VectorBuilder

Overview
VectorBuilder is a global leader in gene delivery technologies. As a trusted partner for thousands of labs and biotech/pharma companies across the globe, VectorBuilder offers a full spectrum of gene delivery solutions covering virtually all
research and clinical needs from bench to bedside.

Offerings
Custom vectors & viruses
VectorBuilder is the world’s largest provider of custom vectors for both viral and non-viral gene delivery. We currently
tailor-build over 80,000 vectors a year for tens of thousands of researchers around the world. Our vector services boast
several important advantages:• Online vector design and ordering platform: This platform is a transformative innovation by
VectorBuilder that allows researchers to design and order custom vectors online, freeing them from thetedious DIY work of cloning vectors and packaging viruses in the lab. This award-winning platform hasa free web-based Vector Design Studio that employs a highly user-friendly, modular design principle. It
enables even novice researchers to easily design custom vectors with just a few mouse clicks and thenorder their vectors online along with related services such as virus packaging and plasmid preparation.
• Comprehensive vector systems: VectorBuilder offers a broad range of vector systems for both viraland non-viral gene delivery, such as AAV, adenovirus (including gutless), lentivirus, MMLV, baculovirus,
VSV, HSV, PiggyBac, Sleeping Beauty, etc. These vectors cover virtually all model organisms andinclude wide-ranging applications, such as overexpression, shRNA knockdown, CRISPR, recombinant
protein expression, and in vitro transcription. Many vectors are specifically designed for clinicalapplications such as antibody production, CAR-T, gene therapy, mRNA vaccines, and oncolytic viraltreatment.
• Vast vector inventory: VectorBuilder has an extensive collection of >700 vector backbones
corresponding to a wide range of viral and non-viral vector systems. We have also amassed >400,000
in-stock vector components including ORFs, shRNAs, and gRNAs corresponding to most known genes
in popular model organisms, as well as commonly used promoters, reporters, linkers, and epitope tags.
Over 80% of the custom vectors we build nowadays are readily assembled from existing backbones
and components, significantly reducing cost and turnaround time.

CRO services
Besides custom vectors and viruses, VectorBuilder offers a wide range of CRO services covering many more genedelivery applications in basic research and drug discovery:• Diverse gene delivery solutions: We offer a comprehensive collection of premade shRNA and gRNAlibraries for multiple species, as well as custom library construction and in vitro and in vivo library
screening services. We also provide stable cell line generation, BAC recombineering, mRNA synthesis/
encapsulation, and reverse genetics rescue of novel viruses. Additionally, we have several COVIDrelated offerings such as spike (S) protein pseudotyping of lentivirus and VSV.

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www.vectorbuilder.com• Preclinical vector testing: VectorBuilder offers preclinical non-GLP and GLP testing of vector efficacy
and safety profiles, including biodistribution, ADME/PK/PD, and toxicology. These services can be
performed in a variety of in vitro and in vivo systems including non-human primate (NHP) models.
• High value-added R&D: We recently launched several high value-added R&D services aimed atdeveloping new delivery strategies and optimizing vector performance for tailored clinical applications,such as AAV capsid evolution, viral G protein evolution, promoter engineering and screening, andexperimental codon optimization for mRNA and viral vectors. These studies can be carried out in NHPs
to maximize their translatability to human use.

CDMO services
VectorBuilder is a full-service CDMO with extensive experience in cGMP vector manufacturing. Operating several stateof-the-art facilities, we have supported many customers along their entire drug-discovery pipelines, going from researchgrade vectors for early discovery, to GMP-like vectors for preclinical testing, to full GMP-grade vectors for clinical trials.
We have provided IND-enabling vectors to a worldwide client base in the US, Europe, Japan, China, and South Korea.
Our CDMO services include process development, analytical development, cell banking, fill/finish, and regulatory support.
Our GMP manufacturing covers the following systems:• Plasmid DNA: VectorBuilder offers GMP-grade plasmid DNA with up to 10 g scale.
• Viral vectors: We offer GMP manufacturing for multiple viral vector systems including AAV, lentivirus,
MMLV, adenovirus, HSV, etc. Either adherent or suspension packaging systems can be used with up to
1,500 L scale.
• mRNA: VectorBuilder's mRNA CDMO services include sequence engineering and optimization, DNA
template construction, mRNA in vitro transcription (using modified nucleotides when appropriate), and
LNP encapsulation.

IP out-licensing
With strong R&D capabilities, VectorBuilder has built an extensive IP portfolio of gene delivery technologies, someof which can be out-licensed. These include new AAV serotypes, novel viral G proteins, engineered tissue-specificpromoters, high-yield recombinant protein producer cell lines, etc.

Mission
VectorBuilder is the future of gene delivery. Our mission is to provide end-to-end solutions for all gene delivery needs
spanning both research and therapy.
We strive to offer innovative and high-quality products and services while maintaining rapid turnaround andexceptional affordability. Our “white-glove” customer care is supported by a PhD-level team with decades of collective
experience to devise the best gene delivery solutions for our customers. As a result, we have received glowing
customer testimonials and numerous citations in high-impact publications.
Operating as a multinational company with branches in North America, Europe, China, Japan, South Korea, Australia,
and Israel, VectorBuilder has served over 50,000 customers from several thousand organizations worldwide, including
universities, research institutions, biotech/pharma companies, and governmental agencies.

