                   
                 TGGACAAGTG GCCACAAACCACCAGAGTGC CCAAACTTTG 
               
               
                   
                   
                 GCGGTGCCTT TTAAGGCACA GGCGCAGACCGGTTGGGTTC 
               
               
                   
                   
                 AAAACCAAGG AATACTTCCG GGTATGGTTT 
               
               
                   
                   
                 GGCAGGACAGAGATGTGTAC CTGCAAGGAC CCATTTGGGC 
               
               
                   
                   
                 CAAAATTCCT CACACGGACG GCAACTTTCA CCCTTCTCCG 
               
               
                   
                   
                 CTGATGGGAG GGTTTGGAAT GAAGCACCCGCCTCCTCAGA 
               
               
                   
                   
                 TCCTCATCAA AAACACACCT GTACCTGCGG 
               
               
                   
                   
                 ATCCTCCAACGGCCTTCAAC AAGGACAAGC TGAACTCTTT 
               
               
                   
                   
                 CATCACCCAG TATTCTACTGGCCAAGTCAG CGTGGAGATC 
               
               
                   
                   
                 GAGTGGGAGC TGCAGAAGGA AAACAGCAAG 
               
               
                   
                   
                 CGCTGGAACC CGGAGATCCA GTACACTTCC AACTATTACA 
               
               
                   
                   
                 AGTCTAATAATGTTGAATTT GCTGTTAATA CTGAAGGTGT 
               
               
                   
                   
                 ATATAGTGAA CCCCGCCCCATTGGCACCAG ATACCTGACT 
               
               
                   
                   
                 CGTAATCTGT AA 
               
               
                   
               
               
                 8 
                 AAV- 
                 ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAA 
               
               
                   
                 PHP.S 
                 CCTTAGTGAAGGAATTCGCGAGTGGTGGGCTTTGAAACCTG 
               
               
                   
                   
                 GAGCCCCTCAACCCAAGGCAAATCAACAACATCAAGACAA 
               
               
                   
                   
                 CGCTCGAGGTCTTGTGCTTCCGGGTTACAAATACCTTGGACC 
               
               
                   
                   
                 CGGCAACGGACTCGACAAGGGGGAGCCGGTCAACGCAGCA 
               
               
                   
                   
                 GACGCGGCGGCCCTCGAGCACGACAAGGCCTACGACCAGC 
               
               
                   
                   
                 AGCTCAAGGCCGGAGACAACCCGTACCTCAAGTACAACCAC 
               
               
                   
                   
                 GCCGACGCCGAGTTCCAGGAGCGGCTCAAAGAAGATACGTC 
               
               
                   
                   
                 TTTTGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAAA 
               
               
                   
                   
                 AGAGGCTTCTTGAACCTCTTGGTCTGGTTGAGGAAGCGGCT 
               
               
                   
                   
                 AAGACGGCTCCTGGAAAGAAGAGGCCTGTAGAGCAGTCTCC 
               
               
                   
                   
                 TCAGGAACCGGACTCCTCCGCGGGTATTGGCAAATCGGGTG 
               
               
                   
                   
                 CACAGCCCGCTAAAAAGAGACTCAATTTCGGTCAGACTGGC 
               
               
                   
                   
                 GACACAGAGTCAGTCCCAGACCCTCAACCAATCGGAGAACC 
               
               
                   
                   
                 TCCCGCAGCCCCCTCAGGTGTGGGATCTCTTACAATGGCTTC 
               
               
                   
                   
                 AGGTGGTGGCGCACCAGTGGCAGACAATAACGAAGGTGCC 
               
               
                   
                   
                 GATGGAGTGGGTAGTTCCTCGGGAAATTGGCATTGCGATTC 
               
               
                   
                   
                 CCAATGGCTGGGGGACAGAGTCATCACCACCAGCACCCGAA 
               
               
                   
                   
                 CCTGGGCCCTGCCCACCTACAACAATCACCTCTACAAGCAA 
               
               
                   
                   
                 ATCTCCAACAGCACATCTGGAGGATCTTCAAATGACAACGC 
               
               
                   
                   
                 CTACTTCGGCTACAGCACCCCCTGGGGGTATTTTGACTTCAA 
               
               
                   
                   
                 CAGATTCCACTGCCACTTCTCACCACGTGACTGGCAGCGAC 
               
               
                   
                   
                 TCATCAACAACAACTGGGGATTCCGGCCTAAGCGACTCAAC 
               
               
                   
                   
                 TTCAAGCTCTTCAACATTCAGGTCAAAGAGGTTACGGACAA 
               
               
                   
                   
                 CAATGGAGTCAAGACCATCGCCAATAACCTTACCAGCACGG 
               
               
                   
                   
                 TCCAGGTCTTCACGGACTCAGACTATCAGCTCCCGTACGTGC 
               
               
                   
                   
                 TCGGGTCGGCTCACGAGGGCTGCCTCCCGCCGTTCCCAGCG 
               
               
                   
                   
                 GACGTTTTCATGATTCCTCAGTACGGGTATCTGACGCTTAAT 
               
               
                   
                   
                 GATGGAAGCCAGGCCGTGGGTCGTTCGTCCTTTTACTGCCTG 
               
               
                   
                   
                 GAATATTTCCCGTCGCAAATGCTAAGAACGGGTAACAACTT 
               
               
                   
                   
                 CCAGTTCAGCTACGAGTTTGAGAACGTACCTTTCCATAGCA 
               
               
                   
                   
                 GCTACGCTCACAGCCAAAGCCTGGACCGACTAATGAATCCA 
               
               
                   
                   
                 CTCATCGACCAATACTTGTACTATCTCTCAAAGACTATTAAC 
               
               
                   
                   
                 GGTTCTGGACAGAATCAACAAACGCTAAAATTCAGTGTGGC 
               
               
                   
                   
                 CGGACCCAGCAACATGGCTGTCCAGGGAAGAAACTACATAC 
               
               
                   
                   
                 CTGGACCCAGCTACCGACAACAACGTGTCTCAACCACTGTG 
               
               
                   
                   
                 ACTCAAAACAACAACAGCGAATTTGCTTGGCCTGGAGCTTC 
               
               
                   
                   
                 TTCTTGGGCTCTCAATGGACGTAATAGCTTGATGAATCCTGG 
               
               
                   
                   
                 ACCTGCTATGGCCAGCCACAAAGAAGGAGAGGACCGTTTCT 
               
               
                   
                   
                 TTCCTTTGTCTGGATCTTTAATTTTTGGCAAACAAGGAACTG 
               
               
                   
                   
                 GAAGAGACAACGTGGATGCGGACAAAGTCATGATAACCAA 
               
               
                   
                   
                 CGAAGAAGAAATTAAAACTACTAACCCGGTAGCAACGGAG 
               
               
                   
                   
                 TCCTATGGACAAGTGGCCACAAACCACCAGAGTGCCCAACA 
               
               
                   
                   
                 GGCGGTTAGGACGTCTTTGGCACAGGCGCAGACCGGCTGGG 
               
               
                   
                   
                 TTCAAAACCAAGGAATACTTCCGGGTATGGTTTGGCAGGAC 
               
               
                   
                   
                 AGAGATGTGTACCTGCAAGGACCCATTTGGGCCAAAATTCC 
               
               
                   
                   
                 TCACACGGACGGCAACTTTCACCCTTCTCCGCTGATGGGAG 
               
               
                   
                   
                 GGTTTGGAATGAAGCACCCGCCTCCTCAGATCCTCATCAAA 
               
               
                   
                   
                 AACACACCTGTACCTGCGGATCCTCCAACGGCCTTCAACAA 
               
               
                   
                   
                 GGACAAGCTGAACTCTTTCATCACCCAGTATTCTACTGGCCA 
               
               
                   
                   
                 AGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAAC 
               
               
                   
                   
                 AGCAAGCGCTGGAACCCGGAGATCCAGTACACTTCCAACTA 
               
               
                   
                   
                 TTACAAGTCTAATAATGTTGAATTTGCTGTTAATACTGAAGG 
               
               
                   
                   
                 TGTATATAGTGAACCCCGCCCCATTGGCACCAGATACCTGA 
               
               
                   
                   
                 CTCGTAATCTGTAA 
               
               
                   
               
               
                 9 
                 AAV- 
                 ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAA 
               
               
                   
                 PHP.eB 
                 CCTTAGTGAAGGAATTCGCGAGTGGTGGGCTTTGAAACCTG 
               
               
                   
                   
                 GAGCCCCTCAACCCAAGGCAAATCAACAACATCAAGACAA 
               
               
                   
                   
                 CGCTCGAGGTCTTGTGCTTCCGGGTTACAAATACCTTGGACC 
               
               
                   
                   
                 CGGCAACGGACTCGACAAGGGGGAGCCGGTCAACGCAGCA 
               
               
                   
                   
                 GACGCGGCGGCCCTCGAGCACGACAAGGCCTACGACCAGC 
               
               
                   
                   
                 AGCTCAAGGCCGGAGACAACCCGTACCTCAAGTACAACCAC 
               
               
                   
                   
                 GCCGACGCCGAGTTCCAGGAGCGGCTCAAAGAAGATACGTC 
               
               
                   
                   
                 TTTTGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAAA 
               
               
                   
                   
                 AGAGGCTTCTTGAACCTCTTGGTCTGGTTGAGGAAGCGGCT 
               
               
                   
                   
                 AAGACGGCTCCTGGAAAGAAGAGGCCTGTAGAGCAGTCTCC 
               
               
                   
                   
                 TCAGGAACCGGACTCCTCCGCGGGTATTGGCAAATCGGGTG 
               
               
                   
                   
                 CACAGCCCGCTAAAAAGAGACTCAATTTCGGTCAGACTGGC 
               
               
                   
                   
                 GACACAGAGTCAGTCCCAGACCCTCAACCAATCGGAGAACC 
               
               
                   
                   
                 TCCCGCAGCCCCCTCAGGTGTGGGATCTCTTACAATGGCTTC 
               
               
                   
                   
                 AGGTGGTGGCGCACCAGTGGCAGACAATAACGAAGGTGCC 
               
               
                   
                   
                 GATGGAGTGGGTAGTTCCTCGGGAAATTGGCATTGCGATTC 
               
               
                   
                   
                 CCAATGGCTGGGGGACAGAGTCATCACCACCAGCACCCGAA 
               
               
                   
                   
                 CCTGGGCCCTGCCCACCTACAACAATCACCTCTACAAGCAA 
               
               
                   
                   
                 ATCTCCAACAGCACATCTGGAGGATCTTCAAATGACAACGC 
               
               
                   
                   
                 CTACTTCGGCTACAGCACCCCCTGGGGGTATTTTGACTTCAA 
               
               
                   
                   
                 CAGATTCCACTGCCACTTCTCACCACGTGACTGGCAGCGAC 
               
               
                   
                   
                 TCATCAACAACAACTGGGGATTCCGGCCTAAGCGACTCAAC 
               
               
                   
                   
                 TTCAAGCTCTTCAACATTCAGGTCAAAGAGGTTACGGACAA 
               
               
                   
                   
                 CAATGGAGTCAAGACCATCGCCAATAACCTTACCAGCACGG 
               
               
                   
                   
                 TCCAGGTCTTCACGGACTCAGACTATCAGCTCCCGTACGTGC 
               
               
                   
                   
                 TCGGGTCGGCTCACGAGGGCTGCCTCCCGCCGTTCCCAGCG 
               
               
                   
                   
                 GACGTTTTCATGATTCCTCAGTACGGGTATCTGACGCTTAAT 
               
               
                   
                   
                 GATGGAAGCCAGGCCGTGGGTCGTTCGTCCTTTTACTGCCTG 
               
               
                   
                   
                 GAATATTTCCCGTCGCAAATGCTAAGAACGGGTAACAACTT 
               
               
                   
                   
                 CCAGTTCAGCTACGAGTTTGAGAACGTACCTTTCCATAGCA 
               
               
                   
                   
                 GCTACGCTCACAGCCAAAGCCTGGACCGACTAATGAATCCA 
               
               
                   
                   
                 CTCATCGACCAATACTTGTACTATCTCTCTAGAACTATTAAC 
               
               
                   
                   
                 GGTTCTGGACAGAATCAACAAACGCTAAAATTCAGTGTGGC 
               
               
                   
                   
                 CGGACCCAGCAACATGGCTGTCCAGGGAAGAAACTACATAC 
               
               
                   
                   
                 CTGGACCCAGCTACCGACAACAACGTGTCTCAACCACTGTG 
               
               
                   
                   
                 ACTCAAAACAACAACAGCGAATTTGCTTGGCCTGGAGCTTC 
               
               
                   
                   
                 TTCTTGGGCTCTCAATGGACGTAATAGCTTGATGAATCCTGG 
               
               
                   
                   
                 ACCTGCTATGGCCAGCCACAAAGAAGGAGAGGACCGTTTCT 
               
               
                   
                   
                 TTCCTTTGTCTGGATCTTTAATTTTTGGCAAACAAGGTACCG 
               
               
                   
                   
                 GCAGAGACAACGTGGATGCGGACAAAGTCATGATAACCAA 
               
               
                   
                   
                 CGAAGAAGAAATTAAAACTACTAACCCGGTAGCAACGGAG 
               
               
                   
                   
                 TCCTATGGACAAGTGGCCACAAACCACCAGAGTGATGGGAC 
               
               
                   
                   
                 TTTGGCGGTGCCTTTTAAGGCACAGGCGCAGACCGGTTGGG 
               
               
                   
                   
                 TTCAAAACCAAGGAATACTTCCGGGTATGGTTTGGCAGGAC 
               
               
                   
                   
                 AGAGATGTGTACCTGCAAGGACCCATTTGGGCCAAAATTCC 
               
               
                   
                   
                 TCACACGGACGGCAACTTTCACCCTTCTCCGCTGATGGGAG 
               
               
                   
                   
                 GGTTTGGAATGAAGCACCCGCCTCCTCAGATCCTCATCAAA 
               
               
                   
                   
                 AACACACCTGTACCTGCGGATCCTCCAACGGCCTTCAACAA 
               
               
                   
                   
                 GGACAAGCTGAACTCTTTCATCACCCAGTATTCTACTGGCCA 
               
               
                   
                   
                 AGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAAC 
               
               
                   
                   
                 AGCAAGCGCTGGAACCCGGAGATCCAGTACACTTCCAACTA 
               
               
                   
                   
                 TTACAAGTCTAATAATGTTGAATTTGCTGTTAATACTGAAGG 
               
               
                   
                   
                 TGTATATAGTGAACCCCGCCCCATTGGCACCAGATACCTGA 
               
               
                   
                   
                 CTCGTAATCTGTAA 
               
               
                   
               
            
           
         
       
     
     II. METHODS 
     A. Methods of Producing AAVs 
     Disclosed herein are methods of identifying a recombinant AAV (rAAV), such as those disclosed herein. The AAV peptide specific to target in vivo environments are identified using multiplexed Cre recombination-based AAV targeted evolution (M-CREATE).  FIG. 1  provides a workflow using M-CREATE. M-CREATE uses an rAAV capsid genome (rAAV-Cap-in-cis-lox or rAAV-ACap-in-cis-lox2 as described in Example 2 below) that couples a full-length AAV Cap gene, controlled by regulatory elements from the AAV Rep gene, with a Cre-invertible switch. The rAAV-ACap-in-cis-lox2 backbone has a bi-directional polyA flanked by two Lox sites (lox71 and lox66). The ACap backbone is nonfunctional as it is missing a portion of the capsid gene. Upon insertion of a library fragment with mutagenesis at a specific site into the capsid, it then becomes a fully functional vector. In a cell expressing the Cre recombinase, the Cre-Lox recombination facilitates inversion of the polyA in addition to flipping the Lox sites to Lox72 and LoxP. (See  FIGS. 1 and 36 ) The randomized 7-mer and 11-mers disclosed herein are generated using PCR, which are then inserted into the rAAV-ACap-in-cis-lox to generate virus libraries with randomized insertions or substitutions in the capsid protein sequence (e.g., at the AA588 589). ( FIG. 1 ). In a first round of in vivo selection, the virus libraries are injected into the blood stream (e.g., intravenously) of transgenic animals expressing Cre recombinase. Tissues obtained from the transgenic animals following injection in in vivo environment (e.g., brain/liver). The inverted reporter expression cassette is detected using selective amplification expression of the reporter gene expression product. The rAAVs in the tissues are isolated and the viral genome around the insertion site is sequenced and aligned with an AAV9 template DNA fragment. The 7-mers and 11-mers that were recovered were enriched in the target in vivo environment, and were cloned into another rAAV-ACap-in-cis-lox2 backbone, and another round of in vivo selection is performed. The 7-mers and 11-mers enriched in the target in vivo environment, and negatively enriched in an off-target in vivo environment are sequenced using suitable methods, such as next generation sequencing.  FIGS. 33-35  provide DNA sequences identified using the methods provided herein. 
     Methods comprise providing a rAAV genome comprising an AAV capsid gene, and a recognition sequence for a Cre recombinase. In some cases, the rAAV genome has two recognition sequences for Cre recombinase that flank a reporter expression cassette. The recognition sequences for Cre recombinase (e.g., LoxP) are oriented such that an inversion of the reporter cassette is facilitated in the presence of Cre recombinase in a cell. Methods comprise transfecting a population of cells expressing the Cre recombinase with the rAAV genome. The Cre recombinase induces an inversion (e.g., “flip” of the reporter gene into a genome of the transgenic animal). In some cases, the rate of inversion (e.g., a level of expression of the reporter gene in a target cell) may be measured using any suitable method, such as quantitative polymerase chain reaction, or immunohistochemistry. The level of expression of the reporter gene is compared to a reference value, which in some cases is the rate of inversion by a reference AAV (e.g., AAV9). Methods disclosed herein provide that an increase in the rate of inversion is detected in a target in vivo environment, as compared to the rate of inversion of a reference AAV. In some cases, a decrease in a rate of inversion is detected in an off-target cell, as compared to the rate of inversion of a reference AAV. The rAAV genome recovered using the methods described herein encodes for an AAV capsid particle (e.g., capsid protein, capsid) with an increased specificity for the target cell, and a decreased specificity for the off-target cell. 
     Disclosed herein are methods of producing a rAAV disclosed herein. In some instances, all elements that are required for AAV production in target cell (e.g., HEK293cells) are transiently transfected into the target cell using suitable methods known in the art. For example, the rAAV of the present disclosure can be product by co-transfecting three plasmid vectors, a first vector with ITR-containing plasmid carrying the transgene (e.g., therapeutic nucleic acid), a second vector that carries the AAV Rep and Cap genes; and (3), a third vector that provides the helper genes isolated from adenovirus. The methods described herein generate high-titer AAV vectors that are free of adenovirus. The Cap gene disclosed here comprises any one of SEQ ID NOS: 10-434, 860-863, 868-949, 1068-5661, 14841-14880, and 14961-15053 which are DNA sequences encoding the modified AAV capsid protein portions of the present disclosure. In some cases, rAAVs of the present disclosure are generated using the methods described in Challis, R. C. et al. Systemic AAV vectors for widespread and targeted gene delivery in rodents. Nat. Protoc. 14, 379 (2019), which is hereby incorporated by reference in its entirety. Briefly, triple transfection of HEK293T cells (ATCC) using polyethylenimine (PEI) is performed, viruses are collected after 120 hours from both cell lysates and media, and purified over iodixanol. 
     Disclosed herein, are methods comprising: (a) introducing into a cell a nucleic acid comprising: (1) a first nucleic acid sequence encoding a therapeutic gene expression product; (2) a second nucleic acid sequence encoding viral genome components comprising: (i) a Replication (Rep) gene encoding a Rep protein; and (ii) a modified capsid (Cap) gene encoding a modified AAV capsid protein described herein, and (3) a third nucleic acid sequence encoding a genome of an AAV helper virus; and (b) assembling a recombinant AAV (rAAV) capsid encapsidating the first nucleic acid, the rAAV capsid comprising a tropism with an increased specificity for a target in vivo environment in a subject and a decreased specificity for an off-target in vivo environment, relative to a tropism of a corresponding parental AAV capsid protein. In some instances, the methods further comprise packing the first nucleic acid sequence encoding the therapeutic gene expression product such that it becomes encapsidated by the modified AAV capsid protein. In some embodiments, the rAAV particles are isolated, concentrated, and purified using suitable viral purification methods, such as those described herein. 
     In a non-limiting example, the rAAVs are generated by triple transfection of precursor cells (e.g., HEK293T) cells using a standard transfection protocol (e.g., PEI). Viral particles are harvested from the media after a period of time (e.g., 72 h post transfection) and from the cells and media at a later point in time (e.g., 120 h post transfection). Virus present in the media is concentrated by precipitation with 8% poly(ethylene glycol) and 500 mM sodium chloride and the precipitated virus is added to the lysates prepared from the collected cells. The viruses are purified over iodixanol (Optiprep, Sigma) step gradients (15%, 25%, 40% and 60%). Viruses are concentrated and formulated in PBS. Virus titers are determined by measuring the number of DNaseI-resistant vector genome copies (VGs) using qPCR and the linearized genome plasmid as a control. 
     The Rep protein can be selected from the group consisting of Rep78, Rep68, Rep52, and Rep40. The genome of the AAV helper virus comprises an AAV helper gene selected from the group consisting of E2, E4, and VA. The second nucleic acid and the first nucleic acid can be in trans. The second nucleic acid and the first nucleic acid can be in cis. 
     The cell can be selected from a group consisting of a human, a primate, a murine, a feline, a canine, a porcine, an ovine, a bovine, an equine, an epine, a caprine and a lupine host cell. In some instances, the cell is a progenitor or precursor cell, such as a stem cell. In some instances, the stem cell is a mesenchymal cell, embryonic stem cell, induced pluripotent stem cell (iPSC), fibroblast or other tissue specific stem cell. The cell can be immortalized. In some cases, the immortalized cell is a HEK293cell. In some instances, the cell is a differentiated cell. Based on the disclosure provided, it is expected that this system can be used in conjunction with any transgenic line expressing a recombinase in the target cell type of interest to develop AAV capsids that more efficiently transduce that target cell population. 
     B. Methods of rAAV-Mediated Delivery of a Heterologous Nucleic Acid 
     Disclosed herein are methods of delivering a heterologous nucleic acid (e.g., therapeutic nucleic acid or transgene disclosed herein) to subject in need thereof. The transgene may be encapsidated by a recombinant AAV (rAAV) capsid protein or rAAV particle such as those described herein. 
     Methods may be ex vivo, e.g., scientific research purposes or for producing adoptive cellular therapies. The subject may be a human primary cell or a mature cell, or cell line. The subject may be a cell from a monkey, hamster, or mouse. In either case, delivery may include contacting the composition with the cell or cell line. 
     Methods may be in vivo, e.g., treating a disease or a condition in a subject in need thereof. In some instances, the subject may be mammal, such as a human or non-human primate, in which case delivery of the composition may comprise administering the composition to the subject. In some embodiments, delivery of the heterologous nucleic acid comprises administering to the subject the composition using any one of the routes of administration described herein. 
     In some embodiments, methods of increasing transduction of a heterologous nucleic acid in a target in vivo environment comprise delivering a rAAV particle described herein, the rAAV engineered to have an increased transduction efficiency in a target in vivo environment (e.g., tissue or cell type). In some instances, the increased transduction efficiency comprises a 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21-fold, 22-fold, 23-fold, 24-fold, 25-fold, 26-fold, 27-fold, 28-fold, 29-fold, 30-fold, 31-fold, 32-fold, 33-fold, 34-fold, 35-fold, 36-fold, 37-fold, 38-fold, 39-fold, 40-fold, 41-fold, 42-fold, 43-fold, 44-fold, 45-fold, 46-fold, 47-fold, 48-fold, 49-fold, 50-fold, 75-fold, or 100-fold increase, or more, relative to a reference AAV. In some instances, the increased transduction efficiency is at least 30-fold. In some instances, the increased transduction efficiency is at least 40-fold. In some instances, the increased transduction efficiency is at least 50-fold. In some instances, the increased transduction efficiency is at least 60-fold. In some instances, the increased transduction efficiency is at least 80-fold. In some instances, the increased transduction efficiency is at least 90-fold. In some instances, the increased transduction efficiency is at least 100-fold. 
     Methods of delivering a heterologous nucleic acid to a target in vivo environment are also provided comprising delivering the rAAV particle described herein that has been engineered to have an increased specificity in a target in vivo environment (e.g., tissue or cell type), as compared to a reference AAV. Methods, in some cases, comprise detecting whether a rAAV possesses more specificity for a target in vivo environment than a reference AAV, includes measuring a level of gene expression product (e.g., RNA or protein) expressed from the heterologous nucleic acid encapsidated by the rAAV in a tissue sample obtained from the target in vivo environment in a subject; and comparing the measured level to a control level (such as, for e.g., the gene expression product expressed from a heterologous nucleic acid encapsidated by a reference AAV (e.g., AAV9)). Suitable methods for measuring expression of a gene expression product luciferase reporter assay and quantitative polymerase chain reaction (qPCR). 
     In some instances, the reference AAV has a serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAVS, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or variant thereof. For example, the reference AAV can have a serotype selected from the group consisting of AAV-PHP.B, AAV-PHP.eB, and AAV-PHP.S. 
     Delivery to the CNS 
     Provided herein are methods of delivering a heterologous nucleic acid to a target in vivo environment comprising delivering a composition to the target in vivo environment selected from the group consisting of a central nervous system (CNS) in a subject, the composition comprising a rAAV particle with a rAAV capsid protein, the rAAV capsid protein encapsidating a viral vector encoding a heterologous nucleic acid (e.g., therapeutic nucleic acid). In some embodiments, the rAAV particle encapsidating the heterologous nucleic acid comprises a rAAV capsid protein engineered with an increased specificity and, in some cases, transduction efficiency when measured in the CNS or the PNS of the subject, even when administered to the subject systemically, as compared to a reference AAV. 
     Methods comprise delivering a rAAV particle comprising an rAAV capsid protein with increased specificity and/or transduction efficiency when measured in the CNS in the subject, as compared to a reference AAV (e.g., AAV9). In some embodiments, delivery is systemic. Alternatively, delivery is direct (e.g., into the affected area of the CNS). 
     The rAAV capsid protein may comprise a substitution of at least one, two, three, four, five, six, seven, eight, nine, ten, or eleven amino acids provided in an amino acid sequence provided in any one of Tables 2-3, in an amino acid sequence of a parental AAV. In some instances, X1 is selected from the group consisting of A, E, D, G, R, S and T. In some instances, the insertion further comprises two amino acids, wherein X2 is selected from the group consisting of A, G, I, L, M, N, Q, R, T, and Y. In some instances, the insertion further comprises three amino acids, wherein X3 is selected from the group consisting of E, K, L, T, and Q. In some instances, the insertion further comprises at least four amino acids, wherein X1 is selected from the group consisting of A, E, D, G, R, S and T, X2 is selected from the group consisting of A, G, I, L, M, N, Q, R, T, and Y, X3 is selected from the group consisting of E, K, L, T, and Q, and X4 is selected from the group consisting of G, I, K, L, M, R, T, and V. In some instances, the insertion further comprises five amino acids wherein X5 is selected from the group consisting of A, D, G, P, L, Q, and V. In some instances, the insertion further comprises at least six amino acids, wherein X6 is selected from the group consisting of F, K, L, N, P, Q, S, and V. In some instances, the insertion further comprises at least seven amino acids, wherein X7 is selected from the group consisting of I, K, L, P, S, and V. 
     Disclosed herein are methods comprising delivering a rAAV particle encapsidating a heterologous nucleic acid to a CNS in a subject, the rAAV particle comprising (i) an increased specificity and/or transduction efficiency of the heterologous nucleic acid for the CNS, wherein the rAAV particle has an rAAV capsid protein comprising an insertion of at least or about three, four, five, six, or seven amino acids of an amino acid sequence TALKPFL, TTLKPFL, TLQIPFK, TMQKPFI, or RYQGDSV, or any amino acid sequence provided in Tables 2-3 or  FIG. 33 , at an amino acid position 588_589 in a parental AAV capsid protein. In some embodiments, the delivering is systemic. In some embodiments, the delivery is direct (e.g., injected into the in vivo environment). In some embodiments, the parental AAV capsid protein is AAV9 capsid protein (for e.g., provided in SEQ ID NO: 1). In some embodiments, delivery is more specific than a delivery of the heterologous nucleic acid by a reference AAV, e.g., AAV9. In some embodiments, the delivery is systemic (e.g., intravenous). In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. 
     Delivery to the Liver 
     In some cases, the methods of delivering a heterologous nucleic acid comprise delivering to a target in vivo environment in a subject a composition, the composition comprising a rAAV particle with a rAAV capsid protein, the rAAV capsid protein encapsidating a viral vector encoding a heterologous nucleic acid (e.g., therapeutic nucleic acid). In some cases, the target in vivo environment is the liver. In some embodiments, the rAAV particle encapsidating the heterologous nucleic acid comprises a rAAV capsid protein engineered with an increased specificity and, in some cases, transduction efficiency when measured in the target in vivo environment of the subject, even when administered to the subject systemically. 
     In some embodiments, methods comprise delivering a rAAV particle comprising an rAAV capsid protein with increased specificity and/or transduction efficiency of the heterologous nucleic acid for the liver in the subject, as compared to a reference AAV (e.g., AAV9). In some embodiments, rAAVs optimized for targeting the liver have amino acid sequences that comprise an amino acid sequence provided in SEQ ID NOS: 950-1031 and 15054-15146 ( FIG. 35 ). 
     The rAAV capsid protein suitable for delivery of the heterologous nucleic acid to the liver can comprise an insertion of at least one amino acid in a parental AAV capsid protein. In some instances, the insertion comprises at least one, two, three, four, five, six, seven, eight, nine, ten, or eleven amino acids provided in an amino acid sequence provided in Table 4, or  FIG. 35 . 
     Disclosed herein are methods comprising delivering a rAAV particle encapsidating a heterologous nucleic acid to the target in vivo environment selected from the group consisting of the liver in a subject, the rAAV particle comprising an increased specificity and/or transduction efficiency of the heterologous nucleic acid for the target in vivo environment, wherein the rAAV particle has an rAAV capsid protein comprising an insertion of at least or about three, four, five, six, seven, eight, nine, ten, or eleven amino acids of an amino acid sequence provided in Table 4, or  FIG. 35 . In some embodiments, delivery is more specific than a delivery of the heterologous nucleic acid by a reference AAV, e.g., AAV9. In some embodiments, methods further comprise reducing or ablating delivery of the heterologous nucleic acid in an off-target in vivo environment, such as the liver, compared to a reference AAV. In some embodiments, delivery is characterized by an increase in efficiency of transduction (e.g., of the heterologous nucleic acid) in the target in vivo environment than a transduction efficiency in the target in vivo environment of the reference AAV. In some embodiments, the delivery is systemic (e.g., intravenous). In some embodiments, the subject is a mammal. In some cases, the mammal is a human. 
     C. Methods of Treatment 
     Disclosed herein are methods of treating a disease or condition, or a symptom of the disease or condition, in a subject, comprising administrating of therapeutically effective amount of one or more compositions (e.g., rAAV particle, AAV vector, pharmaceutical composition) disclosed herein to the subject. In some embodiments, the composition is a rAAV capsid protein described herein. In some embodiments, the composition is an isolated and purified rAAV capsid protein described herein. In some embodiments, the rAAV particle encapsidates an AAV vector comprising a transgene (e.g., therapeutic nucleic acid). In some embodiments, the composition is a rAAV capsid protein described herein conjugated with a therapeutic agent disclosed herein. In some embodiments, the composition is a pharmaceutical composition comprising the rAAV particle and a pharmaceutically acceptable carrier. In some embodiments, the one or more compositions are administered to the subject alone (e.g., standalone therapy). In some embodiments, the one or more compositions are administered in combination with an additional agent. In some embodiments, the composition is a first-line therapy for the disease or condition. In some embodiments, the composition is a second-line, third-line, or fourth-line therapy, for the disease or condition. 
     Provided herein are methods of treating a disease or a condition, or a symptom of the disease or condition, in a subject, comprising: (a) diagnosing a subject with a disease or a condition affecting a target in vivo environment; and (b) treating the disease or the condition by administering to the subject a therapeutically effective amount of a composition disclosed herein (e.g., rAAV particle, AAV vector, pharmaceutical composition), wherein the composition is engineered with an increased specificity for the target in vivo environment. 
     Disclosed herein are methods of treating a disease or a condition, or a symptom of the disease or the condition, afflicting a target in vivo environment in a subject comprising: (a) administering to the subject a composition (e.g., rAAV particle, AAV vector, pharmaceutical composition); and (b) expressing the therapeutic nucleic acid into a target in vivo environment in the subject with an increased specificity and/or transduction efficiency, as compared to a reference AAV. In some cases, the reference AAV is AAV9, or a variant thereof. 
     Methods of treating a disease or condition affecting the central nervous system (CNS) comprise administering a rAAV particle to a CNS in a subject, the rAAV particle comprising an rAAV capsid protein comprising an insertion of at least or about three, four, five, six, seven, eight, nine, ten, or eleven amino acids of an amino acid sequence TALKPFL, TTLKPFL, TLQIPFK, TMQKPFI, RYQGDSV, or TTLKPFS, or any amino acid sequence provided in Tables 2-3, or  FIG. 33 , at an amino acid position 588_589 in a parental AAV capsid protein. In some cases, the insertion is not TLAVPFK, KFPVALT, SVSKPFL, FTLTTPK, MNATKNV, NGGTSSS, TRTNPEA, or YTLSQGW. In some embodiments, the parental AAV capsid protein is AAV9 capsid protein (for e.g., provided in SEQ ID NO: 1. In some instances, the parental AAV capsid protein comprises an amino acid sequence that is at least 95%, 96%, 96.1, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97.0%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98.0%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100.0% identical to SEQ ID NO: 1. In some embodiments, delivery is more specific than a delivery of the heterologous nucleic acid by a reference AAV, e.g., AAV9. In some embodiments, the delivery is systemic (e.g., intravenous). In some embodiments, the subject is a human or a non-human primate. 
     Methods of treating a disease or a condition afflicting a target in vivo environment comprising a liver comprise administering a rAAV particle to the target in vivo environment in a subject, the rAAV particle comprising an rAAV capsid protein comprising a substitution of at least or about three, four, five, six, seven, eight, nine, ten, or eleven, amino acids of an amino acid sequence provided in any one of SEQ ID NOS: 950-1031 and 15054-15146 ( FIG. 35 ). In some embodiments, methods comprise delivering a rAAV particle comprising an rAAV capsid protein with increased specificity for the liver in the subject, as compared to a reference AAV (e.g., AAV9). In some embodiments, rAAVs optimized for targeting the liver have amino acid sequences comprising an amino acid sequence KAYSVQV, PSGSARS, and RTANALG at an amino acid position 588_589 in a parental AAV capsid protein. In some embodiments, the parental AAV capsid protein is AAV9 capsid protein (for e.g., provided in SEQ ID NO: 1). In some instances, the parental AAV capsid protein comprises an amino acid sequence that is at least 95%, 96%, 96.1, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97.0%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98.0%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100.0% identical to SEQ ID NO: 1. In some embodiments, delivery is more specific than a delivery of the heterologous nucleic acid by a reference AAV, e.g., AAV9. In some embodiments, the delivery is systemic (e.g., intravenous or intranasal). In some embodiments, the subject is a human or a non-human primate. 
     Also provided are methods of modulating a target gene expression product, the methods comprising administering to a subject in need thereof a composition (e.g., rAAV particle, AAV vector, pharmaceutical composition) disclosed herein. For example, methods provided herein comprise administering to a subject a rAAV with a rAAV capsid protein encapsidating a viral vector comprising a heterologous nucleic acid that modulates the expression or the activity of the target gene expression product. In some embodiments, the disease or the condition is characterized by an increased or enhanced expression or activity of a gene or gene expression product thereof, as compared to a normal individual. In some cases, administering the therapeutically effective amount of the composition restores the expression or the activity of the gene or gene expression product thereof to a level that is typical in a normal individual. The term “normal individual” refers to an individual that is not afflicted with the disease or the condition characterized by the variation in expression or activity of the gene or gene expression product thereof. 
     Non-limiting examples of genes involved in central nervous system (CNS) diseases or disorders include Sarcoglycan Alpha (SGCA), glutamic acid decarboxylase 65 (GAD65), glutamic acid decarboxylase 67 (GAD67), CLN2 gene, Nerve Growth Factor (NGF), glial cell derived neurotrophic factor (GDNF), Neurturin, Survival Of Motor Neuron 1, Telomeric (SMN1), β-Glucocerebrosidase (GCase), Frataxin (FXN), Huntingtin (HTN), methyl-CpG binding protein 2 (MECP2), peroxisomal biogenesis factor (PEX), progranulin (GRN), an antitubulin agent, copper-zinc superoxide dismutase (SOD1), Glucosylceramidase Beta (GBA), NPC Intracellular Cholesterol Transporter 1 (NPC1), and NPS3. In some embodiments, the peroxisomal biogenesis factor (PEX) is selected from the group consisting of PEX1, PEX2, PEX3, PEX4, PEX5, PEX6, PEX7, PEX10, PEX11(3, PEX12, PEX13, PEX14, PEX16, PEX19, and PEX26. Non-limiting examples of genes implicated in disease or disorder of a particular organ (e.g., lung, heart, liver, muscle, eye) include Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), Factor X (FIX), RPE65, Retinoid Isomerohydrolase (RPE65), Sarcoglycan Alpha (SGCA), and sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2a). In some instances, the expression of a gene or expression or activity of a gene expression product is inhibited by the administration of the composition to the subject. In some instances, the expression of a gene or the expression or the activity of a gene expression product is enhanced by the administration of the composition to the subject. 
     In some cases, the composition is administered at dosage levels sufficient to deliver from about 0.0001 mg/kg to about 100 mg/kg, from about 0.001 mg/kg to about 0.05 mg/kg, from about 0.005 mg/kg to about 0.05 mg/kg, from about 0.001 mg/kg to about 0.005 mg/kg, from about 0.05 mg/kg to about 0.5 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, from about 0.1 mg/kg to about 40 mg/kg, from about 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, or from about 1 mg/kg to about 25 mg/kg, of subject body weight per day, one or more times a day, to obtain the desired therapeutic effect. 