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Table of Contents
AAV Basic Biology

02

AAV Vectors

04

AAV Vector Systems ...................................................................................................04
AAV Vector Design ......................................................................................................05

AAV Offerings for Research

10

AAV Virus Packaging ..................................................................................................10  
AAV Serotype Testing Panel .......................................................................................12  
shRNA (3+1) AAV Packaging ......................................................................................14  
Control AAV Virus ........................................................................................................14
AAV Virus-Like Particles (VLPs)..................................................................................15

AAV GMP Manufacturing

16

Virus Manufacturing ....................................................................................................16
GMP Facilities .............................................................................................................17

AAV CRO Services

18

Library Construction ....................................................................................................18
AAV Capsid Evolution .................................................................................................18
AAV Biodistribution Profiling ........................................................................................20

AAV Knowledge Base22

AAV Serotypes and Tropism .......................................................................................22
Protocols for AAV In Vitro and In Vivo Applications .....................................................23
User Citations .............................................................................................................32

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AAV Basic Biology
Adeno-associated virus (AAV) is a member of Dependoparvovirus
genus and belongs to the viral family Parvoviridae. The AAV virion
is a nonenveloped and relatively small (20 um) capsid, which
has an icosahedral structure and contains a single stranded DNA
genome. Due to its capability to infect dividing and non-dividing
cells, AAV infection is common in humans and other primates but
has not been reported to be pathogenic. When AAV enters hostcells, it can lead to long term expression of a transgene with very
low immunogenicity (unlike wildtype AAV, in host cells the viralgenome of recombinant AAV exists in the form of episomal DNA).
Due to all these characteristics, AAV is an increasingly popularviral vector for applications in gene therapy.

Adeno-associated virus

The AAV Genome
The genome of AAV is a positive or negative-sense single stranded DNA around 4.7 kb in length. Each AAV viral genome
contains two Inverted Terminal Repeats (ITRs) at its ends that are essential to virus replication, capsid assembly, and theintegration of viral DNA into the host cell’s chromosome. AAV viral DNA contains two open reading frames: Rep and Cap.
The Rep ORF is composed of four overlapping genes: Rep78, Rep68, Rep52, and Rep40. The Cap ORF encodes threeoverlapping genes that are required for capsid assembly: VP1, VP2, and VP3. The replication of the AAV genome requires
a DNA polymerase provided by the host cell or a helper virus, since AAV itself lacks a viral DNA polymerase gene.

Figure 1. Wildtype AAV genome (Left) and the structure of an ITR (Right) (adapted from BioRender, 2022 & GonçalvesMA, 2005).

AAV Life Cycle
Wild-type adeno-associated virus requires a helper virus to complete its life cycle. After adeno-associated virus infects
cells, it requires a helper virus, such as adenovirus or HSV, to enter the lysogenic cycle for virus replication. In theabsence of a helper virus, the expression of adeno-associated virus genes is limited. Following entry into the lysogenic
cycle, a part of the adeno-associated virus genome can be integrated into the AAVS1 site of human chromosome 19
through a mechanism involving the ITRs and Rep protein (Rep78). Recombinant AAV commonly used in research does
not contain Rep gene thus, the transduced AAV genome exists in cells in the form of a DNA free body, which is conducive
to long-term expression of the transgene in cells.

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Figure 2. The life cycle of wildtype AAV virus (adapted from Daya S & Berns KI, 2008)

Serotype and Tissue Tropism
Adeno-associated virus is common in humans and other primates. At present, hundreds of AAV variants and serotypes
are known in nature. Among them, AAV2, AAV3, AAV5, and AAV6 are found in human cells, AAV1, AAV4, and AAV7
through 12 are found in other primate cells, with AAV2 being the most studied. Different serotypes of AAV exhibit different
cell or tissue tropism (ability to infect), which is useful in targeting AAV gene therapy to specific cells or tissues. In orderto improve the gene delivery efficiency of AAV, researchers have developed many artificial serotypes of adeno-associatedvirus to improve gene expression, alter target tissue tropism, and increase immune evasion. For example, AAV6.2 isproduced by targeted mutation of AAV6 capsid protein to increase transgene expression and AAV-DJ composed of fusionof a variety of AAV capsid sequences that exhibits broad tropism and increased immune evasion.
Go to page 22 to see more details about serotype and tissue tropism.