     In some cases, the viral genome (vg) concentration of the composition that is administered is between 1.0 ×10 11  vg per kilogram (kg) and 1.0 ×10 16 vg/kg. In some cases, the concentration of infectious particles of at least or about 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 , 10 16 , or 10 17 . In some cases, the concentration of infectious particles is 2×10 7 , 2×10 8 , 2×10 9 , 2×10 10 , 2×10 11 , 2×10 12 , 2×10 13 , 2×10 14 , 2×10 15 , 2×10 16 , or 2×10 17 . In some cases, the concentration of the infectious particles 3×10 7 , 3×10 8 , 3×10 9 , 3×10 10 , 3×10 11 , 3×10 12 , 3×10 13 , 3×10 14 , 3×10 15 , 3×10 16 , or 3×10 17 . In some cases, the concentration of the infectious particles 4×10 7 , 4×10 8 , 4×10 9 , 4×10 10 , 4×10 11 , 4×10 12 , 4×10 13 , 4×10 14 , 4×10 15 , 4×10 16 , or 4×10 17 . In some cases, the concentration of the infectious particles 5×10 7 , 5×10 8 , 5×10 9 , 5×10 10 , 5×10 11 , 5×10 12 , 5×10 13 , 5×10 14 , 5×10 15 , 5×10 16 , or 5×10 17 . In some cases, the concentration of the infectious particles 6×10 7 , 6×10 8 , 6×10 9 , 6×10 10 , 6×10 11 , 6×10 12 , 6×10 13 , 6×10 14 , 6×10 15 , 6×10 16 , or 6×10 17 . In some cases, the concentration of the infectious particles 7×10 7 , 7×10 8 , 7×10 9 , 7×10 10 , 7×10 11 , 7×10 12 , 7×10 13 , 7×10 14 , 7×10 15 , 7×10 16 , or 7×10 17 . In some cases, the concentration of the infectious particles 8×10 7 , 8×10 8 , 8×10 9 , 8×10 10 , 8×10 11 , 8×10 12 , 8×10 13 , 8×10 14 , 8×10 15 , 8×10 16 , or 8×10 17 . In some cases, the concentration of the infectious particles 9×10 7 , 9×10 8 , 9×10 9 , 9×10 10 , 9×10 11 , 9×10 12 , 9×10 13 , 9×10 14 , 9×10 15 , 9×10 16 , or 9×10 17 . 
     In some embodiments, the administering of step is performed once. Alternatively, the administering of step is repeated at least twice. The administering of step may be performed once daily. In some cases, the administering of step comprises intravenous administration. In some cases, the administering comprises pulmonary administration. In some cases, the administering comprises intranasal administration (such as a spray). In some cases, the administering of step comprises injecting the composition into a target in vivo environment. In some cases, the administering of step does not comprise injecting the composition into the target in vivo environment. 
     Subject 
     Disclosed herein methods of delivering at least one of an AAV particle and viral vector to a subject, for example—to treat or prevent a disease or condition in a subject. The subject, in some cases, is a mammal. Non-limiting examples of a mammal include a mouse, rat, guinea pig, rabbit, chimpanzee, or farm animal. In some instances, the mammal is a non-human primate. In some instances, the subject is human. The subject of the present disclosure may not be diagnosed with a disease or condition. Alternatively, the subject may be a patient that is diagnosed with a disease or disorder, or suspected of having the disease or the disorder. 
     Disease or Condition 
     Disclosed herein are methods of treating a disease or condition in a subject by administering a composition comprising a rAAV such as those disclosed herein. At least one advantage of the rAAVs disclosed herein, is that the rAAV may be used to treat virtually any disease or condition that would benefit from a transgene therapy, including but not limited to spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS), Parkinson&#39;s disease, Pompe disease, Huntington&#39;s disease, Alzheimer&#39;s disease, Battens disease, lysosomal storage disorders, glioblastoma multiforme, Rett syndrome, Leber&#39;s congenital amaurosis, Late infantile neuronal ceroid lipofuscinosis (LINCL), chronic pain, stroke, spinal cord injury, traumatic brain injury and lysosomal storage disorders. 
     The disease or the condition may, in some embodiments, be characterized by a reduced or ablated expression or activity of a gene or gene expression product thereof, as compared to a normal individual. In some embodiments, be characterized by an increased or enhanced expression or activity of a gene or gene expression product thereof, as compared to a normal individual. 
     In some cases, the disease or condition is localized to a particular in vivo environment in the subject, e.g., the brain or the liver. The compositions of the present disclosure are particularly useful for the treatment of the diseases or conditions described herein because they specifically target the in vivo environment and deliver a therapeutic nucleic acid engineered to modulate the activity or the expression of a target gene expression product involved with the pathogenesis or pathology of the disease or condition. 
     In some instances, the disease or condition comprises a disease or condition of the central nervous system (CNS). Non-limiting examples of disease of the CNS include Absence of the Septum Pellucidum, Acid Lipase Disease, Acid Maltase Deficiency, Acquired Epileptiform Aphasia, Acute Disseminated Encephalomyelitis, Attention Deficit-Hyperactivity Disorder (ADHD), Adie&#39;s Pupil, Adie&#39;s Syndrome, Adrenoleukodystrophy, Agenesis of the Corpus Callosum, Agnosia, Aicardi Syndrome, Aicardi-Goutieres Syndrome Disorder, AIDS—Neurological Complications, Alexander Disease, Alpers&#39; Disease, Alternating Hemiplegia, Alzheimer&#39;s Disease, Amyotrophic Lateral Sclerosis (ALS), Anencephaly, Aneurysm, Angelman Syndrome, Angiomatosis, Anoxia, Antiphospholipid Syndrome, Aphasia, Apraxia, Arachnoid Cysts, Arachnoiditis, Arnold-Chiari Malformation, Arteriovenous Malformation, Asperger Syndrome, Ataxia, Ataxia Telangiectasia, Ataxias and Cerebellar or Spinocerebellar Degeneration, Atrial Fibrillation and Stroke, Attention Deficit-Hyperactivity Disorder, Autism Spectrum Disorder, Autonomic Dysfunction, Back Pain, Barth Syndrome, Batten Disease, Becker&#39;s Myotonia, Behcet&#39;s Disease, Bell&#39;s Palsy, Benign Essential Blepharospasm, Benign Focal Amyotrophy, Benign Intracranial Hypertension, Bernhardt-Roth Syndrome, Binswanger&#39;s Disease, Blepharospasm, Bloch-Sulzberger Syndrome, Brachial Plexus Birth Injuries, Brachial Plexus Injuries, Bradbury-Eggleston Syndrome, Brain and Spinal Tumors, Brain Aneurysm, Brain Injury, Brown-Sequard Syndrome, Bulbospinal Muscular Atrophy, Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL), Canavan Disease, Carpal Tunnel Syndrome, Causalgia, Cavernomas, Cavernous Angioma, Cavernous Malformation, Central Cervical Cord Syndrome, Central Cord Syndrome, Central Pain Syndrome, Central Pontine Myelinolysis, Cephalic Disorders, Ceramidase Deficiency, Cerebellar Degeneration, Cerebellar Hypoplasia, Cerebral Aneurysms, Cerebral Arteriosclerosis, Cerebral Atrophy, Cerebral Beriberi, Cerebral Cavemous Malformation, Cerebral Gigantism, Cerebral Hypoxia, Cerebral Palsy, Cerebro-Oculo-Facio-Skeletal Syndrome (COFS), Charcot-Marie-Tooth Disease, Charcot-Marie-Tooth syndrome, classical rhizomelic chondrodysplasia punctata (RCDP), Chiari Malformation, Cholesterol Ester Storage Disease, Chorea, Choreoacanthocytosis, Chronic Inflammatory Demyelinating Polyneuropathy (CIDP), Chronic Orthostatic Intolerance, Chronic Pain, Cockayne Syndrome Type II, Coffin Lowry Syndrome, Colpocephaly, Coma, Complex Regional Pain Syndrome, Congenital Facial Diplegia, Congenital Myasthenia, Congenital Myopathy, Congenital Vascular Cavernous Malformations, Corticobasal Degeneration, Cranial Arteritis, Craniosynostosis, Cree encephalitis, Creutzfeldt-Jakob Disease, Cumulative Trauma Disorders, Cushing&#39;s Syndrome, Cytomegalic Inclusion Body Disease, Cytomegalovirus Infection, Dancing Eyes-Dancing Feet Syndrome, Dandy-Walker Syndrome, Dawson Disease, Deafness, De Morsier&#39;s Syndrome, Dejerine-Klumpke Palsy, Dementia, Dementia -Multi -Infarct, Dementia—Semantic, Dementia—Subcortical, Dementia With Lewy Bodies, Dentate Cerebellar Ataxia, Dentatorubral Atrophy, Dermatomyositis, Developmental Dyspraxia, Devic&#39;s Syndrome, Diabetic Neuropathy, Diffuse Sclerosis, Dravet Syndrome, Duchenne muscular dystrophy, Dysautonomia, Dysgraphia, Dyslexia, Dysphagia, Dyspraxia, Dyssynergia Cerebellaris Myoclonica, Dyssynergia Cerebellaris Progressiva, Dystonias, Early Infantile Epileptic Encephalopathy, Empty Sella Syndrome, Encephalitis, Encephalitis Lethargica, Encephaloceles, Encephalopathy, Encephalopathy (familial infantile), Encephalotrigeminal Angiomatosis, Epilepsy, Epileptic Hemiplegia, Erb&#39;s Palsy, Erb-Duchenne and Dejerine-Klumpke Palsies, Essential Tremor, Extrapontine Myelinolysis, Fabry Disease, Fahr&#39;s Syndrome, Fainting, Familial Dysautonomia, Familial Hemangioma, Familial Idiopathic Basal Ganglia Calcification, Familial Periodic Paralyses, Familial Spastic Paralysis, Farber&#39;s Disease, Febrile Seizures, Fibromuscular Dysplasia, Fisher Syndrome, Floppy Infant Syndrome, Foot Drop, Friedreich&#39;s Ataxia, Frontotemporal Dementia, Gaucher Disease, Generalized Gangliosidoses, Gerstmann&#39;s Syndrome, Gerstmann-Straussler-Scheinker Disease, Giant Axonal Neuropathy, Giant Cell Arteritis, Giant Cell Inclusion Disease, glioblastoma, Globoid Cell Leukodystrophy, Glossopharyngeal Neuralgia, Glycogen Storage Disease, Guillain-Barre Syndrome, Hallervorden-Spatz Disease, Head Injury, Headache, Hemicrania Continua, Hemifacial Spasm, Hemiplegia Alterans, Hereditary Neuropathies, Hereditary Spastic Paraplegia, Heredopathia Atactica Polyneuritiformis, Herpes Zoster, Herpes Zoster Oticus, Hirayama Syndrome, Holmes-Adie syndrome, Holoprosencephaly, HTLV-1 Associated Myelopathy, Hughes Syndrome, Huntington&#39;s Disease, Hydranencephaly, Hydrocephalus, Hydrocephalus—Normal Pressure, Hydromyelia, Hypercortisolism, Hypersomnia, Hypertonia, Hypotonia, Hypoxia, Immune-Mediated Encephalomyelitis, Inclusion Body Myositis, Incontinentia Pigmenti, Infantile Hypotonia, Infantile Neuroaxonal Dystrophy, Infantile Phytanic Acid Storage Disease, Infantile Refsum Disease, Infantile Spasms, Inflammatory Myopathies, Iniencephaly, Intestinal Lipodystrophy, Intracranial Cysts, Intracranial Hypertension, Isaacs&#39; Syndrome, Joubert Syndrome, Kearns-Sayre Syndrome, Kennedy&#39;s Disease, Kinsbourne syndrome, Kleine-Levin Syndrome, Klippel-Feil Syndrome, Klippel-Trenaunay Syndrome (KTS), Kliiver-Bucy Syndrome, Korsakoff s Amnesic Syndrome, Krabbe Disease, Kugelberg-Welander Disease, Kuru, Lambert-Eaton Myasthenic Syndrome, Landau-Kleffner Syndrome, Lateral Femoral Cutaneous Nerve Entrapment, Lateral Medullary Syndrome, Learning Disabilities, Leigh&#39;s Disease, Lennox-Gastaut Syndrome, Lesch-Nyhan Syndrome, Leukodystrophy, Levine-Critchley Syndrome, Lewy Body Dementia, Lipid Storage Diseases, Lipoid Proteinosis, Lissencephaly, Locked-In Syndrome, Lou Gehrig&#39;s Disease, Lupus—Neurological Sequelae, Lyme Disease—Neurological Complications, Machado- Joseph Disease, Macrencephaly, Megalencephaly, Melkersson-Rosenthal Syndrome, Meningitis, Meningitis and Encephalitis, Menkes Disease, Meralgia Paresthetica, Metachromatic Leukodystrophy, Microcephaly, Migraine, Miller Fisher Syndrome, Mini Stroke, Mitochondrial Myopathy, Moebius Syndrome, Monomelic Amyotrophy, Motor Neuron Diseases, Moyamoya Disease, Mucolipidoses, Mucopolysaccharidoses, Multi-Infarct Dementia, Multifocal Motor Neuropathy, Multiple Sclerosis, Multiple System Atrophy, Multiple System Atrophy with Orthostatic Hypotension, Muscular Dystrophy, Myasthenia -Congenital, Myasthenia Gravis, Myelinoclastic Diffuse Sclerosis, Myoclonic Encephalopathy of Infants, Myoclonus, Myopathy, Myopathy- Congenital, Myopathy -Thyrotoxic, Myotonia, Myotonia Congenita, Narcolepsy, Neuroacanthocytosis, Neurodegeneration with Brain Iron Accumulation, Neurofibromatosis, Neuroleptic Malignant Syndrome, Neurological Complications of AIDS, Neurological Complications of Lyme Disease, Neurological Consequences of Cytomegalovirus Infection, Neurological Manifestations of Pompe Disease, Neurological Sequelae Of Lupus, Neuromyelitis Optica, Neuromyotonia, Neuronal Ceroid Lipofuscinosis, Neuronal Migration Disorders, Neuropathy- Hereditary, Neurosarcoidosis, Neurosyphilis, Neurotoxicity, Nevus Cavernosus, Niemann-Pick Disease, O′Sullivan-McLeod Syndrome, Occipital Neuralgia, Ohtahara Syndrome, Olivopontocerebellar Atrophy, Opsoclonus Myoclonus, Orthostatic Hypotension, Overuse Syndrome, Pain -Chronic, Pantothenate Kinase-Associated Neurodegeneration, Paraneoplastic Syndromes, Paresthesia, Parkinson&#39;s Disease, Paroxysmal Choreoathetosis, Paroxysmal Hemicrania, Parry -Romberg, Pelizaeus-Merzbacher Disease, Pena Shokeir II Syndrome, Perineural Cysts, Periodic Paralyses, Peripheral Neuropathy, Periventricular Leukomalacia, Persistent Vegetative State, Pervasive Developmental Disorders, Phytanic Acid Storage Disease, Pick&#39;s Disease, Pinched Nerve, Piriformis Syndrome, Pituitary Tumors, Polymyositis, Pompe Disease, Porencephaly, Post-Polio Syndrome, Postherpetic Neuralgia, Postinfectious Encephalomyelitis, Postural Hypotension, Postural Orthostatic Tachycardia Syndrome, Postural Tachycardia Syndrome, Primary Dentatum Atrophy, Primary Lateral Sclerosis, Primary Progressive Aphasia, Prion Diseases, Progressive Hemifacial Atrophy, Progressive Locomotor Ataxia, Progressive Multifocal Leukoencephalopathy, Progressive Sclerosing Poliodystrophy, Progressive Supranuclear Palsy, Prosopagnosia, Pseudo-Torch syndrome, Pseudotoxoplasmosis syndrome, Pseudotumor Cerebri, Psychogenic Movement, Ramsay Hunt Syndrome I, Ramsay Hunt Syndrome II, Rasmussen&#39;s Encephalitis, Reflex Sympathetic Dystrophy Syndrome, Refsum Disease, Refsum Disease—Infantile, Repetitive Motion Disorders, Repetitive Stress Injuries, Restless Legs Syndrome, Retrovirus-Associated Myelopathy, Rett Syndrome, Reye&#39;s Syndrome, Rheumatic Encephalitis, Riley-Day Syndrome, Sacral Nerve Root Cysts, Saint Vitus Dance, Salivary Gland Disease, Sandhoff Disease, Schilder&#39;s Disease, Schizencephaly, Seitelberger Disease, Seizure Disorder, Semantic Dementia, Septo-Optic Dysplasia, Severe Myoclonic Epilepsy of Infancy (SMEI), Shaken Baby Syndrome, Shingles, Shy-Drager Syndrome, Sjogren&#39;s Syndrome, Sleep Apnea, Sleeping Sickness, Sotos Syndrome, Spasticity, Spina Bifida, Spinal Cord Infarction, Spinal Cord Injury, Spinal Cord Tumors, Spinal Muscular Atrophy, Spinocerebellar Atrophy, Spinocerebellar Degeneration, Steele-Richardson-Olszewski Syndrome, Stiff-Person Syndrome, Striatonigral Degeneration, Stroke, Sturge-Weber Syndrome, Subacute Sclerosing Panencephalitis, Subcortical Arteriosclerotic Encephalopathy, Short-lasting, Unilateral, Neuralgiform (SUNCT) Headache, Swallowing Disorders, Sydenham Chorea, Syncope, Syphilitic Spinal Sclerosis, Syringohydromyelia, Syringomyelia, Systemic Lupus Erythematosus, Tabes Dorsalis,Tardive Dyskinesia, Tarlov Cysts, Tay-Sachs Disease, Temporal Arteritis, Tethered Spinal Cord Syndrome, Thomsen&#39;s Myotonia, Thoracic Outlet Syndrome, Thyrotoxic Myopathy, Tic Douloureux, Todd&#39;s Paralysis, Tourette Syndrome, Transient Ischemic Attack, Transmissible Spongiform Encephalopathies, Transverse Myelitis, Traumatic Brain Injury, Tremor, Trigeminal Neuralgia, Tropical Spastic Paraparesis, Troyer Syndrome, Tuberous Sclerosis, Vascular Erectile Tumor, Vasculitis Syndromes of the Central and Peripheral Nervous Systems, Von Economo&#39;s Disease, Von Hippel-Lindau Disease (VHL), Von Recklinghausen&#39;s Disease, Wallenberg&#39;s Syndrome, Werdnig-Hoffman Disease, Wernicke-Korsakoff Syndrome, West Syndrome, Whiplash, Whipple&#39;s Disease, Williams Syndrome, Wilson Disease, Wolman&#39;s Disease, and X-Linked Spinal and Bulbar Muscular Atrophy. 
     In some instances, the disease or condition comprises a liver disease or disorder, or is associated with a liver disease or disorder. Non-limiting examples include disorders of bile acid synthesis (e.g., Wilson disease, Progressive familial intrahepatic cholestasis type 3), disorders of carbohydrate metabolism (e.g., Hereditary fructose intolerance, Glycogen storage disease type IV), disorders of amino acids metabolism (e.g., tyrosinemia type I), Urea cycle disorders (e.g., argininosuccinate lyase deficiency, citrin deficiency (CTLN2, NICCD)), disorders of lipid metabolism (e.g., cholesteryl ester storage disease), and others including but not limited to Alpha-1 antitrypsin deficiency, cystic fibrosis, hereditary hemochromatosis, Alström syndrome, and congenital hepatic fibrosis. 
     In some instances, the disease or condition is a disease or condition is of the liver Non-limiting examples of liver diseases or disorders include Alagille Syndrome, Alcohol-Related Liver Disease, Alpha-1 Antitrypsin Deficiency, Autoimmune Hepatitis, Benign Liver Tumors, Biliary Atresia, Cirrhosis, Crigler-Najjar Syndrome, Galactosemia, Gilbert Syndrome, Hemochromatosis, Hepatic Encephalopathy, Hepatitis A, Hepatitis B, Hepatitis C, Hepatorenal Syndrome, Intrahepatic Cholestasis of Pregnancy (ICP), Lysosomal Acid Lipase Deficiency (LAL-D), Liver Cysts, Liver Cancer, Newborn Jaundice, Non-Alcoholic Fatty Liver Disease, Primary Biliary Cholangitis (PBC), Primary Sclerosing Cholangitis (PSC), Reye Syndrome, Type I Glycogen Storage Disease, and Wilson Disease. 
     Provided here, are methods of treating a disease or a condition associated with an aberrant expression or activity of a target gene or gene expression product thereof, the method comprising modulating the expression or the activity of a target gene or gene expression product in a subject by administering a rAAV encapsidating a heterologous nucleic acid of the present disclosure. In some instances, administration is systemic administration. In some instances, the expression or the activity of the target gene or gene expression product is decreased, relative to that in a normal (non-diseased) individual; and administering the rAAV to the subject is sufficient to increase the expression of the activity of the target gene or gene expression product to that of a normal individual. In some instances, the expression or the activity of the gene or gene expression product is increased, relative to that in a normal individual; and administering the rAAV to the subject is sufficient to decrease the expression or the activity of the target gene or gene expression product. In a non-limiting example, a subject diagnosed with Alzheimer&#39;s disease, which is caused, in some cases, by a gain-of-function of a Presenilin 1 and/or Presenilin 2 (encoded by the gene PSEN1 and PSEN2, respectively) is administered a rAAV disclosed herein encapsidating a therapeutic nucleic acid that is a silencing RNA (siRNA), or other RNAi with a loss-of-function effect on PSEN1 mRNA. 
     Also provided are methods of treating or preventing a disease or condition disclosed herein in a subject comprising administering to the subject a therapeutically effective amount of an AAV vector comprising a nucleic acid sequence encoding a therapeutic gene expression product described herein. The AAV vector may be encapsidated in the modified capsid protein or AAV viral particle described herein. In some instances, the therapeutic gene expression product is effective to modulate the activity or expression of a target gene or gene expression product. 
     Formulations, Dosages, and Routes of Administration 
     In general, methods disclosed herein comprise administering a therapeutic rAAV composition by systemic administration. In some instances, methods comprise administering a therapeutic rAAV composition by oral administration. In some instances, methods comprise administering a therapeutic rAAV composition by intraperitoneal injection. In some instances, methods comprise administering a therapeutic rAAV composition in the form of an anal suppository. In some instances, methods comprise administering a therapeutic rAAV composition by intravenous (“i.v.”) administration. It is conceivable that one may also administer therapeutic rAAV compositions disclosed herein by other routes, such as subcutaneous injection, intramuscular injection, intradermal injection, transdermal injection percutaneous administration, intranasal administration, intralymphatic injection, rectal administration intragastric administration, intraocular administration, intracerebro-ventricularl administration, intrathecally, or any other suitable parenteral administration. In some instances, methods comprise administering a therapeutic rAAV composition by topical administration, example, by brushing or otherwise contacting the rAAV composition to a region of the subject (e.g., eardrum, bladder). In some embodiments, routes for local delivery closer to site of injury or inflammation are preferred over systemic routes. Routes, dosage, time points, and duration of administrating therapeutics may be adjusted. In some embodiments, administration of therapeutics is prior to, or after, onset of either, or both, acute and chronic symptoms of the disease or condition. 
     An effective dose and dosage of pharmaceutical compositions to prevent or treat the disease or condition disclosed herein is defined by an observed beneficial response related to the disease or condition, or symptom of the disease or condition. Beneficial response comprises preventing, alleviating, arresting, or curing the disease or condition, or symptom of the disease or condition. In some embodiments, the beneficial response may be measured by detecting a measurable improvement in the presence, level, or activity, of biomarkers, transcriptomic risk profile, or intestinal microbiome in the subject. An “improvement,” as used herein refers to shift in the presence, level, or activity towards a presence, level, or activity, observed in normal individuals (e.g. individuals who do not suffer from the disease or condition). In instances wherein the therapeutic rAAV composition is not therapeutically effective or is not providing a sufficient alleviation of the disease or condition, or symptom of the disease or condition, then the dosage amount and/or route of administration may be changed, or an additional agent may be administered to the subject, along with the therapeutic rAAV composition. In some embodiments, as a patient is started on a regimen of a therapeutic rAAV composition, the patient is also weaned off (e.g., step-wise decrease in dose) a second treatment regimen. 
     In some embodiments, pharmaceutical compositions in accordance with the present disclosure may be administered at dosage levels sufficient to deliver from about 0.0001 mg/kg to about 100 mg/kg, from about 0.001 mg/kg to about 0.05 mg/kg, from about 0.005 mg/kg to about 0.05 mg/kg, from about 0.001 mg/kg to about 0.005 mg/kg, from about 0.05 mg/kg to about 0.5 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, from about 0.1 mg/kg to about 40 mg/kg, from about 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, or from about 1 mg/kg to about 25 mg/kg, of subject body weight per day, one or more times a day, to obtain the desired therapeutic, diagnostic, or prophylactic, effect. It will be understood that the above dosing concentrations may be converted to vg or viral genomes per kg or into total viral genomes administered by one of skill in the art. 
     In some cases, a dose of the pharmaceutical composition may comprise a concentration of infectious particles of at least or about 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 , 10 16 , or 10 17 . In some cases, the concentration of infectious particles is 2×10 7 , 2×10 8 , 2×10 9 , 2×10 10 , 2×10 11 , 2×10 12 , 2×10 13 , 2×10 14 , 2×10 15 , 2×10 16 , or 2×10 17 . In some cases, the concentration of the infectious particles is 3×10 7 , 3×10 8 , 3×10 9 , 3×10 10 , 3×10 11 , 3×10 12 , 3×10 13 , 3×10 14 , 3×10 15 , 3×10 16 , or 3×10 17 . In some cases, the concentration of the infectious particles is 4×10 7 , 4×10 8 , 4×10 9 , 4×10 10 , 4×10 11 , 4×10 12 , 4×10 13 , 4×10 14 , 4×10 15 , 4×10 16 , or 4×10 17 . In some cases, the concentration of the infectious particles is 5×10 7 , 5×10 8 , 5×10 9 , 5×10 10 , 5×10 11 , 5×10 12 , 5×10 13 , 5×10 14 , 5×10 15 , 5×10 16 , or 5×10 17 . In some cases, the concentration of the infectious particles is 6×10 7 , 6×10 8 , 6×10 9 , 6×10 10 , 6×10 11 , 6×10 12 , 6×10 13 , 6×10 14 , 6×10 15 , 6×10 16 , or 6×10 17 . In some cases, the concentration of the infectious particles is 7×10 7 , 7×10 8 , 7×10 9 , 7×10 10 , 7×10 11 , 7×10 12 , 7×10 13 , 7×10 14 , 7×10 15 , 7×10 16 , or 7×10 17 . In some cases, the concentration of the infectious particles is 8×10 7 , 8×10 8 , 8×10 9 , 8×10 10 , 8×10 11 , 8×10 12 , 8×10 13 , 8×10 14 , 8×10 15 , 8×10 16 , or 8×10 17 . In some cases, the concentration of the infectious particles is 9×10 7 , 9×10 8 , 9×10 9 , 9×10 10 , 9×10 11 , 9×10 12 , 9×10 13 , 9×10 14 , 9×10 15 , 9×10 16 , or 9×10 17 . 
     Disclosed herein, in some embodiments are formulations of pharmaceutically-acceptable excipients and carrier solutions suitable for delivery of the rAAV compositions described herein, as well as suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens. In some embodiments, the amount of therapeutic gene expression product in each therapeutically-useful composition may be prepared is such a way that a suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable. In some instances, the rAAV compositions are suitably formulated pharmaceutical compositions disclosed herein, to be delivered either intraocularly, intravitreally, parenterally, subcutaneously, intravenously, intracerebro-ventricularly, intramuscularly, intrathecally, orally, intraperitoneally, by oral or nasal inhalation, or by direct injection to one or more cells, tissues, or organs by direct injection. 
     In some embodiments, the pharmaceutical forms of the AAV-based viral compositions suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin. 
     In some cases, for administration of an injectable aqueous solution, for example, the solution may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, and the general safety and purity standards as required by FDA Office of Biologics standards. 
     Disclosed herein are sterile injectable solutions comprising the rAAV compositions disclosed herein, which are prepared by incorporating the rAAV compositions disclosed herein in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Injectable solutions may be advantageous for systemic administration, for example by intravenous administration. 
     Also provided herein are formulations in a neutral or salt form. Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as injectable solutions, drug-release capsules, and the like. 
     Pulmonary administration may be advantageously achieved via the buccal administration. In some embodiments, formulations may comprise dry particles comprising active ingredients. In such embodiments, dry particles may have a diameter in the range from about 0.5 nm to about 7 nm or from about 1 nm to about 6 nm. In some embodiments, formulations may be in the form of dry powders for administration using devices comprising dry powder reservoirs to which streams of propellant may be directed to disperse such powder. In some embodiments, self-propelling solvent/powder dispensing containers may be used. In such embodiments, active ingredients may be dissolved and/or suspended in low-boiling propellant in sealed containers. Such powders may comprise particles wherein at least 98% of the particles by weight have diameters greater than 0.5 nm and at least 95% of the particles by number have diameters less than 7 nm. Alternatively, at least 95% of the particles by weight have a diameter greater than 1 nm and at least 90% of the particles by number have a diameter less than 6 nm. Dry powder compositions may include a solid fine powder diluent such as sugar and are conveniently provided in a unit dose form. Low boiling propellants generally include liquid propellants having a boiling point of below 65 ° F. at atmospheric pressure. Generally, propellants may constitute 50% to 99.9% (w/w) of the composition, and active ingredient may constitute 0.1% to 20% (w/w) of the composition. Propellants may further comprise additional ingredients such as liquid non-ionic and/or solid anionic surfactant and/or solid diluent (which may have particle sizes of the same order as particles comprising active ingredients). 
     Pharmaceutical compositions formulated for pulmonary delivery may provide active ingredients in the form of droplets of solution and/or suspension. Such formulations may be prepared, packaged, and/or sold as aqueous and/or dilute alcoholic solutions and/or suspensions, optionally sterile, comprising active ingredients, and may conveniently be administered using any nebulization and/or atomization device. Such formulations may further comprise one or more additional ingredients including, but not limited to, a flavoring agent such as saccharin sodium, a volatile oil, a buffering agent, a surface-active agent, and/or a preservative such as methylhydroxybenzoate. Droplets provided by this route of administration may have an average diameter in the range from about 0.1 nm to about 200 nm. Formulations described herein useful for pulmonary delivery may also be useful for intranasal delivery. In some embodiments, formulations for intranasal administration comprise a coarse powder comprising the active ingredient and having an average particle size from about 0.2 μm to 500 μm. Such formulations are administered in the manner in which snuff is taken, i.e. by rapid inhalation through the nasal passage from a container of the powder held close to the nose. 
     Formulations suitable for nasal administration may, for example, comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) of active ingredient, and may comprise one or more of the additional ingredients described herein. A pharmaceutical composition may be prepared, packaged, and/or sold in a formulation suitable for buccal administration. Such formulations may, for example, be in the form of tablets and/or lozenges made using conventional methods, and may, for example, comprise 0.1% to 20% (w/w) active ingredient, the balance comprising an orally dissolvable and/or degradable composition and, optionally, one or more of the additional ingredients described herein. Alternately, formulations suitable for buccal administration may comprise powders and/or an aerosolized and/or atomized solutions and/or suspensions comprising active ingredients. Such powdered, aerosolized, and/or aerosolized formulations, when dispersed, may comprise average particle and/or droplet sizes in the range of from about 0.1 nm to about 200 nm, and may further comprise one or more of any additional ingredients described herein. 
     Suitable dose and dosage administrated to a subject is determined by factors including, but not limited to, the particular therapeutic rAAV composition, disease condition and its severity, the identity (e.g., weight, sex, age) of the subject in need of treatment, and can be determined according to the particular circumstances surrounding the case, including, e.g., the specific agent being administered, the route of administration, the condition being treated, and the subject or host being treated. 
     The amount of AAV compositions and time of administration of such compositions will be within the purview of the skilled artisan having benefit of the present teachings. It is likely, however, that the administration of therapeutically-effective amounts of the disclosed compositions may be achieved by a single administration, example, a single injection of sufficient numbers of infectious particles to provide therapeutic benefit to the patient undergoing such treatment. This is made possible, at least in part, by the fact that certain target cells (e.g., neurons) do not divide, obviating the need for multiple or chronic dosing. 
     Alternatively, in some circumstances, it may be desirable to provide multiple, or successive administrations of the AAV vector compositions, either over a relatively short, or a relatively prolonged period of time, as may be determined by the medical practitioner overseeing the administration of such compositions. For example, the number of infectious particles administered to a mammal may be on the order of about 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , or even higher, infectious particles/ml given either as a single dose, or divided into two or more administrations as may be required to achieve therapy of the particular disease or disorder being treated. In fact, in certain embodiments, it may be desirable to administer two or more different AAV vector compositions, either alone, or in combination with one or more other therapeutic drugs to achieve the desired effects of a particular therapy regimen. In various embodiments, the daily and unit dosages are altered depending on a number of variables including, but not limited to, the activity of the therapeutic rAAV composition used, the disease or condition to be treated, the mode of administration, the requirements of the individual subject, the severity of the disease or condition being treated, and the judgment of the practitioner. 
     In some embodiments, the administration of the therapeutic rAAV composition is hourly, once every 2 hours, 3 hours, 4 hours, 5 hours, 6 hours,? hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, or 5 years, or 10 years. The effective dosage ranges may be adjusted based on subject&#39;s response to the treatment. Some routes of administration will require higher concentrations of effective amount of therapeutics than other routes. 
     Although not anticipated given the advantages of the present disclosure, in certain embodiments wherein the patient&#39;s condition does not improve, upon the doctor&#39;s discretion the administration of therapeutic rAAV composition is administered chronically, that is, for an extended period of time, including throughout the duration of the patient&#39;s life in order to ameliorate or otherwise control or limit the symptoms of the patient&#39;s disease or condition. In certain embodiments wherein a patient&#39;s status does improve, the dose of therapeutic rAAV composition being administered may be temporarily reduced or temporarily suspended for a certain length of time (i.e., a “drug holiday”). In specific embodiments, the length of the drug holiday is between 2 days and 1 year, including by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, or more than 28 days. The dose reduction during a drug holiday is, by way of example only, by 10%-100%, including by way of example only 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 100%. In certain embodiments, the dose of drug being administered may be temporarily reduced or temporarily suspended for a certain length of time (i.e., a “drug diversion”). In specific embodiments, the length of the drug diversion is between 2 days and 1 year, including by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, or more than 28 days. The dose reduction during a drug diversion is, by way of example only, by 10%-100%, including by way of example only 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 100%. After a suitable length of time, the normal dosing schedule is optionally reinstated. 
     In some embodiments, once improvement of the patient&#39;s conditions has occurred, a maintenance dose is administered if necessary. Subsequently, in specific embodiments, the dosage or the frequency of administration, or both, is reduced, as a function of the symptoms, to a level at which the improved disease, disorder or condition is retained. In certain embodiments, however, the patient requires intermittent treatment on a long-term basis upon any recurrence of symptoms. 
     Toxicity and therapeutic efficacy of such therapeutic regimens are determined by standard pharmaceutical procedures in cell cultures or experimental animals, including, but not limited to, the determination of the LD50 and the EDS°. The dose ratio between the toxic and therapeutic effects is the therapeutic index and it is expressed as the ratio between LD50 and EDS°. In certain embodiments, the data obtained from cell culture assays and animal studies are used in formulating the therapeutically effective daily dosage range and/or the therapeutically effective unit dosage amount for use in mammals, including humans. In some embodiments, the dosage amount of the therapeutic rAAV composition described herein lies within a range of circulating concentrations that include the ED50 with minimal toxicity. In certain embodiments, the daily dosage range and/or the unit dosage amount varies within this range depending upon the dosage form employed and the route of administration utilized. 
     Additional Therapeutic 
     A therapeutic nucleic acid may be used alone or in combination with an additional therapeutic agent (together, “therapeutic agents”). In some cases, an “additional therapeutic agent” as used herein is administered alone. The therapeutic agent may be administered together or sequentially in a combination therapy. The combination therapy may be administered within the same day, or may be administered one or more days, weeks, months, or years apart. In some cases, a therapeutic nucleic acid provided herein is administered if the subject is determined to be non-responsive to a first line of therapy. 
     The additional therapeutic agent can comprise a small molecule. The additional therapeutic agent can comprise an antibody, or antigen-binding fragment. The additional therapeutic agent can comprise a cell-based therapy. Exemplary cell-based therapies include without limitation immune effector cell therapy, chimeric antigen receptor T-cell (CAR-T) therapy, natural killer cell therapy and chimeric antigen receptor natural killer (NK) cell therapy. Either NK cells, or CAR-NK cells, or a combination of both NK cells and CAR-NK cells can be used in combination with the methods disclosed herein. In some embodiments, the NK cells and CAR-NK cells are derived from human induced pluripotent stem cells (iPSC), umbilical cord blood, or a cell line. The NK cells and CAR-NK cells can comprise a cytokine receptor and a suicide gene. The cell-based therapy can comprises a stem cell therapy. The stem cell therapy may be embryonic or somatic stem cells. The stem cells may be isolated from a donor (allogeneic) or isolated from the subject (autologous). The stem cells may be expanded adipose-derived stem cells (eASCs), hematopoietic stem cells (HSCs), mesenchymal stem (stromal) cells (MSCs), or induced pluripotent stem cells (iPSCs) derived from the cells of the subject. 
     III. KITS 
     Disclosed herein are kits comprising compositions disclosed herein. Also disclosed herein are kits for the treatment or prevention of a disease or conditions of the central nervous system (CNS), or target organ or environment (e.g., liver). In some instances, the disease or condition is cancer, a pathogen infection, pulmonary disease or condition, neurological disease, muscular disease, or an immune disorder, such as those described herein. In one embodiment, a kit can include a therapeutic or prophylactic composition containing an effective amount of a composition of a rAAV particle encapsidating a recombinant AAV vector encoding a therapeutic nucleic acid (e.g., therapeutic nucleic acid) and a recombinant AAV (rAAV) capsid protein of the present disclosure. In another embodiment, a kit can include a therapeutic or prophylactic composition containing an effective amount of cells modified by the rAAV described herein (“modified cell”), in unit dosage form that express therapeutic nucleic acid. In some embodiments, a kit comprises a sterile container which can contain a therapeutic composition; such containers can be boxes, ampules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments. 
     In some cases, rAAV are provided together with instructions for administering the rAAV to a subject having or at risk of developing the disease or condition (e.g., disease of the CNS, PNS, liver, and the like). Instructions can generally include information about the use of the composition for the treatment or prevention of the disease or condition. 
     In some cases, a kit can include allogenic cells. In some cases, a kit can include cells that can comprise a genomic modification. In some cases, a kit can comprise “off-the-shelf” cells. In some cases, a kit can include cells that can be expanded for clinical use. In some cases, a kit can contain contents for a research purpose. 
     In some cases, the instructions include at least one of the following: description of the therapeutic rAAV composition; dosage schedule and administration for treatment or prevention of the disease or condition disclosed herein; precautions; warnings; indications; counter-indications; overdosage information; adverse reactions; animal pharmacology; clinical studies; and/or references. The instructions can be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container. In some cases, instructions provide procedures for administering the rAAV to the subject alone. In some cases, instructions provide procedures for administering the rAAV to the subject at least about 1 hour (hr), 2 hr, 3 hr, 4 hr, 5 hr, 6 hr, 7 hr, 8 hr, 9 hr,10 hr, 11 hr, 12 hr, 13 hr, 14 hr, 15 hr, 16 hr, 17 hr, 18 hr, 19 hr, 20 hr, 21 hr, 22 hr, 23 hr, 24 hr, 25 hr, 26 hr, 27 hr, 28 hr, 29 hr, 30 hr, or up to 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days after or before administering an additional therapeutic agent disclosed herein. In some instances, the instructions provide that the rAAV is formulated for intravenous injection. 
     IV. DEFINITIONS 
     The terminology used herein is for the purpose of describing particular cases only and is not intended to be limiting. As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof are used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising.” 
     The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the given value. Where particular values are described in the application and claims, unless otherwise stated the term “about” should be assumed to mean an acceptable error range for the particular value. 
     As used herein “consisting essentially of” when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination for the stated purpose. Thus, a composition consisting essentially of the elements as defined herein would not exclude other materials or steps that do not materially affect the basic and novel characteristic(s) of the claimed disclosure, such as compositions for treating skin disorders like acne, eczema, psoriasis, and rosacea. 
     The terms “homologous,” “homology,” or “percent homology” are used herein to generally mean an amino acid sequence or a nucleic acid sequence having the same, or similar sequence to a reference sequence. Percent homology of sequences can be determined using the most recent version of BLAST, as of the filing date of this application. 
     The terms “increased,” or “increase” are used herein to generally mean an increase by a statically significant amount. In some embodiments, the terms “increased,” or “increase,” mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 10%, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, standard, or control. Other examples of “increase” include an increase of at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 1000-fold or more as compared to a reference level. 
     The terms, “decreased” or “decrease” are used herein generally to mean a decrease by a statistically significant amount. In some embodiments, “decreased” or “decrease” means a reduction by at least 10% as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease (e.g., absent level or non-detectable level as compared to a reference level), or any decrease between 10-100% as compared to a reference level. In the context of a marker or symptom, by these terms is meant a statistically significant decrease in such level. The decrease can be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, and is preferably down to a level accepted as within the range of normal for an individual without a given disease. 
     The terms “subject” is any organism. In some instances, the organism is a mammal. Non-limiting examples of mammal include, any member of the mammalian class: humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like. In one aspect, the mammal is a human. The term “animal” as used herein comprises human beings and non-human animals. In one embodiment, a “non-human animal” is a mammal, for example a rodent such as rat or a mouse. In some instances, the subject is a patient, which as used herein, may refer to a subject diagnosed with a particular disease or disorder. 
     The term “gene,” as used herein, refers to a segment of nucleic acid that encodes an individual protein or RNA (also referred to as a “coding sequence” or “coding region”), optionally together with associated regulatory region such as promoter, operator, terminator and the like, which may be located upstream or downstream of the coding sequence. 
     The term “adeno-associated virus,” or “AAV” as used herein refers to the adeno-associated virus or derivatives thereof. Non-limited examples of AAV&#39;s include AAV type 1 (AAV1), AAV type 2 (AAV2), AAV type 3 (AAV3), AAV type 4 (AAV4), AAV type 5 (AAV5), AAV type 6 (AAV6), AAV type 7 (AAV7), AAV type 8 (AAV8), AAV type 9 (AAV9), AAV type 10 (AAV10), AAV type 11 (AAV11), AAV type 12 (AAV12), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV. In some instances, the AAV is described as a “Primate AAV,” which refers to AAV that infect primates. Likewise an AAV may infect bovine animals (e.g., “bovine AAV”, and the like). In some instances, the AAV is wildtype, or naturally occurring. In some instances, the AAV is recombinant. 
     The term “AAV capsid” as used herein refers to a capsid protein or peptide of an adeno-associated virus. In some instances, the AAV capsid protein is configured to encapsidate genetic information (e.g., a transgene, therapeutic nucleic acid, viral genome). In some instances, the AAV capsid of the instant disclosure is a modified AAV capsid, relative to a corresponding parental AAV capsid protein. 
     The term “tropism” as used herein refers to a quality or characteristic of the AAV capsid that may include specificity for, and/or an increase or a decrease in efficiency of, expressing the encapsidated genetic information into one in in vivo environment, relative to a second in vivo environment. An in vivo environment, in some instances, is a cell-type. An in vivo environment, in some instances, is an organ or organ system. 
     The term “AAV vector” as used herein refers to nucleic acid polymer encoding genetic information related to the virus. The AAV vector may be a recombinant AAV vector (rAAV), which refers to an AAV vector generated using recombinatorial genetics methods. In some instances, the rAAV vector comprises at least one heterologous polynucleotide (e.g. a polynucleotide other than a wild-type or naturally occurring AAV genome such as a transgene). 
     The term “AAV particle” as used herein refers to an AAV virus, virion, AAV capsid protein or component thereof. In some cases, the AAV particle is modified relative to a parental AAV particle. 
     The term “gene product” of “gene expression product” refers to an expression product of a polynucleotide sequence such as, for e.g., a polypeptide, peptide, protein or RNA, including interfering RNA (e.g., siRNA, miRNA, shRNA) and messenger RNA (mRNA). 
     The terms “operatively linked” or “operably linked” refers to a location of two or more elements being close together, and in some cases, next to one other (e.g., genetic elements such as a promoter, enhancer, termination signal sequence, polyadenylation sequence, and the like) that enables a functional relationship between the two or more elements. In one non-limiting example, a promoter that is operatively linked to a coding region enables the initiation of transcription of the coding sequence. 
     The term “heterologous” as used herein refers to a genetic element (e.g., coding region) or gene expression product (e.g., RNA, protein) that is derived from a genotypically distinct entity from that of the rest of the entity to which it is being compared. 
     The term “endogenous” as used herein refers to a genetic element (e.g., coding region) or gene expression product (e.g., RNA, protein) that is naturally occurring in or associated with an organism or a particular cell within the organism. 
     A “detectable moiety” as used herein refers to a moiety that can be covalently or noncovalently attached to a compound or biomolecule that can be detected for instance, using techniques known in the art. In embodiments, the detectable moiety is covalently attached. The detectable moiety may provide for imaging of the attached compound or biomolecule. The detectable moiety may indicate the contacting between two compounds. Exemplary detectable moieties are fluorophores, antibodies, reactive dies, radio-labeled moieties, magnetic contrast agents, and quantum dots. Exemplary fluorophores include fluorescein, rhodamine, GFP, coumarin, FITC, Alexa fluor, Cy3, Cy5, BODIPY, and cyanine dyes. Exemplary radionuclides include Fluorine-18, Gallium-68, and Copper-64. Exemplary magnetic contrast agents include gadolinium, iron oxide and iron platinum, and manganese. 
     The terms “treat,” “treating,” and “treatment” as used herein refers to alleviating or abrogating a disorder, disease, or condition; or one or more of the symptoms associated with the disorder, disease, or condition; or alleviating or eradicating a cause of the disorder, disease, or condition itself. Desirable effects of treatment can include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishing any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state and remission or improved prognosis. 
     The term “therapeutically effective amount” refers to the amount of a compound or therapy that, when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the symptoms of a disorder, disease, or condition of the disease; or the amount of a compound that is sufficient to elicit biological or medical response of a cell, tissue, system, animal, or human that is being sought by a researcher, veterinarian, medical doctor, or clinician. 
     The term “pharmaceutically acceptable carrier,” “pharmaceutically acceptable excipient,” “physiologically acceptable carrier,” or “physiologically acceptable excipient” refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material. A component can be “pharmaceutically acceptable” in the sense of being compatible with the other ingredients of a pharmaceutical formulation. It can also be suitable for use in contact with the tissue or organ of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications, commensurate with a reasonable benefit/risk ratio. See, Remington: The Science and Practice of Pharmacy, 21st Edition; Lippincott Williams &amp; Wilkins: Philadelphia, Pa., 2005; Handbook of Pharmaceutical Excipients, 5th Edition; Rowe et al., Eds., The Pharmaceutical Press and the American Pharmaceutical Association: 2005; and Handbook of Pharmaceutical Additives, 3rd Edition; Ash and Ash Eds., Gower Publishing Company: 2007; Pharmaceutical Preformulation and Formulation, Gibson Ed., CRC Press LLC: Boca Raton, Fla., 2004). 
     The term “pharmaceutical composition” refers to a mixture of a compound disclosed herein with other chemical components, such as diluents or carriers. The pharmaceutical composition can facilitate administration of the compound to an organism. Multiple techniques of administering a compound exist in the art including, but not limited to, oral, injection, aerosol, parenteral, and topical administration. 
     Non-limiting examples of “sample” include any material from which nucleic acids and/or proteins can be obtained. As non-limiting examples, this includes whole blood, peripheral blood, plasma, serum, saliva, mucus, urine, semen, lymph, fecal extract, cheek swab, cells or other bodily fluid or tissue, including but not limited to tissue obtained through surgical biopsy or surgical resection. In various embodiments, the sample comprises tissue from the large and/or small intestine. In various embodiments, the large intestine sample comprises the cecum, colon (the ascending colon, the transverse colon, the descending colon, and the sigmoid colon), rectum and/or the anal canal. In some embodiments, the small intestine sample comprises the duodenum, jejunum, and/or the ileum. Alternatively, a sample can be obtained through primary patient derived cell lines, or archived patient samples in the form of preserved samples, or fresh frozen samples. 
     The term “in vivo” is used to describe an event that takes place in a subject&#39;s body. 
     The term “ex vivo” is used to describe an event that takes place outside of a subject&#39;s body. An ex vivo assay is not performed on a subject. Rather, it is performed upon a sample separate from a subject. An example of an ex vivo assay performed on a sample is an “in vitro” assay. 
     The term “in vitro” is used to describe an event that takes places contained in a container for holding laboratory reagent such that it is separated from the biological source from which the material is obtained. In vitro assays can encompass cell-based assays in which living or dead cells are employed. In vitro assays can also encompass a cell-free assay in which no intact cells are employed. 
     The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. 
     V. EXAMPLES 
     Example 1 
     Method of Producing an rAAV 
     A recombinant AAV (rAAV) is produced. Three plasmid vectors are triple-transfected into an immortalized HEK293 using a standard transfection protocol (e.g., with PEI). The first vector contains a transgene cassette flanked by inverted terminal repeat (ITR) sequences from a parental AAV virus. The transgene cassette has a promoter sequence and that drives transcription of a heterologous nucleic acid in the nucleus of the target cell. The second vector contains nucleic acids encoding the AAV Rep gene, as well as a modified Cap gene e.g., AAV2/9 REP-AAP-ACap). Modified Cap gene comprises any one of the DNA sequences provided in  FIGS. 33-35 , which are the DNA sequences encoding the modified AAV capsid proteins of the present disclosure. The third vector contains nucleic acids encoding helper virus proteins needed for viral assembly, and packaging of the heterologous nucleic acid into the modified capsid structure. 
     Viral particles are harvested from the media after 72 h post transfection and from the cells and media at 120 h post transfection. Virus present in the media is concentrated by precipitation with 8% poly(ethylene glycol) and 500 mM sodium chloride and the precipitated virus is added to the lysates prepared from the collected cells. The viruses are purified over iodixanol (Optiprep, Sigma) step gradients (15%, 25%, 40% and 60%). Viruses are concentrated and formulated in PBS. Virus titers are determined by measuring the number of DNasel-resistant vector genome copies (VGs) using qPCR and the linearized genome plasmid as a control. 
     Example 2 
     Methods of Identifying Variant AAV Capsid Proteins 
     Plasmid 
     Library generation. The rAAV-ACap-in-cis-Lox2 plasmid ( FIG. 36 ) is a modification of the rAAV-ACap-in-cis-Lox plasmid. For 7-mer-i library fragment generation, the pCRII-9Cap-XE plasmid was used as a template. The AAV2/9 REP-AAP-ACap plasmid ( FIG. 36 ) was modified from the AAV2/9 REP-AAP plasmid. 
     The rAAV-ACap-in-cis-Lox2 plasmid consists of three major elements that are flanked by AAV2 ITRs. (i) UBC ubiquitous promoter driving the expression of fluorescent protein, mNeongreen, followed by a synthetic polyadenylation sequence. The mCherry expression cassette of the previous version of the plasmid was replaced by mNeonGreen cassette. (ii) A portion of AAV2 rep gene that has the splicing sequences and AAVS p41 promoter (1680-1974 residues of GenBank AF085716.1) followed by AAV9 cap gene. The prior version of this plasmid, rAAV-ACap-in-cis-Lox, has a short 12 bp sequence between restriction sites Xbal and Agel at AA 450 and 592 of the AAV9 Cap gene. This was replaced by a 723 bp sequence of mRuby2 gene in-frame (acts as filler DNA) in the newer version of the plasmid. (iii) SV40 polyadenylation sequence that is flanked by lox71 and lox66 sites. The minor changes were introduced to the prior version of the plasmid to facilitate ease of cloning and to visualize mammalian cell transfection. The Lox sites in these rAAV plasmids show modest levels of Cre-independent flipping. This was minimized during PCR-based capsid recovery by lowering the number of amplification cycles to a point where we cannot recover any rAAV capsids from the control DNA extracted from wild-type mice (i.e., lacking Cre expression) that were injected with the library. The pCRII-9Cap-XE plasmid contains the AAV9 capsid gene sequence from AAs 450-592 and is flanked by Xbal and Agel restriction sites. 
     The AAV2/9 REP-AAP-ACap plasmid has the five previously existing stop codons of AAV2/9 REP-AAP in addition to the deletion of AAs 450-592 of the AAV9 capsid sequence. These modifications did not affect vector production. The deletion of the overlapping fragment between the REP-AAP and rAAV-ACap-in-cis-Lox2 plasmids minimizes recombination between plasmids that could potentially generate AAV9 wild-type capsids during co-transfection in vector production. 
     Capsid Characterization 
     AAV capsids. The AAV capsid variants with 7-mer insertions or 11-mer substitutions were made between positions 587-597 of AAV-PHP.B capsid using the pUCmini-iCAP-PHP.B backbone (Addgene ID: 103002). 
     ssAAV genomes. To characterize the AAV capsid variants, the single stranded (ss) rAAV genomes were used. Genomes such as pAAV:CAG-mNeonGreen27 (equivalent plasmid, pAAV: CAG-eYFP35; Addgene ID: 104055), pAAV:CAG-NLS-EGFP26 (equivalent version with one NLS is on Addgene ID 104061), pAAV:CAG-DIO-EYFP35 (Addgene ID: 104052), pAAV: GfABC1D-2xNLS-mTurquoise235 (Addgene ID: 104053), and pAAV-Ple261-iCre30 (Addgene ID 49113) were used. 
     pAAV:CAG-mNeonGreen2 genome consists of a ubiquitous CMV-(3-Actin-intron-(3-Globin (CAG) hybrid promoter driving the expression of a fluorescent protein, mNeonGreen (equivalent plasmid, pAAV: CAG-eYFP3; Addgene ID: 104055). pAAV:CAG-NLS-EGFP1 consists of NLS sequences at the N- and C-termini of EGFP and is driven by the CAG promoter. An equivalent version with one NLS is on Addgene (ID 104061). pAAV:CAG-DIO-EYFP3 (Addgene ID: 104052) consists of a EYFP gene built in the reverse direction of the CAG promoter, and it is flanked by a pair of Cre-Lox sites (Lox P and Lox 2272) on either ends. 
     In cells expressing Cre, the Cre-lox pair inverts EYFP enabling transcription and translation, followed by excision in the lox site to prevent re-inversion. pAAV: GfABC1D-2xNLS-mTurquoise23, referred to elsewhere as pAAV:GFAP-2xNLS-mTurquoise2 (Addgene ID: 104053), consists of NLS sequences at the N- and C-termini of mTurquoise2 and is driven by the astrocyte-specific promoter GfABC1D4. pAAV:Ple261-iCre5 (Addgene ID 49113) contains an endothelial-cell-specific promoter driving the expression of iCre. 
     pAAV:CAG-XFP (mNeongreen) was packaged for characterizing AAV variants. However, when performing quantification of cell-types: neurons, astrocytes and oligodendrocytes, CAG-NLS-EGFP was used to restrict the expression to nucleus for easier quantification using microscope images. GFAP-NLS-mTurq2 is used to quantify astrocytes. CAG-DIO-EYFP is used for Cre driver lines, due to the presence of lox sites in this plasmid. 
     The self-complementary genome from Dr. Guangping Gao, scAAV:CB6-EGFP genome has a hybrid ubiquitous CB6 promoter (975 bp) comprising a CMV enhancer (cytomegalovirus immediate early enhancer), a chicken-f3-actin promoter and hybrid intron, that drives the expression of EGFP. The genome has a rabbit globin poly A (127 bp) following the EGFP gene. The scAAV:CAG-EGFP (Addgene ID:83279), vector uses a ubiquitous CMV-β-Actin-intron-β-Globin (CAG) hybrid promoter to drive the expression of EGFP. 
     Round-I AAV Capsid Library Generation 
     Mutagenesis strategy. The 7-mer randomized insertion was designed using the NNK saturation mutagenesis strategy, involving degenerate primers (from Integrated DNA Technologies, Inc.) containing mixed bases. N can be A, C, G, or T bases and K can be G, or T. Using this strategy, combinations of all 20 AAs at each position of the 7-mer peptide using 33 codons were obtained, resulting in a library size of 1.28 billion at the level of AA combinations. The mutagenesis strategy for the 3-mer-s PHP.B library is described in Chan, K. Y. et al. Engineered AAVs for efficient noninvasive gene delivery to the central and peripheral nervous systems.  Nat. Neurosci.  20, 1172-1179 (2017). 
     Library cloning. The 480 bp AAV capsid fragment (450-592 AAs) with the 7-mer random insertion between AAs 588 and 589 was generated by conventional PCR methods with a mixed base degenerate primer. The library fragment was amplified from the pCRII-9Cap-XE template by Q5 Hot Start High-Fidelity 2× Master Mix (NEB; M0494S) with forward primer, XF: 5′-ACTCATCGACCAATACTTGTACTATCTCTCTAGAAC-3′ and reverse primer, 7×MNN-588i: 5′-GTATTCCTTGGTTTTGAACCCAACCGGTCTGCGCCTGTGCMNNMNNMNNMNNMNN MNNMNNTTGGGCACTCTGGTGGTTTGTG-3′. To avoid PCR-induced biases resulting from point mutations, recombination, and template switching, PCR amplification was limited to 15-20 cycles and the reactions were scaled up to get the required yield. The resulting PCR products were run on a 1% agarose gel and extracted with a Zymoclean Gel DNA Recovery kit (Zymo Research; D4007). It is critical to avoid AAV contamination during this step by taking precautionary measures like using a clean gel-running box and freshly prepared 1×TAE buffer. 
     rAAV-ACap-in-cis-Lox2 plasmid (6960 bp) was linearized with the restriction enzymes Agel and Xbal by following the NEB recommended protocol for double digestion. The digested plasmid was run on a 0.8%-1% agarose gel to extract the linearized backbone (6237 bp) with a Zymoclean Gel DNA Recovery kit. 
     The amplified library fragment was assembled into the linearized vector with the NEBuilder HiFi DNA Assembly Master Mix (NEB; E2621S) and a 1:2 molar ratio of vector to insert, to assemble at 50° C. for 60 min. 
     Library purification. The assembled library was then subjected to Plasmid Safe (PS) DNase (Epicentre; E3105K) treatment to purify the assembled product by degrading the un-assembled DNA fragments from the mixture. For the R1 library, around 3U of PS DNase per 1 μg of input DNA was used in a 30 min reaction at 37° C. Alternatively, Exonuclease V (RecBCD) was used following the NEB recommended protocol (NEB; M0345S). Both procedures yielded comparable results. The resulting mixture was further purified with a DNA Clean and Concentrator kit (Zymo Research; D4013). 
     Library yield. With an assembly efficiency of 15%-20% post-PS treatment, a yield of about 15-20 ng per 100 ng of input DNA per 20 μL reaction was obtained. For building the 7-mer-i DNA library, approximately 5-6 μg of input DNA was used to obtain around 800 ng of assembled library. 
     Quality control. To validate successful assembly of the library, 1 ng of the final assembled library was transformed into  E. coli  SURE 2 Supercompetent Cells (Integrated Sciences; 200152). Colonies on an LB/Agar plate containing carbenicillin antibiotic after overnight incubation at 37° C. were identified. The DNA library was sequenced around the insertion site (Laragen; Sanger Sequencing). A non-biased library shows multiple nucleotide peaks of equal diversity (25% each of A, T, G, C) across every base position of the diversified region. To verify that the ITRs were intact, Smal digestion was carried out as per the NEB recommended protocol (NEB; R0141S). To validate successful transfection and assess the vector-production yield per 150 mm dish, 10 ng of 7-mer-i library was used to transfect HEK293 producer cells. Uniform expression of mNeonGreen protein across HEK cells was observed, and an average yield of 0.1-1×10 11  vg was obtained per 150 mm dish. Using the average yield per dish, the vector production for in vivo selection was scaled up (See  FIG. 45 ). 
     Round-2 AAV Capsid DNA Library 
     PCR pool design. To maintain proportionate pooling, the fraction of each sample/library that needs to be pooled based on an individual library&#39;s diversity was mathematically determined. This process involved estimation of the diversity precluding noise and consideration of amplification of this diversity across samples by determining the area under the curve for the interval of high-confidence variants that falls in the higher RC range. The area under the curve (AUC) was estimated using the composite Simpson&#39;s rule by plotting all the recovered variants in a library (X-coordinate) to their read counts (RCs or copy number from deep sequencing data, Y-coordinate) (see  FIG. 40 ). To determine the definite intervals for AUC, the data was sorted based on the decreasing order of the RCs. Noticeably, the distribution has two phases, with a steadier slope of variants in the higher RC range, followed by a steep drop in the slope of the curve (˜50-1000 fold lower RCs). By observation, this steeper side of the curve is predominant in sequencing errors/ PCR mutations, hence this error dominant slope otherwise called noise from was precluded from the AUC estimation. When comparing composite Simpson&#39;s rule with another function, such as composite trapezoidal rule, the difference was miniscule. 
     This area is then used to determine the fraction of an individual library that needs to be pooled into PCR pool library using the formula: [Area under the curve/ total number of libraries pooled]. 
     The pooled sample was used as a template for further amplification with 12 cycles of 98° C. for 10 s, 60° C. for 20 s, and 72° C. for 30 s by Q5 polymerase, using the primers 588-R2lib-F: 5′-CACTCATCGACCAATACTTGTACTATCTCTCT-3′ and 588-R2lib-R: 5′-GTATTCCTTGGTTTTGAACCCAACCG-3′. Similar to R1 library generation, the PCR product was assembled into the rAAV-ACap-in-cis-Lox2 plasmid and the virus was produced. 
     The R1 libraries used to build R2 were the Cre-Lox flipped rAAV DNA from half of the mouse brains (˜0.3 g) and portion of spinal cords (0.1-0.2 g) from all Cre lines. The amount of tissue processed here was sufficient for complete capsid library recovery. The differentially pooled and amplified libraries (by PCR pool or synthetic pool) were assembled using gibson assembly with a follow-up PS or Exonuclease V treatment (as described in R1 library generation). Successful library generation was validated by transformation, Sanger sequencing, and an ITR Smal digest. 
     For vector production, about 10 ng of the purified and assembled library was used to transfect each 150 mm dish of 293T cells, and a yield of about 6×10 11  vg per 150 mm dish was obtained (i.e. the R2 yield was six times that of R1, unsurprisingly given these sequences have already produced well enough to survive R1 selection). 
     Synthetic pool design. As described in the PCR pool strategy, high-confidence variants were chosen with RCs above the error-dominant noise slope from the plot of library distribution (see  FIG. 40 ). This came to about 9000 sequences from all brain and spinal cord samples of all Cre lines. A similar primer design as mentioned in the description of the R1 library generation was used. Primers XF: 5′-ACTCATCGACCAATACTTGTACTATCTCTCTAGAAC-3′ and 11-mer-588i: 5′-GTATTCCTTGGTTTTGAACCCAACCGGTCTGCGCXXXXXXMNNMNNMNNMNNMNN MNNMNNXXXXXXACTCTGGTGGTTTGTG-3′, where “XXXXXXMNNMNNMNNMNNMNNMNNMNNXXXXXX” was replaced with unique nucleotide sequence of a 7-mer tissue recovered variant (7×MNN) along with modification of two adjacent codons flanking on either end of the 7-mer insertion site (6xX), which are residues 587-588 “AQ” and residues 589-590 “AQ” on AAV9 capsid. To select sequences for synthesis, the R1 brain and spinal cord libraries were chosen and used the same threshold limit for RC as described in the PCR pool strategy. This came to about 9000 sequences from all brain and spinal cord samples of all Cre lines. Since spike-in library has 11-mer mutated variants, the same primer design where “XXXXXXMNNMNNMNNMNNMNNMNNMNNXXXXXX” was replaced with a specific nucleotide sequence of a 11-mer variant. A duplicate of each sequence in this library was designed with different codons optimized for mammals. The primers were designed using a custom built python based script (code will be made available in Github). The custom-designed oligopool was synthesized in an equimolar ratio by Twist Biosciences. The oligopool was used to amplify the pCRII-XE Cap9 template over 13 cycles of 98° C. for 10 s, 60° C. for 20 s, and 72° C. for 30 s. To obtain a higher yield for large-scale library preparation, the product of the first PCR was used as a template for the second PCR using the primers XF and 588-R2lib-R (described above) and amplified for 13 cycles. As described in the description of the R1 library generation, the PCR product was assembled into an rAAV backbone and processed and purified for virus production. About 10 ng per 150 mm dish of HEK293 cells produced about 6×10 11  vg of virus library. 
     AAV Viral Library Production and Purification 
     To prevent capsid mosaic formation of the 7-mer-i library in HEK293 producer cells, only 10 ng of assembled library per 150 mm dish were transfected along with other required reagents for AAV vector production. In addition to the 10 ng of library transfection per 150 mm dish of 293T producer cells, three plasmids were transfected: AAV2/9 REP-AAP-ACap, pUC18 and pHelper (genes encoding adenoviral proteins for AAV replication) at a ratio of 1:1:2. The plasmid pUC18 acts as a filler DNA to compensate for the low amount of library DNA in order to maintain the N:P ratio required for optimal transfection using polyethylenimine (PEI, Polysciences; 24765-1) transfection). The cells and culture media were harvested at 60 h post-transfection to collect the viral particles. rAAV harvest and purification were performed as per the protocol. The small amount of library DNA per plate and early cell harvest time are critical for reducing the possibility of mosaic capsid assemblies during vector production (similar considerations seen in prior reports). 
     For 7-mer-i library, the production was scaled up to 60 dishes (˜1.8×10 7  cells/dish) and with ˜10% transfected with the library, resulted in ˜1×10 8  total transformants. For an NNK 7mer library with ˜1×10 8  total transformants, the number of unique variants is 9.99×10 7 . 
     For the rAAV DNA extraction from purified rAAV viral library, ˜10% of the purified viral library was used to extract the viral genome by proteinase K treatment. In order to degrade any contaminating DNA from the purified library, it was treated with DNase I enzyme (5 μl of 10 U/μl) (Sigma-Aldrich; 4716728001) in 100 μl of DNase I buffer and incubated for 1 hat 37° C. The enzyme was inactivated by adding 5 μl of 0.5 M EDTA at 70° C. for 10 min. Following DNase I treatment, the capsid protein shell was digested by adding 120 μl of proteinase solution containing 5 μl of 20 μg/μl of proteinase K and incubated at 50° C. overnight. To inactivate the proteinase K, the mixture was boiled at 95° C. The extracted rAAV library DNA was then concentrated and purified using phenol chloroform and ethanol. An equal volume of Phenol:Chloroform:Isoamyl Alcohol 25:24:1, pH8.0 (˜250 μl; ThermoFisher Scientific; 15593031) was added and vortexed for 30 s. The mixture is incubated for 5 min at room temperature (RT) before centrifugation at 15,000 rpm for 10 min at 4° C. The upper aqueous phase was separated and mixed with an equal volume of chloroform and vortexed for 30 s. Following 5 min incubation at RT, centrifuge at 15,000 rpm for 10 min at 4° C. The upper aqueous phase was separated and one-tenth volume of 3M sodium acetate (pH 5.2) along with 2 μl Co-Precipitant Pink (Bioline; BIO-37075) and 2.5 volumes of ice cold 100% ethanol was added before vortexing for 30 s. The mixture was incubated for at least 1 hr at ˜20° C. before centrifugation at 15,000 rpm for 15 min at 4° C. The pellet was air dried and resuspended in TE buffer. The DNA concentration was determined using the Qubit ssDNA assay. 
     Animals 
     All animal procedures performed in this study were approved by the California Institute of Technology Institutional Animal Care and Use Committee (IACUC). C57BL/6J (000664), Tek-Cre (8863), SNAP25-Cre (23525), GFAP-Cre (012886), Synl-Cre (3966), and Ai14 (007908) mice lines used in this study were purchased from the Jackson Laboratory (JAX). For in vivo library selection, 6- to 8-week-old adult male and female mice were intravenously injected with the viral libraries. Both genders were used for capsid selection to recover capsid variants with minimal gender bias. The IV injection of rAAVs was into the retro-orbital sinus of adult mice. For testing the transduction phenotypes of rAAVs, 6- to 8-week-old C57BL/6J or Tek-Cre or Ai14 adult male mice were randomly assigned. The experimenter was not blinded for any of the experiments performed in this study. 
     In Vivo Selection 
     The 7-mer-i viral library selections were carried out in different lines of Cre transgenic adult mice: Tek-Cre, SNAP25-Cre, and GFAP-Cre for the R1 selections, and those three plus Syn1-Cre for the R2 selections. Male and female adult mice were intravenously administered with a viral vector dose of 2×10 11  vg/mouse for the R1 selection, and a dose of 1×10 12  vg/mouse for the R2 selection. The dose was determined based on the virus yield which was different across selection rounds ( FIG. 45 ). Both genders were used to recover capsid variants with minimal gender bias. Two weeks post-injection, mice were euthanized and all organs including brain were collected, snap frozen on dry ice, and stored at −80° C. 
     rAAV Genome Extraction from Tissue 
     Optimization. For rAAV genome extraction from tissues, both the Trizol method (Life Technologies; 15596) and the QlAprep Spin Miniprep kit (Qiagen, Inc; 27104) were used according to the manufacturers&#39; recommended protocols, and found the Trizol method to be more efficient. The total rAAV genome recovery from 0.1 g of mouse liver was quantified by quantitative PCR using the primers mNeonGreen-F: 5′-CGACACATGAGTTACACATCTTTGGCTC-3′ and mNeonGreen-R: 5′-GGAGGTCACCCTTGGTGGACTTC-3′, which binds to the mNeonGreen gene of the ssAAV-ACap-in-cis-Lox2 genome. As an internal control, the amount of mitochondrial DNA (a measure of the recovery of smaller genomes) was quantified using primers Mito-F: 5′-CCCAGCTACTACCATCATTCAAGT-3′ and Mito-R: 5′-GATGGTTTGGGAGATTGGTTGATGT-3′. Although the percentage of viral DNA per 1 ng total extracted DNA was about 1.5 fold higher with the QlAprep kit than with the Trizol method, the overall recovery was lower with the QlAprep kit. 
     The extracted viral genome was digested with a restriction enzyme, such as SmaI (found within the ITRs), to improve rAAV genome recovery by PCR. This was analyzed by quantitative PCR with Cre+primers, CapF-56: 5′-ATTGGCACCAGATACCTG ACTCGTAA-3′, Cre+R-58: 5′-CAAGTAAAACCTCTACAAATGTGGTAAAATCG-3′ and Cre-primers, CapF-56 (see above) and Cre-R-57: 5′-GTCCAAACTCATCAATGTATCTTATCATGTCTG-3′. 
     rAAV genome extraction with the Trizol method. Half of a frozen brain hemisphere (0.3 g approx.) was homogenized with a 2 ml glass homogenizer (Sigma Aldrich; D8938) or a motorized plastic pestle (Fisher Scientific;12-141-361, 12-141-363) (for smaller tissues) and processed as described in prior work. The extracted DNA was then treated with 3-6 μl of 10 μg/μl RNase Cocktail Enzyme Mix (ThermoFisher Scientific; AM2286) to remove RNA and digested with Smal restriction enzyme. The treated mixture was then purified with a Zymo DNA Clean and Concentrator kit (D4033). From deep sequencing data analysis, it was observed that the amount of tissue processed for rAAV genome recovery is sufficient. 
     rAAV genome recovery by Cre-dependent PCR. rAAV genomes with Lox sites flipped by Cre recombination were selectively recovered and amplified using PCR with primers that yield a PCR product only if the Lox sites are flipped (See  FIG. 37 ). The primers 71F: 5′-CTTCCAGTTCAGCTACGAGTTTGAGAAC-3′ and CDF/R: 5′-CAAGTAAAACCTCTACAAATGTGGTAAAATCG-3′ were used and amplified the Cre-recombined genomes over 25 cycles of 98° C. for 10 s, 58° C. for 30 s, and 72° C. for 1 min, using Q5 DNA polymerase 
     Total rAAV genome recovery by PCR (Cre-independent). To recover all rAAV genomes from a tissue, the primers XF (5′-ACTCATCGACCAATACTTGTACTATCTCTCTAGAAC-3′) and 588-R2lib-R (5′-GTATTCCTTGGTTTTGAACCCAACCG-3′) were used to amplify the genomes over 25 cycles of 98° C. for 10 s, 60° C. for 30 s, and 72° C. for 30 min, using Q5 DNA polymerase. 
     Sample Preparation for NGS 
     To analyze selections using deep sequencing, the DNA library was processed, the virus library, and the tissue libraries post-in vivo selection to add flow cell adaptors around the diversified 7-mer insertion region (See  FIG. 37 ). 
     Preparation of rAAV DNA and Viral DNA library. The Gibson-assembled rAAV DNA library and the DNA extracted from the viral library were amplified by Q5 DNA polymerase using the primers 588i-lib-PCR1-6bpUID-F: 5′-CACGACGCTCTTCCGATCTAANNNNNNAGTCCTATGGACAAGTGGCCACA-3′ and 588i-lib-PCR1-R: 5′-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTCCTTGGTTTTGAACCCAACCG-3′ that are positioned around 50 bases from the randomized 7-mer insertion on the capsid, and that contain the Read1 and Read2 flow cell sequences on the 5′ end. 
     The primer, 588i-lib-PCR1-6bpUID-F: 5′ CACGACGCTCTTCCGATCTAANNNNNNAGTCCTATGGACAAGTGGCCACA-3′ used to minimally amplify DNA and virus libraries for NGS has 6 nucleotides long UID (unique identifier) “NNNNNN” that sits after 19 nucleotides of Read-1 sequence used in NGS “5′-CACGACGCTCTTCCGATCT”and linker “AA”. The sequence after UID “AGTCCTATGGACAAGTGGCCACA” is the region that anneals to the AAV9 capsid. UID is an optional feature for NGS data analysis to identify potential PCR amplification errors. However, this feature wasn&#39;t utilized in the NGS data analysis in this study to maintain consistency with the primers used in rAAV genome recovery from tissues which lacks this UID feature (primers 71F: 5′ -CTTCCAGTTCAGCTACGAGTTIG.AGAAC-3 and CDF/R: 5′ CAAGTAAAACCTCT ACA A ATGTGGTAA AATCG-3). The UID or any kind of overhangs seemed to affect the PCR based recovery from tissue. Presumably, the primer thermostability has a key role to play in very low amount of extracted rAAV genomes from tissues. 
     Using 5-10 ng of template DNA in a 50 μl reaction, the DNA was minimally amplified for 4 cycles of 98° C. for 10 s, 60° C. for 30 s, and 72° C. for 10 s. The mixture was then purified with a PCR purification kit. The eluted DNA was then used as a template in a second PCR to add the unique indices (single or dual) via the recommended primers (NEB; E7335S, E7500S, E7600S) in a 12-cycle reaction using the same temperature cycle as described above. The samples were then sent for deep sequencing following additional processing and validation. 
     The PCR products post indices addition were run on a freshly prepared 2% low-melting-point agarose gel (ThermoFisher Scientific; 16520050) for better separation and recovery of the approx. 120 bp DNA band on the gel. Before sending the sample for NGS, the nucleotide diversity at the randomized 7-mer position was verified by Sanger sequencing. If needed, an optional PCR was carried out to send sufficient sample for Sanger sequencing using 15-20 cycles of 98° C. for 10 s, 60° C. for 30 s, and 72° C. for 10 s with the primers NGS-QC-F: 5′-AATGATACGGCGACCACCGAG-3′ and NGS-QC-R: 5′-CAAGCAGAAGACGGCATACGA-3′. Upon validation, the libraries were sent for deep sequencing using the Illumina HiSeq 2500 System (Millard and Muriel Jacobs Genetics and Genomics Laboratory, Caltech; Integrative Genomics Core, City of Hope). 
     Preparation of rAAV tissue DNA library. The PCR-amplified rAAV DNA library from tissue (see section: In vivo selection (i) (c)) was further amplified with a 1:100 dilution of this DNA as a template to the primers 1527: 5′-ACACTCTTTCCCTACACGACGCTCTTCCGATCTGACAAGTGGCCACAAACCACCAG-3′ and 1532: 5′-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCCTTGGTTTTGAACCCAACCG-3′ that are positioned around 50 bases from the randomized 7-mer insertion on the capsid, and that contain the Read1 and Read2 sequences on the 5′ end. The DNA was amplified by Q5 Hot Start High-Fidelity 2X Master Mix or NEBNext Ultra II Q5 Master Mix (NEB; M0544) for 10 cycles of 98° C. for 10 s, 59° C. for 30 s, and 72° C. for 10 s. The mixture was purified with a PCR purification kit. The eluted DNA was then used as a template in a second PCR to add the unique indices (single or dual) using the recommended primers (NEB; E7335S, E7500S, E7600S) in a 10-cycle reaction with the same temperature cycle as described above (for DNA and virus library preparation). The extracted DNA was validated by Sanger sequencing and sent for deep sequencing as described in the previous section. 
     In Vivo Characterization of AAV Vectors 
     Cloning AAV capsid variants. The AAV capsid variants were cloned into a pUCmini-iCAP-PHP.B backbone (Addgene ID: 103002) using overlapping forward and reverse primers with 11-mer substitution (in case of 7-mer-i variants, the flanking amino acids from AAV9 capsid AA587-588 “AQ” and AA589-590 “AQ” were subjected to codon modification) that spans from the Mscl site (at position 581 AA) to the Agel site (at position 600 AA) on the pUCmini plasmid. The primers were designed for all capsid variants using a custom python script (Code will be made available on github), and since they cover the entire fragment insertion, these primers are self-annealed and amplified using PCR to create a dsDNA fragment without the use of a template DNA. They are amplified by Q5 Hot Start High-Fidelity 2X Master Mix for 20 cycles of 98° C. for 10 s, 60° C. for 30 s, and 72° C. for 10 s. This fragment was then assembled into the MscI/AgeI digested pUCmini-iCAP-PHP.B backbone by the Gibson assembly method. There is a second Mscl site on the backbone; however, this was blocked by methylation. The assembled plasmids were then transformed into NEB Stable Competent  E. coli  (New England Biolabs, Inc; C3040H), and colonies were selected on carbenicillin/ampicillin-LB agar plates. 
     List of primers used to clone AAV-PHP variants: The variants from 7-mer-i and 3-mer-s libraries were cloned as 11-mer substitution. 
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Primers For AAV-PHP Variants 
               
            
           
           
               
               
               
               
               
            
               
                 Variant 
                 Amino 
                 Nucleotide 
                 Forward primer 
                   
               
               
                 Name 
                 acid 
                 sequence 
                 (5′-3′) 
                 Reverse primer (5′-3′) 
               
               
                   
               
               
                 AAV- 
                 AQTALKP 
                 GCCCAAA 
                 GGAGTCCTATGGAC 
                 TTCCTTGGTTTTGAAC 
               
               
                 PHP.V1 
                 FLAQ 
                 CCGCCCTC 
                 AAGTGGCCACAAA 
                 CCAACCGGTCTGCGC 
               
               
                   
                   
                 AAACCCTT 
                 CCACCAGAGTGCCC 
                 CTGTGCGAGGAAGGG 
               
               
                   
                   
                 CCTCGCAC 
                 AAACCGCCCTCAAA 
                 TTTGAGGGCGGTTTGG 
               
               
                   
                   
                 AG 
                 CCC (SEQ ID NO: 
                 GC (SEQ ID NO: 1047) 
               
               
                   
                   
                   
                 1032) 
                   
               
               
                   
               
               
                 AAV- 
                 AQTTLKP 
                 GCCCAAA 
                 GGAGTCCTATGGAC 
                 TTCCTTGGTTTTGAAC 
               
               
                 PHP.V2 
                 FLAQ 
                 CCACCCTC 
                 AAGTGGCCACAAA 
                 CCAACCGGTCTGCGC 
               
               
                   
                   
                 AAACCCTT 
                 CCACCAGAGTGCCC 
                 CTGTGCGAGGAAGGG 
               
               
                   
                   
                 CCTCGCAC 
                 AAACCACCCTCAAA 
                 TTTGAGGGTGGTTTGG 
               
               
                   
                   
                 AG 
                 CCC (SEQ ID NO: 
                 GC (SEQ ID NO: 1048) 
               
               
                   
                   
                   
                 1033) 
                   
               
               
                   
               
               
                 AAV- 
                 AQTLQIPF 
                 GCCCAAA 
                 GGAGTCCTATGGAC 
                 TTCCTTGGTTTTGAAC 
               
               
                 PHP.B4 
                 KAQ 
                 CGTTGCAG 
                 AAGTGGCCACAAA 
                 CCAACCGGTCTGCGC 
               
               
                   
                   
                 ATTCCTTT 
                 CCACCAGAGTGCCC 
                 CTGTGCCTTAAAAGG 
               
               
                   
                   
                 TAAGGCA 
                 AAACGTTGCAGATT 
                 AATCTGCAACGTTTGG 
               
               
                   
                   
                 CAG 
                 CCT (SEQ ID NO: 
                 GC (SEQ ID NO: 1049) 
               
               
                   
                   
                   
                 1034) 
                   
               
               
                   
               
               
                 AAV- 
                 AQTLQLP 
                 GCCCAAA 
                 GGAGTCCTATGGAC 
                 TTCCTTGGTTTTGAAC 
               
               
                 PHP.B5 
                 FKAQ 
                 CCCTCCAA 
                 AAGTGGCCACAAA 
                 CCAACCGGTCTGCGCT 
               
               
                   
                 (SEQ ID 
                 CTCCCCTT 
                 CCACCAGAGTGCCC 
                 TGGGCTTTGAAGGGG 
               
               
                   
                 NO: 1062) 
                 CAAAGCC 
                 AAACCCTCCAACTC 
                 AGTTGGAGGGTTTGG 
               
               
                   
                   
                 CAA (SEQ 
                 CCC (SEQ ID NO: 
                 GC (SEQ ID NO: 1050) 
               
               
                   
                   
                 ID NO: 
                 1035) 
                   
               
               
                   
                   
                 1068) 
                   
                   
               
               
                   
               
               
                 AAV- 
                 AQTLQQP 
                 GCCCAAA 
                 GGAGTCCTATGGAC 
                 TTCCTTGGTTTTGAAC 
               
               
                 PHP.B6 
                 FKAQ 
                 CTTTGCAG 
                 AAGTGGCCACAAA 
                 CCAACCGGTCTGCGC 
               
               
                   
                 (SEQ ID 
                 CAGCCGTT 
                 CCACCAGAGTGCCC 
                 CTGTGCCTTAAACGGC 
               
               
                   
                 NO: 1063) 
                 TAAGGCA 
                 AAACTTTGCAGCAG 
                 TGCTGCAAAGTTTGG 
               
               
                   
                   
                 CAG (SEQ 
                 CCG (SEQ ID NO: 
                 GC (SEQ ID NO: 1051) 
               
               
                   
                   
                 ID NO: 
                 1036) 
                   
               
               
                   
                   
                 1069) 
                   
                   
               
               
                   
               
               
                 AAV- 
                 AQSIERPF 
                 GCCCAAA 
                 GGAGTCCTATGGAC 
                 TTCCTTGGTTTTGAAC 
               
               
                 PHP.B7 
                 KAQ 
                 GCATCGA 
                 AAGTGGCCACAAA 
                 CCAACCGGTCTGCGC 
               
               
                   
                   
                 AAGACCCT 
                 CCACCAGAGTGCCC 
                 CTGTGCTTTGAAGGGT 
               
               
                   
                   
                 TCAAAGC 
                 AAAGCATCGAAAG 
                 CTTTCGATGCTTTGGG 
               
               
                   
                   
                 ACAG 
                 ACCC (SEQ ID NO: 
                 C (SEQ ID NO: 1052) 
               
               
                   
                   
                   
                 1037) 
                   
               
               
                   
               
               
                 AAV- 
                 AQTMQKP 
                 GCCCAAA 
                 GGAGTCCTATGGAC 
                 TTCCTTGGTTTTGAAC 
               
               
                 PHP.B8 
                 FIAQ 
                 CCATGCAA 
                 AAGTGGCCACAAA 
                 CCAACCGGTCTGCGC 
               
               
                   
                   
                 AAACCCTT 
                 CCACCAGAGTGCCC 
                 CTGTGCGATGAAGGG 
               
               
                   
                   
                 CATCGCAC 
                 AAACCATGCAAAA 
                 TTTTTGCATGGTTTGG 
               
               
                   
                   
                 AG 
                 ACCC (SEQ ID NO: 
                 GC (SEQ ID NO: 1053) 
               
               
                   
                   
                   
                 1038) 
                   
               
               
                   
               
               
                 AAV- 
                 AQRYQGD 
                 GCCCAAA 
                 GGAGTCCTATGGAC 
                 TTCCTTGGTTTTGAAC 
               
               
                 PHP.C1 
                 SVAQ 
                 GGTATCAG 
                 AAGTGGCCACAAA 
                 CCAACCGGTCTGCGC 
               
               
                   
                   
                 GGTGATTC 
                 CCACCAGAGTGCCC 
                 CTGTGCAACAGAATC 
               
               
                   
                   
                 TGTTGCAC 
                 AAAGGTATCAGGG 
                 ACCCTGATACCTTTGG 
               
               
                   
                   
                 AG 
                 TGAT (SEQ ID NO: 
                 GC (SEQ ID NO: 1054) 
               
               
                   
                   
                   
                 1039) 
                   
               
               
                   
               
               
                 AAV- 
                 AQWSTNA 
                 GCCCAAT 
                 GGAGTCCTATGGA 
                 TTCCTTGGTTTTGAA 
               
               
                 PHP.C2 
                 GYAQ 
                 GGTCGAC 
                 CAAGTGGCCACAA 
                 CCCAACCGGTCTGCG 
               
               
                   
                   
                 AAACGCT 
                 ACCACCAGAGTGC 
                 CCTGTGCGTAACCAG 
               
               
                   
                   
                 GGTTACG 
                 CCAATGGTCGACA 
                 CGTTTGTCGACCATT 
               
               
                   
                   
                 CACAG 
                 AACGCT (SEQ ID 
                 GGGC (SEQ ID NO: 
               
               
                   
                   
                   
                 NO: 1040) 
                 1055) 
               
               
                   
               
               
                 AAV- 
                 AQERVGF 
                 GCCCAAG 
                 GGAGTCCTATGGA 
                 TTCCTTGGTTTTGAA 
               
               
                 PHP.C3 
                 AQAQ 
                 AGCGTGT 
                 CAAGTGGCCACAA 
                 CCCAACCGGTCTGCG 
               
               
                   
                   
                 AGGTTTC 
                 ACCACCAGAGTGC 
                 CCTGTGCCTGTGCGA 
               
               
                   
                   
                 GCACAGG 
                 CCAAGAGCGTGTA 
                 AACCTACACGCTCTT 
               
               
                   
                   
                 CACAG 
                 GGTTTC (SEQ ID 
                 GGGC (SEQ ID NO: 
               
               
                   
                   
                   
                 NO: 1041) 
                 1056) 
               
               
                   
               
               
                 AAV-PHP.N 
                 AQTLAVP 
                 GCGCAGA 
                 GGAGTCCTATGGAC 
                 TTCCTTGGTTTTGAAC 
               
               
                   
                 FSNP (SEQ 
                 CCCTAGCT 
                 AAGTGGCCACAAA 
                 CCAACCGGTCTGCGC 
               
               
                   
                 ID NO: 
                 GTCCCTTT 
                 CCACCAGAGTGCGC 
                 AGGGTTCGAAAAAGG 
               
               
                   
                 1064) 
                 TTCGAACC 
                 AGACCCTAGCTGTC 
                 GACAGCTAGGGTCTG 
               
               
                   
                   
                 CT (SEQ ID 
                 CCT (SEQ ID NO: 
                 CGC (SEQ ID NO: 1057) 
               
               
                   
                   
                 NO: 1070) 
                 1042) 
                   
               
               
                   
               
               
                 AAV- 
                 AQARQM 
                 GCCCAAG 
                 GGAGTCCTATGGAC 
                 TTCCTTGGTTTTGAAC 
               
               
                 PHP.X1 
                 DLSAQ 
                 CCAGACA 
                 AAGTGGCCACAAA 
                 CCAACCGGTCTGCGC 
               
               
                   
                 (SEQ ID 
                 AATGGAC 
                 CCACCAGAGTGCCC 
                 CTGTGCGCTGAGGTCC 
               
               
                   
                 NO: 1065) 
                 CTCAGCGC 
                 AAGCCAGACAAAT 
                 ATTTGTCTGGCTTGGG 
               
               
                   
                   
                 ACAG (SEQ 
                 GGAC (SEQ ID NO: 
                 C (SEQ ID NO: 1058) 
               
               
                   
                   
                 ID NO: 
                 1043) 
                   
               
               
                   
                   
                 1071) 
                   
                   
               
               
                   
               
               
                 AAV- 
                 AQTNKVG 
                 GCCCAAA 
                 GGAGTCCTATGGAC 
                 TTCCTTGGTTTTGAAC 
               
               
                 PHP.X2 
                 NIAQ (SEQ 
                 CCAACAA 
                 AAGTGGCCACAAA 
                 CCAACCGGTCTGCGC 
               
               
                   
                 ID NO: 
                 AGTCGGC 
                 CCACCAGAGTGCCC 
                 CTGTGCGATGTTGCCG 
               
               
                   
                 1066) 
                 AACATCGC 
                 AAACCAACAAAGT 
                 ACTTTGTTGGTTTGGG 
               
               
                   
                   
                 ACAG (SEQ 
                 CGGC (SEQ ID NO: 
                 C (SEQ ID NO: 1059) 
               
               
                   
                   
                 ID NO: 
                 1044) 
                   
               
               
                   
                   
                 1072) 
                   
                   
               
               
                   
               
               
                 AAV- 
                 AQQNVTK 
                 GCCCAAC 
                 GGAGTCCTATGGA 
                 TTCCTTGGTTTTGAA 
               
               
                 PHP.X3 
                 GVAQ 
                 AGAACGT 
                 CAAGTGGCCACAA 
                 CCCAACCGGTCTGCG 
               
               
                   
                   
                 AACGAAG 
                 ACCACCAGAGTGC 
                 CCTGTGCCACACCCT 
               
               
                   
                   
                 GGTGTGG 
                 CCAACAGAACGTA 
                 TCGTTACGTTCTGTT 
               
               
                   
                   
                 CACAG 
                 ACGAAG (SEQ ID 
                 GGGC (SEQ ID NO: 
               
               
                   
                   
                   
                 NO: 1045) 
                 1060) 
               
               
                   
               
               
                 AAV- 
                 AQLNAIK 
                 GCCCAAC 
                 GGAGTCCTATGGA 
                 TTCCTTGGTTTTGAA 
               
               
                 PHP.X4 
                 NIAQ (SEQ 
                 TCAACGC 
                 CAAGTGGCCACAA 
                 CCCAACCGGTCTGCG 
               
               
                   
                 ID NO: 
                 TATCAAG 
                 ACCACCAGAGTGC 
                 CCTGTGCGATGTTCT 
               
               
                   
                 1067) 
                 AACATCG 
                 CCAACTCAACGCT 
                 TGATAGCGTTGAGTT 
               
               
                   
                   
                 CACAG 
                 ATCAAG (SEQ ID 
                 GGGC (SEQ ID NO: 
               
               
                   
                   
                 (SEQ ID 
                 NO: 1046) 
                 1061) 
               
               
                   
                   
                 NO: 1073) 
               
               
                   
               
            
           
         
       
     
     AAV vector production. Using an optimized protocol, AAV vectors were produced from 5-10 150 mm plates, which yielded sufficient amounts for administration to adult mice. 
     AAV vector administration. AAV vectors were administered intravenously to adult male mice (6-8 weeks of age) via retro-orbital injection at doses of 1-10×10 11  vg. The AAV doses are determined by the experimental needs. CAG-NLS-GFP related experiments for quantification were done at medium dose of 1×10 11  vg given this was the dose previously determined for AAV-PHP.eB characterization. Otherwise, the non-NLS genome related experiments were done at 3×10 11  vg, with the exception of Cre-driver lines (GFAP-Cre or Tek-Cre), or a lower strength promoter containing genome (GFAP-NLS-mTurq) where the dose was 1×10 12  vg. The high dose was chosen to understand the full potential of the new vectors in these systems. 
     All experiments with vectors carrying CAG, a strong ubiquitous promoter, were incubated for 3 weeks. The 4 week incubations are those that involved expression from Cre driver lines or cell-type specific promoter where it is generally recommended for a longer wait time. The 2 week incubations are those where the vectors carried self-complementary genomes with strong ubiquitous promoters. 
     Tissue processing. After 3 weeks of expression (unless noted otherwise), the mice were anesthetized with Euthasol (pentobarbital sodium and phenytoin sodium solution, Virbac AH) and transcardially perfused with 30-50 mL of 0.1 M phosphate buffered saline (PBS) (pH 7.4), followed by 30-50 ml of 4% paraformaldehyde (PFA) in 0.1 M PBS. After this procedure, all organs were harvested and post-fixed in 4% PFA at 4° C. overnight. The tissues were then washed and stored at 4° C. in 0.1 M PBS and 0.05% sodium azide. All solutions used for this procedure were freshly prepared. For the brain and liver, 100- i .tm thick sections were cut on a Leica VT1200 vibratome. 
     For vascular labeling, the mice were anesthetized and transcardially perfused with 20 mL of ice-cold PBS, followed by 10 mL of ice-cold PBS containing Texas Red-labeled Lycopersicon Esculentum (Tomato) Lectin (1:100, Vector laboratories, TL-1176), and then placed in 30 mL of ice-cold 4% PFA for fixation. 
     Immunohistochemistry. Tissue sections—typically 100-μm thick—were first incubated in blocking buffer (10% normal donkey serum, 0.1% Triton X-100, and 0.01% sodium azide in 0.1 M PBS, pH 7.4) with primary antibodies at appropriate dilutions for 24 h at room temperature on a rocker. The primary antibodies used in this study were rabbit S100 (1:400, Abcam, ab868), rabbit Olig2 (1:400; Abcam, ab109186), rabbit NeuN (1:400, Abcam, ab177487), and rabbit GLUT-1 (1:400; Millipore Sigma, 07-1401). After primary antibody incubation, the tissues were washed 1-3 times with wash buffer 1 (0.1% Triton X-100 in 0.1 M PBS buffer, pH 7.4) over a period of 5-6 h in total. The tissues were then incubated in blocking buffer with the secondary antibodies at appropriate dilutions for 12-24 h at room temperature and then washed in three times in 0.1 M PBS, pH 7.4 over a total duration of 5-6 h. The secondary antibody used in this study was Alexa Fluor 647 AffiniPure donkey anti-rabbit IgG (H+L) (Jackson ImmunoResearch Lab, 711-605-152). When performing nuclear staining, 4′,6-Diamidine-2′-phenylindole dihydrochloride (DAPI, Sigma Aldrich, 10236276001) is used at a 1:1000 dilution in 0.1 M PBS, pH 7.4 and incubated with tissues for 15 minutes followed by a single wash for 10 minutes in 0.1 M PBS, pH 7.4. The DAPI and/or antibody-stained tissue sections were mounted with ProLong Diamond Antifade Mountant (ThermoFisher Scientific, P36970). 
     Hybridization chain reaction (HCR) based RNA labeling in tissues. Fluorescence in situ hybridization chain reaction (FITC-HCR) was used to label excitatory neurons with VGLUT1 and inhibitory neurons with GAD1 to characterize the AAV capsid variant AAV-PHP.N in brain tissue using an adapted third-generation HCR protocol. To characterize the AAV capsid variant AAV-PHP.N in brain tissue, HCR method was sought to label excitatory and inhibitory neurons. Fluorescence in situ hybridization chain reaction (FITC-HCR) was used to label excitatory neurons with VGLUT1 and inhibitory neurons with GAD1. Adapting the third-generation HCR, 13 probe sets were designed for each target by using custom-made software (https://github.com/GradinaruLab/HCRprobe). After 3 weeks of expression, the mice were transcardially perfused and fixed as described earlier (Section D. Tissue processing). To minimize RNase enzyme exposure in fixed tissues, following overnight fixation in 4% PFA, the tissues were washed and stored at 4° C. in 0.1 M RNase-free PBS and 0.05% sodium azide. The harvested brains were henceforth handled with care to avoid exposure to RNase using reagents such as RNAlater stabilization solution/RNase-free PBS/ RNaseZap (ThermoFisher Scientific, AM7021, AM9624, AM9780). Once the harvested brains were sagittally sliced to 100-pm thick sections, FITC-HCR was performed to detect both genes. Tissue slices were permeabilized with 0.1% Triton X-100 in 0.1 M RNase-free PBS for 1 h at RT and pre-hybridized in hybridization solution (10% dextran sulfate and 10% ethylene carbonate in 2xSSC buffer (saline-sodium citrate)) for &gt;30 min at 37° C. The designed probes were diluted in hybridization solution to get a final concentration of 2 nM. The tissue sections were then subjected to hybridization with the probes overnight at 37° C. Following this, the sections were washed with pre-warmed wash buffer (10% ethylene carbonate in 2xSSC) at 37° C. for 30 min twice, followed by 2xSSC at RT for 30 min twice. Amplification with hairpin pairs (Molecular Technologies, CA) were performed in amplification buffer (10x dextran sulfate in 2xSSC); hairpins were snap-cooled at 95° C. for 90 s, followed by RT for 30 min, and diluted with amplification buffer (60 nM). Tissues were then incubated in this amplification buffer with hairpins overnight at RT with gentle agitation. Once the amplification was done, samples were briefly washed with 2xSSC and mounted in Prolong Diamond for imaging. 
     Imaging and image processing. All images in this study were acquired either with a Zeiss LSM 880 confocal microscope using the objectives Fluar 5×0.25 M27, Plan-Apochromat 10×0.45 M27 (working distance 2.0 mm), and Plan-Apochromat 25×0.8 Imm Con DIC M27 multi-immersion; or with a Keyence BZ-X700 microscope. The acquired images were processed in Zen Black 2.3 SP1 (Zeiss), BZ-X Analyzer (Keyence), Illustrator CC 2018 (Adobe), Photoshop CC 2018 (Adobe), and Imaris (Bitplane). To prevent any imaging artifacts resulting from multiple fluorescence spectral overlap, the fluorescence excitation and emission spectra were kept distinct following the recommended linear unmixed acquisition of individual colors. A far-red fluorescent dye was chosen for any additional marker staining to keep the imaging parameters distinct from in vivo fluorescent expression thereby preventing any spectral overlap across detector channels. The tissues were routinely monitored for auto fluorescence or imaging artifacts before acquisition, and imaging parameters were adjusted if needed. The imaging parameters were cross-checked with tissues lacking in vivo transduction to avoid any imaging artifacts. The regions used for the images were closely matched across experimental groups to minimize bias during comparisons. 
     Tissue clearing and imaging of thick tissues. To demonstrate the ability of PHP.V1 to transduce the vasculature across thick tissues, such as half of a mouse-brain hemisphere or a femur bone, tissue from Tek-Cre mice was assessed after 4 weeks&#39; of expression. 
     The brain hemisphere was stained with the primary antibody, Anti-GFP (Ayes Labs, GFP-1020), and the secondary antibody, goat anti-Chicken IgY, Alexa Fluor 633 (ThermoFisher Scientific, A-21103), and cleared via the iDISCO protoco1 38 . For imaging, a commercial light-sheet microscope (Lavision BioTec) with a custom objective lens (4×) was used. The resulting image files were reorganized by a custom MATLAB script to allow stitching with TeraStitcher. For 3-D visualization, Imaris (Bitplane) was used. 
     To image the mouse femur bone, the bones were sectioned to 300 μm thickness for antibody penetration and stained with the primary antibody, Anti-GFP, and the secondary antibody, Alexa Fluor 488 donkey anti-chicken IgY (Jackson ImmunoResearch Lab, 703-545-155), and then cleared via the TDE (2-2′-thiodiethanol) clearing method 41 . The images were acquired with a confocal microscope (Zeiss LSM 880) and visualized in Imaris software. 
     Tissue processing and imaging for quantification of rAAV transduction in vivo. For quantification of rAAV transduction, 6- to 8-week-old male mice were intravenously injected with the virus, which was allowed to express for 3 weeks (unless specified otherwise). The mice were randomly assigned to groups and the experimenter was not blinded. The mice were perfused and the organs were fixed in PFA. The brains and livers were cut into 100-μm thick sections and immunostained with different cell-type-specific antibodies, as described above. The images were acquired either with a 25× objective on a Zeiss LSM 880 confocal microscope or with a Keyence BZ-X700 microscope; images that are compared directly across groups were acquired and processed with the same microscope and settings. 
     For quantification of PHP.B-family variant transduction in tissues, the images were acquired using 25x objective with lx digital zoom on a Zeiss LSM 880 confocal microscope. With n=3 mice per variant, images were acquired across 4 brain regions—cortex, striatum, ventral midbrain and thalamus, and tissues were stained with 3 cell type markers (NeuN, Olig2, and S100). For each mouse, 2 images per brain region per cell type marker were acquired, and the mean were plotted. 
     For PHP.N transduction analysis, the images were acquired using 20× objective on Keyence BZ-X700 microscope. With n=3 mice, images across 4 brain regions—cortex, striatum, ventral midbrain and thalamus were acquired to cover the entire brain regions for 3 cell type markers (NeuN, Olig2, and S100). This involved 6-8 images to cover cortex, thalamus and striatum, and 2 images to cover ventral midbrain per mouse per cell-type marker. For each mouse, across each region, the mean from the images were plotted. 
     For PHP.V GLUT1 + transduction analysis, the images were acquired using 25× objective with lx digital zoom on a Zeiss LSM 880 confocal microscope. Each distinct blood vessel in the image with GLUT1 + staining and XFP expression was determined as positive for transduction. Quantification of expression from the CAG-mNeonGreen vector was performed across the cortex (n=3 per group). Each data point is drawn from the mean of 3-2 images per mouse. Different brain regions were quantified for Tek-Cre and Ai14 mouse experiments with n=2 per group. For cortex, cerebellum, striatum and ventral midbrain, the mean was plotted from 3-4 images per mouse per region. 
     In Vitro Characterization of AAV Vectors 
     Human Brain Microvascular Endothelial Cells (HBMEC) (ScienCell Research Laboratories, Cat. 1000) were cultured as per the instructions provided by the vendor. HBMEC were cultured from a frozen stock vial in fibronectin-coated T-75 flask (7000-9000 cells/cm 2  seeding density) using the Endothelial Cell Medium (Cat. 1001). The cells were subcultured in fibronectin-coated 48-well plates (0.95 cm 2  growth area) at the recommended seeding density and incubated at 37° C. for ˜24-48 h till the cells were completely adherent with - 18  70-80% confluence. The viral vectors packaging pAAV-CAG-mNeongreen were added to the cell culture at a dose of either 1×10 8  or 1×10 10  vg per well (3 wells per dose per vector). The media was changed 24 hours later and the culture was assessed for fluorescence expression at 3 days&#39; post infection. Per vendor recommendation, the culture media was changed every other day to maintain the cell culture. 
     Data Analysis 
     Quantification of rAAV vector transduction in mouse tissue. Manual counting was performed with the Adobe Photoshop CC 2018 Count Tool for cell types in which expression and/or antibody staining covered the whole cell morphology. The Keyence Hybrid Cell Count software (BZ-H3C) was used where the software could reliably detect distinct cells in an entire dataset. To maintain consistency in counting across different markers and groups, one person was assigned to quantify across all groups in all brain areas. 
     Manual counting was performed for GLUT-1-stained blood vessels and expression of the ssAAV:CAG-mNeonGreen and ssAAV:CAG-DIO-EYFP, where the efficiency was calculated as the percentage of XFP+vessels relative to the GLUT-1 staining. Manual counting was also performed to quantify nuclear or soma stained cells, including NeuN-, Olig2-, and S100-stained cells. The efficiency was calculated as the percentage of XFP+cells relative to cell-marker+cells. 
     Keyence Hybrid Cell Count software (BZ-H3C) was used to quantify expression of nuclear localized AAV genomes in liver hepatocytes that co-localized with the DNA stain, DAPI; and also for the study involving ssAAV:GFAP-2xNLS-mTurquoise2 genomes with S100 cell marker. 
     The mean fluorescence intensity across microscopic images were quantified using ImageJ software. The images were processed for background subtraction and using the Threshold operation, the mean fluorescence intensity was measured. 
     NGS data alignment and processing. The raw fastq files from NGS runs were processed with custom built scripts (codes will be made available on Github) that align the data to AAV9 template DNA fragment containing the diversified region 7xNNK (for R1) or 11xNNN (for R2 since it was synthesized as 11xNNN). The pipeline to process these datasets involved filtering the dataset to remove the low-quality reads by using the deep sequencing quality score for each sequence. The variant sequences were then recovered from the sequencing reads by searching for the flanking template sequences, and extracting the nucleotides of the diversified region (perfect string match algorithm). The quality of the aligned data was further investigated to remove any erroneous sequences (such as ones with stop codons). The raw data was plotted (as shown in Supplementary  FIG. 1 e   ) to study the quality of recovery across every library. Based on the RC distribution, we adapted a thresholding method to remove plausible erroneous mutants that may have resulted from PCR or NGS based errors. The assumption is that if there is a PCR mutation or NGS error on the recovered parent sequence, the parent must have existed at least one round earlier than the erroneous sequence, and thus a difference in RCs should exist. 
     For R1 tissue libraries, a steep drop was observed in the slope of the distribution curve following a long tail of low count sequences, and were found to be rich in sequences that are variations of the parents in the higher counts range. A threshold for RCs was manually set to remove such erroneous mutants. The thresholded data were then processed differently based on the experimental needs as described elsewhere using custom Python based scripts. 
     For R2 tissue libraries from PCR pool and synthetic pool, given the smaller library size compared to R1, the data was thresholded in two steps. Only the tissue recovered sequences that were present in the respective input DNA and virus library were considered (after removing lower count variants from input libraries following the same principle as R1 tissue libraries). This step partially removed the long tail of low count reads. As a second step, the thresholding that was described for R1 tissue libraries was applied. 
     While it is plausible that true variants may be lost during thresholding, this method minimized false positives as the low count mutants in tissue and virus libraries often seemed to have very high enrichment score (as RCs are normalized to input library). In other words, thresholding allowed selective investigation on positively and negatively enriched variants that had a higher-confidence in their NGS RCs. 
     As an alternative to the manual thresholding method, an optional error correction method called “Collapsing” was built to further validate the outcome from filtered datasets. This method starts at the lowest count variants (variants of count 1) and searches for potential parent variants that are off by one nucleotide but have at least 2-fold higher counts (fold change=(2 ΔCT ) where CT is PCR cycle threshold). This error correction method then transfers the counts of these potential erroneous sequences to their originating sequences and repeats recursively until all sequences have been considered. On applying this error correction to the thresholded data, an additional ˜0.002-0.03% of sequences were captured (compared to &gt;19% captured by thresholding), confirming that the thresholding strategy was largely successful. 
     NGS data analysis. The aligned data were then further processed via a custom data-processing pipeline, with scripts written in Python (available on Github). The enrichment scores of variants (Total =N) across different libraries were calculated from the read counts (RCs) according to the following formula: Enrichment score =log10 [(Variant I RC in tissue libraryl/ Sum of variants N RC in libraryl)/(Variant I RC in virus library/ Sum of variants N RC in virus library)] 
     To consistently represent library recovery between R1 and R2 selected variants, the enrichment score of the variants in R1 selection was estimated. The standard score of variants in a specific library was calculated using this formula: Standard score=(read count_i-mean)/standard deviation. Where read count_i is raw copy number of a variant i, Mean is the mean of read counts of all variants across a specific library, Standard deviation is the standard deviation of read counts of all variants across a specific library. 
     Since the DNA and virus libraries were not completely sampled unlike the tissue libraries, we assigned an estimated RC for variants that were not present in the input library but were present in the output library. For instance, R1 virus library is the input library to the R1 tissue libraries. The estimated RC is defined as a number that is lower than the lowest RC in the library with the assumption that these variants were found at a relatively lower abundance than the variants recovered from the deep sequencing. In virus libraries, since RC of 1.0 was the lowest, we assigned all missing variants an estimated RC of 0.9. We use this method to calculate the enrichment score of the R1 tissue libraries which is normalized to R1 virus library ( FIG. 1 d   ). This was done to represent libraries across two selection rounds consistently. Although, the individual enrichment score among R1 variants didn&#39;t add a significant value to the variants selected for R2 selection as described in the criteria to separate signal vs noise in R1 using the RCs. 
     Heatmap generation. The relative AA distributions of the diversified regions are plotted as heatmaps. The plots were generated using the Python Plotly plotting library. The heatmap values were generated from custom scripts written in Python, using functions in the custom “pepars” Python package. Each heatmap uses both an expected (input) distribution of amino acid sequences and an output distribution. The output distribution must be a list of sequences and their count, and the input distribution can be either a list of sequences and their count, or an expected amino acid frequency from a template, such as NNK. For both input and output, the total count of amino acids in each position is tallied in accordance to each sequence&#39;s count and then divided by the total sum of counts, giving a frequency of each amino acid at each position. Then, the log2 fold change is calculated between the output and the input. For amino acids with a count of 0 in either the input or output, no calculation is performed. In order to distinguish between statistically significant amino acid biases, a statistical test was performed using the statsmodels Python library. For the case where there are two amino acid counts, a two-sided, two-proportion z-test was performed; for comparing the output amino acid count to an expected input frequency from a template, a one-proportion z-test was performed. All p-values were then corrected for multiple comparisons using Bonferroni correction. Only bias differences below a significance threshold of le-4 are then outlined on the heatmap; all other (insignificant) squares are left empty. 
     Clustering analysis. Using custom scripts written in MATLAB (version R2017b; MathWorks) the reverse Hamming distances representing the number of shared AAs between two peptides was determined. Cytoscape (version 3.7.1 53 ) software was then used to cluster the variants. The AA frequency plot representing the highlighted cluster was created using Weblogo (Version 2.8.2). The reverse Hamming distances (representing the number of shared AAs between two peptides) was determined for all unique capsid variants with greater than 10 count and greater than 2.5-fold enrichment after R2 selection. This process iteratively compares each variant with all other variants within the group. Capsid variants were then clustered by their reverse Hamming distances using Cytoscape. The minimum reverse Hamming distance for visualization was chosen manually based on sequence similarity. 
     For the amino acid frequency plots, the number on the bottom represents the position of the diversified motif starting from 1. The size of the amino acid in the stack reflects the proportion of unique clones in which the AA appears at that specific position in the motif. The color code is based on the AA properties. The positively charged residues K, R, and H are in blue. The negatively charged residues D and E are in red. The amide containing polar residues Q, and N are in magenta. The polar residues T, and S, are in green. The hydrophobic residues A, L, V, I, P, F, M, and W are in black. 
     Example 3 
     Multiplexed-CREATE Allows Detailed Characterization of the Capsid Libraries During Round-1 Selection 
     To identify variants that enrich in specific cell types or organs parallel selections across multiple targets were performed, and the enrichment or depletion of each capsid variant across those targets was mapped. 
     During DNA and virus library generation there is potential for accumulation of biases that over-represent certain capsid variants, obscuring their true enrichment during in vivo selection. These biases may result from PCR amplification bias in the DNA library or sequence bias in the efficiency of virus production across various steps: capsid assembly, genome packaging and stability during purification. This was investigated with the 7-mer-i library, a randomized 7-mer library inserted between positions 588-589 of AAV9 ( FIGS. 1 and 2 ) in rAAV-ACap9-in-cis-Lox2 plasmid ( FIG. 36 ). Sequencing libraries after DNA assembly and virus purification to a depth of 10-20 million (M) reads was adequate to capture the bias among variants during virus production ( FIG. 3 ; despite ˜1% variant overlap among these libraries;  FIGS. 38 and 39 ), demonstrating that even permissive sites like 588-589 will impose biological constraints on sampled sequence space. The DNA library had a uniform distribution of 9.6 M unique variants within ˜10 M total reads (read count (RC) mean=1.0, S.D. =0.074), indicating minimal bias. In contrast, the virus library had 3.6 M unique variants within ˜20 M depth (RC mean=4.59, S.D. =11.15) indicating enrichment of a subset of variants during viral production. 
     For in vivo selection, the 7-mer-i viral library was intravenously injected at a dose of 2×10 11  vg per adult transgenic mouse expressing Cre in different brain cell types: GFAP-Cre mice for astrocytes, SNAP25-Cre mice for neurons, and Tek-Cre mice for endothelial cells (n=2 mice per Cre transgenic line, see Methods). Two weeks after intravenous (IV) injection, the brain and liver tissues were harvested, with the latter serving as a control organ since AAV9 transduces it with high efficiency. The rAAV genomes were extracted from tissues and the capsids that transduced Cre-expressing cells were selectively amplified ( FIGS. 40-44 ). Upon deep sequencing, ˜8×10 4  unique nucleotide variants recovered from brain tissues and &lt;50 variants in spinal cords (˜48% of which were identified in virus library) were observed across the transgenic lines, and each variant was represented with an enrichment score reflecting the 
                                                    Design Parameters   Synthetic pool design   PCR pool design                        
change in relative abundance between the brain and the starting virus library ( FIG. 4 ).
 
     Two features of this dataset stand out. First, the recovered variants in brain tissue were disproportionately represented among the fraction of the transformed capsid library observed by sequencing after viral production demonstrating how production biases skew selection results. Second, the distribution of capsid read counts (RCs) revealed that more than half of the unique recovered variants after selection appear at remarkably low read counts. These variants may either be unintended mutants from experimental manipulation or AAV9-like variants with low basal level of CNS transduction ( FIG. 40 ). 
     Example 4 
     A Novel Round-2 Library Design Improves the Selection Outcome 
     Concerned that the sequence bias during viral production and recovery would propagate across selection rounds despite post-hoc enrichment scoring, an unbiased library was designed based on the round-1 (R1) output (synthetic pool library) via oligo pools (Twist Bioscience). This library was compared to a library PCR amplified directly from the recovered R1 DNA (PCR pool library) ( FIG. 5 , Table 7). 
     Table 7: Comparison between the two methods for R2 selection. The table summarizes the pros and cons of selection design parameters by the synthetic pool and PCR pool R2 selection methods. 
     
       
         
           
               
               
               
             
               
                   
               
             
            
               
                 Carryover of 
                 No, likelihood 
                 Yes, potential 
               
               
                 R1 selection 
                 of false 
                 to minimize 
               
               
                 bias among variants 
                 positives is low 
                 by normalization 
               
               
                 Carryover of R1 selection 
                 No 
                 Yes 
               
               
                 induced mutants 
                   
                   
               
               
                 Confidence in library 
                 High, using alternate 
                 Low 
               
               
                 performance 
                 codon replicates 
                   
               
               
                 Customize library or add 
                 Yes, in an 
                 Yes, with greater 
               
               
                 internal controls 
                 unbiased manner 
                 risk of bias 
               
               
                 Control library size 
                 Yes, without reducing 
                 Yes, with libraries 
               
               
                   
                 libraries or pooling 
                 reduced for pooling 
               
               
                 Cost for R2 library 
                 High 
                 Low 
               
               
                 generation 
               
               
                   
               
            
           
         
       
     
     The synthetic pool library design comprised: (1) equimolar amounts of ˜8950 capsid variants present at high read counts in at least one of the R1 selections from brain and spinal cord ( FIG. 40 ); ( 2 ) alternative codon replicates of those ˜8950 variants (optimized for mammalian codons) to reduce false positives; and (3) a “spike-in” library of controls ( FIGS. 74 and 75 ), resulting in a total library size of 18,000 nucleotide variants. 
     As anticipated, both round-2 (R2) virus libraries produced a high titer (˜6×10 11  vg per 10 ng of R2 DNA library per 150 mm dish;  FIGS. 45 ), and ˜99% of variants from the R2 DNA were found after viral production ( FIG. 6 ). However, the distribution of the DNA and virus libraries from both designs differed significantly. The PCR pool library carries forward the R1 selection biases ( FIGS. 7, 46, and 47 ) where the abundance reflects prior enrichment across tissues in R1 as well as bias from viral production and sample mixing. Comparatively, the synthetic pool DNA library is more evenly distributed, minimizing bias amplification across selection rounds. 
     For in vivo selection, a dose of 1×10 12  vg per adult transgenic mouse was administered into three of the previously used lines (n=2 mice per Cre transgenic line—GFAP, SNAP25, Tek), as well as the Syn-Cre line (for neurons). Two weeks after IV injection, rAAV genomes from brain samples were extracted, selectively amplified, and deep sequenced (as in R1). The synthetic pool library produced a greater number of positively enriched capsid variants than the PCR pool brain library (e.g. ˜1700 versus ˜700 variants/tissue library at amino acid (AA) level in GFAP-Cre) ( FIGS. 8 and 48 ). In the synthetic pool, ˜90% of the variants from the spike-in library were positively enriched as expected ( FIG. 48 , middle panel;  FIG. 74 ). 
     The degree of correlation for enrichment scores of variants recovered from both PCR and synthetic pool libraries varies in each Cre transgenic line, demonstrating the presence of noise within experiments ( FIG. 49 ). The synthetic pool&#39;s codon replicate feature addresses this predicament by pinpointing the level of enrichment needed within each selection to rise above noise ( FIGS. 9, 50, and 51 ). This is a significant advantage over the PCR pool design, allowing researchers to confidently interpret enrichment scores in a given selection. 
     The degree of enrichment at which correlation breaks down appears to vary with Cre-line. A downside of PCR pool is that there is no way to tell whether it or synthetic pool is the more ‘true’ enrichment score or even that there may be cause for concern regarding certain enrichment values. The correlation among positively enriched variants between the two methods was found to improve with the magnitude of positive enrichment. For each experiment there is a level of enrichment below which the scores become irreproducible, or noisy.  FIG. 50  demonstrates that neither PCR pool nor Synthetic pool is inherently more ‘true’ at lower enrichment scores. This is because Synthetic pool methodology with its codon replicates has a self-contained control to determine an enrichment level below which enrichment value has no further predictive power. The term ‘noise’ has been used herein to refer to regions of enrichment in a particular experiment below which values lose their reproducibility and predictive power. Being able to experimentally determine enrichment signal above noise allows researchers to focus their attention and data analyses on enrichment levels that are internally reproducible and thereby avoid selecting false positive variants or drawing invalid conclusions. 
     Thus, if one is interested in only the highest enriched variants for a particular tissue, PCR pool design coupled with enrichment normalization to virus library may not drastically differ from synthetic pool design over one additional round of selection for a subset of in vivo selections (such as Tek-Cre or SNAP-Cre). Without additional validation, however, it is difficult to predict whether a given in vivo system will perform akin to Tek-Cre. This becomes critical in a multiplexed selection study where target-specific variants may not garner the highest enrichments in one particular in vivo selection. 
     Example 5 
     Analysis of AAV Capsid Libraries After Round-2 Selections 
     Whereas the AA distribution of the DNA library closely matched the Oligopool design, virus production selected for a motif with Asn (N) at position 2, β-branched AAs (I, T, V) at position 4, and positively charged AAs (K, R) at position 5 ( FIGS. 10, 52 ). Fitness for BBB crossing resulted in a very different pattern. In comparison to the R2 virus library, highly enriched variants share preferences, for example, proline (P) in position 5, and phenylalanine (F) in position 6. 
     The distribution of the positively enriched variants from brain across all peripheral organs was then determined ( FIG. 11 , left). About 60 variants that are highly enriched in brain are comparatively depleted across all other organs ( FIG. 11 , middle). Encouraged by the expected behavior of spike-in control variants (AAV9, PHP.B, PHP.eB), eleven novel variants were chosen for further validation ( FIG. 11 , right), including several that would have been overlooked if the choice had been based on PCR pool or CREATE (Table 8). 
     
       
         
           
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 Ranking of AAV-PHP capsids across methods.Ranks of selected variants 
               
               
                 among all capsids recovered from R2 Tek-Cre selection by synthetic pool enrichment score 
               
               
                 (representing M-CREATE), PCR pool enrichment score (representing closer to M-CREATE), or 
               
               
                 PCR pool read counts (representing CREATE), the highest ranks of which starts from 1, and 
               
               
                 “Not recovered” represent absence of the variant from R2 sequencing data. 
               
            
           
           
               
               
               
               
            
               
                   
                 Synthetic pool 
                 PCR pool 
                 PCR pool 
               
               
                 AAV 
                 enrichment 
                 enrichment 
                 read count 
               
               
                 Variants 
                 rank 
                 rank 
                 rank 
               
               
                   
               
               
                 PHP.V1 
                  1 
                  4 
                  3 
               
               
                 PHP.V2 
                  2 
                  1 
                  1 
               
               
                 PHP.B4 
                  4 
                 10 
                  56 
               
               
                 PHP.B7 
                  6 
                 13 
                  36 
               
               
                 PHP.B8 
                  3 
                  7 
                  23 
               
               
                 PHP.C1 
                 13 
                 34 
                  74 
               
               
                 PHP.C2 
                 12 
                 20 
                 293 
               
               
                 PHP.C3 
                 16 
                 Not recovered 
                 Not recovered 
               
               
                   
               
            
           
         
       
     
     These variants were chosen due to their enrichments and where they fall in sequence space. The positively enriched variants were found to cluster into distinct families based on sequence similarity. In agreement with the heatmaps discussed above, the most enriched variants form a distinct family across selections that share a common motif: T in position 1, L in position 2, P in positive 5, F in position 6, and K or L in position 7 ( FIGS. 12, 53 ). This AA pattern closely resembles the previously identified variant, AAV-PHP.B—TLAVPFK. Given the sequence similarity among members, we predicted that they may similarly cross the BBB and target the central nervous system. 
     The ability to twice recover the AAV-PHP.B sequence family from completely independently constructed and selected libraries confirms that the viral library&#39;s sequence space coverage was broad enough to recover a family of variants sharing a common motif. Unlike CREATE which identified only one variant, AAV-PHP.B, M-CREATE yielded a diverse PHP.B-like family that hints toward important chemical features of this motif. The sequence diversity within this family suggests that isolating AAV-PHP.B was not simply good fortune in previous experiments (considering a theoretical starting library size of ˜1.3 billion), and that this is a dominant family for this particular experiment. 
     Example 6 
     Capsid Recovery from Round-2 Selection Yields a Pool of AAV9 Variants with Enhanced BBB Entry and CNS Ttransduction 
     Given the dominance of the PHP.B-family in this particular selection, the most enriched member was tested: TALKPFL ( FIGS. 12 and 13 ) henceforth referred to as AAV-PHP.V1. Somewhat surprisingly given its sequence similarity to AAV.PHP.B, the tropism of AAV-PHP.V1 is biased toward transducing brain vascular cells ( FIGS. 14, 54 ). When delivered intravenously, AAV-PHP.V1 carrying a fluorescent reporter under the control of the ubiquitous CAG promoter transduces ˜60% of GLUT1 +  cortical brain vasculature compared to ˜20% with AAV-PHP.eB and almost no transduction with AAV9 ( FIGS. 14 and 16 ). In addition to the vasculature, AAV-PHP.V1 also transduced ˜60% of cortical S100 +  astrocytes ( FIG. 17 ). However, AAV-PHP.V1 is not as efficient for astrocyte transduction as the previously reported AAV-PHP.eB (when packaged with an astrocyte specific GfABC1D promoter,  FIG. 55 ). 
     For applications requiring endothelial cell-restricted transduction via intravenous delivery, AAV-PHP.V1 vectors can be used in three different systems: (1) in endothelial cell-type specific Tek-Cre mice with a Cre-dependent expression vector ( FIG. 15  (left) and  FIG. 18 ), (2) in fluorescent reporter mice where Cre is delivered with an endothelial cell-type specific MiniPromoter (Ple261) ( FIG. 15  (right) and  FIGS. 19 and 56  through the left column of  FIGS. 58 ), and ( 3 ) in wild-type mice by packaging a self-complementary genome (scAAV) containing a ubiquitous promoter (Right column of  FIG. 58 ). The mechanism of endothelial cell-specific transduction by AAV-PHP.V1 using scAAV genomes is unclear, but shifts in vector tropism when packaging scAAV genomes have been reported for another capsid. 
     Given the dramatic difference in tropism between AAV-PHP.V1 and AAV-PHP.B/eB, we tested several additional variants within the PHP.B-like family. One variant, AAV-PHP.V2—TTLKPFL, differed by only one AA from AAV-PHP.V1, has a similar tropism ( FIGS. 59-61 ). AAV-PHP.V2 was found at high abundance in R1 selection across all brain libraries and was highly enriched in R2 ( FIGS. 4, 11  (right panel), 12, 13, and 40). Given its sequence similarity, similar tropism was expected to that of AAV-PHP.V1. This was validated in vivo in C57BL/6J adult mice (ssAAV-PHP.V2:CAG-mNeongreen genome, 3×10 11  vg dose per adult mice, n=3,  FIG. 59 ), in Tek-Cre mice (ssAAV-PHP.V2:CAG-DIO-EYFP genome, 1×10 12  vg dose per adult mouse, n=2,  FIG. 60 ), and in GFAP-Cre mice (ssAAV-PHP.V2:CAG-DIO-EYFP, 1×10 12  vg dose per adult mouse, n=2,  FIG. 61 ). 
     Three other variants with sequences of roughly equal deviation from both AAV.PHP.V1 and AAV.PHP.B, AAV-PHP.B4—TLQIPFK, AAV-PHP.B7—SIERPFK, and AAV-PHP.B8—TMQKPFI ( FIGS. 12, 13, 20, and 21 ), have PHP.B-like tropism with biased transduction toward neurons and astrocytes ( FIGS. 21 and 62-64 ). Similar variants among the spike-in library, AAV-PHP.B5—TLQLPFK and AAV-PHP.B6—TLQQPFK, also shared this tropism ( FIGS. 13, 20, 21, and 62 ). 
     To evaluate the performance of the spike-in library, two highly enriched variants similarly placed in sequence space were chosen: AAV-PHP.B6—TLQLPFK and AAV-PHP.B7—TLQQPFK ( FIGS. 48  (middle panel), and 53) that were previously identified in the 3-mer-s PHP.B library but never validated in vivo. At a modest dose of 1×10 11  vg in C57BL/6J adult mice, these variants also display PHP.B-like tropism ( FIGS. 20-21 and 62 ). 
     A series of variants selected to verify M-CREATE&#39;s predictive power outside this family were then investigated: (1) A highly enriched variant with a completely unrelated sequence, AAV-PHP.C1—RYQGDSV ( FIGS. 12, 13, 20, and 21 ), transduced astrocytes at a similar efficiency and neurons at lower efficiency compared to other tested variants from B-family ( FIG. 21 ). ( 2 ) Two variants found in high abundance in the R2 synthetic pool virus library and negatively enriched in brain (with both codon replicates in agreement), AAV-PHP.X1—ARQMDLS and AAV-PHP.X2—TNKVGNI ( FIG. 46 , right), poorly transduced the CNS ( FIG. 63 ). (3) Two variants that were found in higher abundance in brain libraries from the PCR pool R2, AAV-PHP.X3—QNVTKGV and AAV-PHP.X4—LNAIKNI also failed to outperform AAV9 in the brain ( FIG. 65 ). 
     Collectively, the characterization of these AAV variants demonstrates several key points. First, within a diverse sequence family, there is room for both functional redundancy and the emergence of novel tropisms. Second, highly enriched sequences outside the dominant family are also likely to possess enhanced function. Third, buoyed by codon replicate agreement in the synthetic pool, a variant&#39;s enrichment across tissues may be predictive. Fourth, while the synthetic pool R2 library contains a subset of the sequences that are in the PCR pool R2 and may thereby lack some enhanced variants, the excluded PCR pool population is enriched in false positives. 
     The ability to confidently predict in vivo transduction from a pool of 18,000 variants across mice is a significant advance in the selection process and demonstrates the power of M-CREATE for the evolution of individual vectors. 
     Example 7 
     Re-Investigation of Capsid Selection that Yielded AAV.PHP.eB Reveals Variant that Specifically Transduces Neurons 
     Using NGS, a 3-mer-s (s-substitution) PHP.B library generated by the prior CREATE methodology that yielded AAV-PHP.eB 27  was reinvestigated ( FIG. 24 ). The brain libraries were deep sequenced using Cre-dependent PCR and a R2 liver library from wild-type mice (processed via PCR for all capsid sequences regardless of Cre-mediated inversion) and identified 150-200 positively enriched capsids in brain tissue ( FIGS. 25, 66, and 67 ). Briefly, the re-investigated 3-mer-s PHP.B library diversified positions 587-597 of the AAV-PHP.B capsid (equivalent of 587-590 AA on AAV9) in portions of three consecutive AAs, (40,000 total variants) ( FIG. 24 ). Selections were performed in three Cre-transgenic lines: Vglut2-IRES-Cre for glutamatergic neurons, Vgat-IRES-Cre for GABAergic neurons, and GFAP-Cre for astrocytes. 
     Variants that were positively enriched in brain and negatively enriched in liver show a significant bias towards certain AAs: G, D, E at position 1; G, S at position 2 (which includes the AAV-PHP.eB motif, DG); and S, N, P at position 9, 10, 11 ( FIGS. 26 and 68 ). Variants that were positively enriched in the brain were clustered according to their sequence similarities and ranked by their negative enrichment in liver (represented by node size in clusters). A distinct family referred to as N emerged with a common motif “SNP” at positions 9-11 on PHP.B backbone ( FIGS. 27 and 69 ). 
     The core variant of the N-family cluster: AQTLAVPFSNP was found in high abundance in R1 and R2 selections, had higher enrichment score in Vglut2 and Vgat brain tissues compared to GFAP, and had negative enrichment in liver tissue ( FIGS. 25 and 66-69 ). Unlike AAV-PHP.eB, this variant (AAV-PHP.N) specifically transduced NeuN +  neurons even when packaged with a ubiquitous CAG promoter, although the transduction efficiency varied across brain regions (from ˜10-70% in NeuN +  neurons, including both VGLUT1 +  excitatory and GAD1 + inhibitory neurons,  FIGS. 28, 29, 70, and 71 ). 
     Thus, by re-examining the 3-mer-s library several novel variants were identified, including one with notable cell-type-specific tropism. While Vglut2-Cre and Vgat-Cre mice were used for in vivo selection, no variants stood out for neuronal subtype-specific transduction of excitatory and inhibitory populations from initial investigations on the NGS dataset. It is possible that a biological solution to this (stringent) selection was not present in the library. 
     Example 8 
     Investigation of Capsid Families Beyond C57BL/6J Mouse Strain 
     The enhanced CNS tropism of AAV-PHP.eB is absent in a subset of mouse strains. It is highly efficient in C57BL/6J, FVB/NCrl, DBA/2, and SJL/J, with intermediate enhancement in 129S1/SvimJ, and no enhancement in BALB/cJ and several additional strains. This pattern holds for the two newly identified variants from the PHP.B family, AAV-PHP.V1 and AAV-PHP.N ( FIG. 30 , Table 9), which did not transduce the CNS in BALB/cJ, yet transduced the FVB/NJ strain ( FIG. 31 ). AAV-PHP.V1 transduced Human Brain Microvascular Endothelial Cell (HBMEC) culture, resulting in increased mean fluorescent intensity compared to AAV9 and AAV-PHP.eB ( FIG. 72 ) however, suggesting the potential for mechanistic complexity. 
     
       
         
           
               
             
               
                 TABLE 9 
               
             
            
               
                   
               
               
                 AAV-PHP vectors identified by CREATE and M-CREATE. The table provides 
               
               
                 a summary of the variants that have been identified so far using CREATE 
               
               
                 and M-CREATE, along with their tropism and the evolutionary steps 
               
               
                 from the parent capsid that was involved in their discovery. 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                   
                   
                 Rounds of 
               
               
                 AAV 
                 Reference/ 
                   
                   
                 evolution from 
               
               
                 Variants 
                 Selection method 
                 Tropism 
                 Production 
                 parent capsid 
               
               
                   
               
               
                 PHP.B, 
                 Deverman et al, 
                 Broad CNS 
                 Good 
                 1 round from 
               
               
                 B2, B3 
                 2016/CREATE 
                 transduction 
                   
                 AAV9 
               
               
                 PHP.A 
                 Deverman et al, 
                 Astrocyte 
                 Poor; prone to 
                 1 round from 
               
               
                   
                 2016/CREATE 
                 transduction 
                 precipitate upon 
                 AAV9 
               
               
                   
                   
                   
                 storage at 4° C. 
                   
               
               
                 PHP.eB 
                 Chan et al, 
                 Enhanced Broad 
                 Good 
                 2 rounds from 
               
               
                   
                 2017/CREATE 
                 CNS transduction 
                   
                 AAV9 or 
               
               
                   
                   
                   
                   
                 1 round from 
               
               
                   
                   
                   
                   
                 PHP.B 
               
               
                 PHP.S 
                 Chan et al, 
                 Sensory neuron 
                 Good 
                 1 round from 
               
               
                   
                 2017/CREATE 
                 transduction 
                   
                 AAV9 
               
               
                 PHP.V1, 
                 Current study/ 
                 BBB Vascular 
                 Good 
                 1 round from 
               
               
                 V2 
                 M-CREATE 
                 cells and 
                   
                 AAV9 
               
               
                   
                   
                 astrocytes 
                   
                   
               
               
                   
                   
                 transduction 
                   
                   
               
               
                 PHP.B4, 
                 Current study/ 
                 Broad CNS 
                 Good 
                 1 round from 
               
               
                 B7, B8, 
                 M-CREATE 
                 transduction 
                   
                 AAV9 
               
               
                 PHP.B5, 
                 Current study/ 
                 Broad CNS 
                 Good 
                 2 rounds from 
               
               
                 B6 
                 M-CREATE and 
                 transduction 
                   
                 AAV9 or 
               
               
                   
                 CREATE 
                   
                   
                 1 round from 
               
               
                   
                   
                   
                   
                 PHP.B 
               
               
                 PHP.C1, 
                 Current study/ 
                 Broad CNS 
                 Good; PHP.C1 
                 1 round from 
               
               
                 C2, C3 
                 M-CREATE 
                 transduction 
                 prone to 
                 AAV9 
               
               
                   
                   
                 across mouse 
                 precipitate upon 
                   
               
               
                   
                   
                 strains 
                 storage at 4° C. 
                   
               
               
                 PHP.N 
                 Current study/ 
                 Neuron 
                 Average 
                 2 rounds from 
               
               
                   
                 M-CREATE and 
                 transduction 
                   
                 AAV9 or 
               
               
                   
                 CREATE 
                   
                   
                 1 round from 
               
               
                   
                   
                   
                   
                 PHP.B 
               
               
                   
               
            
           
         
       
     
     Importantly, M-CREATE revealed many non-PHP.B-like sequence families that enriched through selection for transduction of cells in the CNS. We tested the previously mentioned AAV-PHP.C1: RYQGDSV, as well as AAV-PHP.C2: WSTNAGY, and AAV-PHP.C3: ERVGFAQ ( FIG. 30 ). These showed enhanced BBB crossing irrespective of mouse strain, with roughly equal CNS transduction in BALB/cJ and C57BL/6J ( FIGS. 32 and 73 ). Collectively, these preliminary studies suggest that M-CREATE is capable of finding capsid variants with diverse mechanisms of BBB entry that lack strain-specificity. 
     Example 8 
     Exemplary Insertion of Variant AAV Capsid Protein Sequence 
     Demonstration of 7 amino acid peptide insertion in AAV capsid: Peptide sequence “TLQIPFK” (SEQ ID: 435) is positioned between AA 588-589 of AAV capsid. The insertion sequence is underlined and bold. 
     
       
         
           
               
            
               
                 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGY 
               
               
                   
               
               
                 KYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEF 
               
               
                   
               
               
                 QERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSP 
               
               
                   
               
               
                 QEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGS 
               
               
                   
               
               
                 LTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALP 
               
               
                   
               
               
                 TYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQR 
               
               
                   
               
               
                 LINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDY 
               
               
                   
               
               
                 QLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYF 
               
               
                   
               
               
                 PSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRT 
               
               
                   
               
               
                 INGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSE 
               
               
                   
               
               
                 FAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGR 
               
               
                   
               
               
                 DNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQ  AQAQT 
               
               
                   
               
               
                 GWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHP 
               
               
                   
               
               
                 PPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSK 
               
               
                   
               
               
                 RWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL 
               
            
           
         
       
     
     Demonstration of 11 amino acid peptide insertion in AAV capsid: 
     Peptide sequence “DGTTLKPFLAQ” (SEQ ID: 867) is positioned by replacing AA 587-590 of AAV capsid. The inserted sequence is underlined and highlighted. 
     
       
         
           
               
            
               
                 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGY 
               
               
                   
               
               
                 KYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEF 
               
               
                   
               
               
                 QERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSP 
               
               
                   
               
               
                 QEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGS 
               
               
                   
               
               
                 LTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALP 
               
               
                   
               
               
                 TYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQR 
               
               
                   
               
               
                 LINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDY 
               
               
                   
               
               
                 QLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYF 
               
               
                   
               
               
                 PSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRT 
               
               
                   
               
               
                 INGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSE 
               
               
                   
               
               
                 FAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGR 
               
               
                   
               
               
                 DNVDADKVMITNEEEIKTTNPVATESYGQVATNHQS  AQT 
               
               
                   
               
               
                 GWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHP 
               
               
                   
               
               
                 PPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSK 
               
               
                   
               
               
                 RWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL. 
               
            
           
         
       
     
     Example 9 
     Treating Huntington&#39;s Disease (HD) 
     A subject having Huntington&#39;s disease is identified. The subject is then systemically administered a first amount of a viral vector that includes a polynucleotide that encodes for a Zinc finger protein (ZFP) engineered to represses the transcription of the Huntingtin (HTT) gene. The vector will be encapsidated by a modified AAV capsid protein with an amino acid sequence provided in  FIG. 33  or provided in Tables 2-4, so as to allow proper targeting of the ZFP to the nervous system, among other organs. If needed, the subject is administered a second or third dose of the vector, until a therapeutically effective amount of the ZFP is expressed in the subject in the nervous system. 
     Example 10 
     Phase 1A Clinical Trial 
     A phase 1A clinical trial is performed to evaluate the safety, tolerability, pharmacokinetics, and pharmacodynamics of an one-time intravenous injection of test composition comprising viral vector encapsidated by a modified capsid protein with any of the amino acid sequences provided in  FIG. 33  or Tables 2-4, in subjects with late Huntington&#39;s Disease (HD). Eligible subjects are men and women between 21 and 65 years of age. 
     Inclusion Criteria: Eligible subjects are men and women between 21 and 65 years of age. Subjects that (i) sign and date Sign and date International Classification of Functioning, Disability and Health (ICF); (2) male or female participant aged ≥21 and ≤65; (3) participants who submit medical report (PCR) attesting Huntington&#39;s disease with a number of CAG repeats on chromosome 4, greater than or equal to 40 and less than or equal to 50 (if the participant has not performed the examination and/or if he does not have the report available, a new exam should be done); (4) Score 5 points or more in motor assessment UHDRS scale (Unified Huntington&#39;s Disease Rating Scale) at the time of enrollment; (5) Score between 8 and 11 points in the functional capacity of the UHDRS scale at the time of enrollment. 
     Exclusion Criteria: (1) Any medical observation data (clinical and physical) that medical research judge as a risk for subject if enrollment at the study; (2) any laboratory exam data that medical research judge as a risk for subject if enrollment at the study; (4) history of epilepsy; (5) diagnostic of major cognitive impairment; (6) active decompensated psychiatric disease; (7) current or prior history of neoplasia; (8) current history of gastrointestinal, hepatic, renal, endocrine, pulmonary, hematologic, immune, metabolic pathology or severe and uncontrolled cardiovascular disease; (8) diagnostic of any active infection, be it viral, bacterial, fungal, or caused by another pathogen; (9) participants who have contraindication to undergo any of the tests performed in this study, for example, have pacemakers or surgical clip; (10) history of alcohol or illegal drugs abusers; (11) history of 1 or more episodes of suicide in the two years before Visit V-4; (12) active smoker or have stopped smoking less than six months prior to enrollment; (13) test positive in at least one of the serological tests: HIV 1 and 2 (Anti-HIV-1,2), HTLV I and II, HBV (HBsAg, anti-HBc), HCV (anti-HCV-Ab) and VDRL (Treponema pallidum); (14) history of drug allergy, including contrasts for imaging, or bovine products; (15) in use or expected use of immunosuppressive drugs or prohibited medicines for the first three months after the first administration of the investigational product; (16) any clinical changes that is interpreted by the medical researcher as a risk to participant&#39;s enrollment. 
     Experimental: 
     Placebo. One-time injection of placebo at Week 0. 
     Test High Dose. One-time injection of test composition 2×10{circumflex over ( )}10 vg at Week 0. 
     Test Middle Dose. One-time injection of test composition 6×10{circumflex over ( )}9 vg at Week 0. 
     Test Low Dose. One-time injection of test composition 2×10{circumflex over ( )}9 vg at Week 0. 
     Test Lowest Dose. One-time injection of test composition 2×10{circumflex over ( )}8 vg at Week 0. 
     Primary Outcome Measures: Safety of the test composition by periodic monitoring changes at adverse events, vital signs, laboratory tests, ECG and incidence of benign and malignant neoplasms [ Time Frame: five years ]. The safety of the investigational product will be evaluated in detail from periodic evaluations contemplating monitoring changes of: (1) adverse events including type, frequency, intensity, seriousness, severity, and action taken related to the investigational product study; (2) vital signs (BP, HR, axillary temperature), physical and medical examination (BMI, weight, height, medical condition—cardiovascular, pulmonary, digestive, musculoskeletal and peripheral, with emphasis on the neurological assessment and others); (2) laboratory tests included hematologic, biochemical, urologic and serological analysis; (3) electrocardiogram (ECG) of 12 derivations; (4) and incidence and classification of benign and malignant neoplasms in the following organs/systems: CNS, lung, liver, spleen, pancreas, prostate, testicle, urinary, hematological and skeletal system through the laboratory tests, magnetic resonance imaging, computerized tomography and ultrasonography. 
     Secondary Outcome Measures: Preliminary efficacy of Cellavita HD by global clinical response (CIBIS) and UHDRS improvement [ Time Frame: five years ] will be evaluated by statistical comparison of the results of each UHDRS scale component: motor, cognitive and behavior. The global clinical response will be assessed by statistical comparison between baseline score observed by the Investigator before and after Cellavita HD treatment. Preliminary efficacy of Cellavita HD by comparison of the inflammatory markers [ Time Frame: one year ] will be evaluated by statistical comparison of the inflammatory markers included IL-4, IL-6, IL-10 (interleukin IL) and TNF-alpha (tumoral necrosis factor alpha). Immunological Response of Cellavita HD [Time Frame: one year]. The immunological response induced by Cellavita HD will be evaluated by statistical comparison between baseline results of CD4+ and CD8+ proliferation and the other evaluated times. Preliminary efficacy of Cellavita HD by comparison of the CNS assessment [Time Frame: one year]. Will be evaluated by statistical comparison of the CNS assessment through magnetic resonance image at cortical thickness measurements, volumes of different brain structures, especially the basal ganglia, with special attention to caudate and metabolic changes identified in proton spectroscopy. Risk of suicidal ideation by Hamilton Depression Rating Scale (HDRS) [Time Frame: five years] will be evaluated by suicidal domain. The classificatory pontuation may correspond to mild depression (score: 8 to 13), moderate depression (score: 19-22) and severe depression (score: &gt;23). 
     While preferred instances of the present examples have been shown and described herein, it will be obvious to those skilled in the art that such instances are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the instances of the disclosure described herein may be employed in practicing the disclosure. It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby. 
     While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.