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AAV Vectors
AAV Vector Systems
VectorBuilder offers a variety of AAV vector systems:

Mammalian Gene Expression
ssAAV and scAAV Vector

Mammalian Non-Coding RNA Expression
ssAAV Vector

Chimeric Baculovirus-AAV Vector

Mammalian Inducible Gene Expression(Tet-Based)

Mammalian Conditional Gene
Expression (Cre-Lox Based)

Tet-On (All-In-One) AAV Vector

Cre-On Gene Expression AAV Vector

TRE Driven GOI Expression AAV Vector

Cre-Off Gene Expression AAV Vector

Tet Regulatory Protein Expression AAV Vector

Cre-Switch Gene Expression AAV Vector

Mammalian CRISPR Gene Editing
gRNA and Cas9 Coexpression AAV

Mammalian shRNA Knockdown
U6-Based shRNA ssAAV Vector

Vector (Single SagRNA)
U6-Based shRNA scAAV Vector
Single gRNA AAV Vector
Single miR30-shRNA AAV Vector
Dual gRNA AAV Vector
Dual miR30-shRNA AAV Vector
Cas9 Expression AAV Vector
Triple miR30-shRNA AAV Vector

Mammalian CRISPR Gene Regulation

Quad miR30-shRNA AAV Vector

CRISPR-Based Gene Activation AAV Vector (dSaCas9-SAM System)▶▷

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AAV Vector Design
ssAAV or scAAVThe AAV genome is a linear single-stranded DNA (ssDNA). When target cells are transduced with ssAAV, the singlestranded viral DNA is not transcribed until its complementary strand is synthesized. Due to this, the second-strand
synthesis tends to be the rate-limiting step for the expression of a given transgene. Our scAAV transfer vector isengineered from ssAAV with two important differences. First, the trs (terminal resolution site) located in the 3' ITR isdeleted in scAAV. As a result, scAAV has a tendency of forming a double-stranded DNA molecule during replication thatis the concatenation of two full single-stranded genomes, one plus strand and the other minus strand. This moleculecan form a self-complementary intramolecular dsDNA genome. When scAAV viral particles enter host cells, this selfcomplementary intramolecular dsDNA genome can skip second-strand synthesis, to quickly express genes carried on the
scAAV vector. Therefore, scAAV has faster and increased transgene expression relative to ssAAV. Second, due to factthat wildtype AAV can carry up to about 4.7 kb of single-stranded DNA genome yet each scAAV DNA molecule packaged
into a viral particle is the concatemer of two single-stranded genomes of opposite strands, the cargo capacity of scAAV interms of the length of the 5' ITR to 3' ITR transgene that can be properly packaged into mature virus is only about half thatof ssAAV.

Figure 3. Virus genome formation of ssAAV and scAAV (adapted from McCarty DM, 2008).

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The Cargo Capacity of AAV Vectors
AAV has the smallest cargo capacity of any of our viral vector systems. AAV can accommodate a maximum of 4.7 kb ofsequence between the ITRs, which leaves ~4.2 kb of cargo space for a user's DNA of interest for ssAAV and ~2.2 kb for
scAAV.
5' ITRpUC ori

5' ITR

Promoter

Promoter

Kozak
Kozak

pUC ori

ORF
ORF

AmpiciIIin

Regulatory Element

SV40 late pA
AmpiciIIin

BGH pA
3' ITR

ssAAV

scAAV

The Advantages of AAV Gene Delivery
Safety: AAV is the safest viral vector system available. AAV is inherently replication-deficient, and is not known
to cause any human diseases.
Low risk of host genome disruption: Upon transduction into host cells, AAV vectors remain as episomal
DNA in the nucleus. The lack of integration into the host genome can be a desirable feature for in vivo humanapplications, as it reduces the risk of host genome disruption that might lead to cancer.
High viral titer: Our AAV vector can be packaged into high titer virus. When AAV virus is obtained through ourvirus packaging service, titer can reach >1013 genome copy per ml (GC/ml).
Broad tropism: A wide range of cell and tissue types from commonly used mammalian species such as
human, mouse, and rat can be readily transduced with our AAV vector when it is packaged into the appropriate
serotype. Some cell types may be difficult to transduce, depending on the serotype used.
Effectiveness in vitro and in vivo: Our vector is often used to transduce cells in live animals and it can also be
used effectively in vitro.

Choose Promoter
For AAV vectors that use Pol II RNA polymerase for transcription, either a ubiquitous or tissue specific promoter can beapplied to drive the expression of a GOI. However, it has been observed that the transcriptional efficiency among thesepromoters is significantly varied in different cell lines. VectorBuilder offers many popular promoters that users can choose
from when designing their AAV vectors.

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Mammalian Ubiquitous Promoters
Ubiquitous promoters are widely used to construct viral vectors due to their generally high transcriptional activity
when driving the expression of a target gene. Details of these ubiquitous promotes are listed in the table below:
Name

Annotations

Size (bp)

CMV

Strong promoter; may have variable strength in some cell types.

589

CMV+intron

Strong promoter; may have variable strength in some cell types; presence of thebeta-globin intron facilitates the nuclear export of mRNA by splicing and is predicted 1242
to enhance gene expression in eukaryotes.

EF1A

Strong promoter

1179

EFS

Medium-strength promoter

232

CAG

Strong promoter

1733

CBh

Strong promoter

789

CBA

Strong promoter

850

SFFV

Strong promoter; drives high levels of gene expression, particularly in cell types ofthe myeloid lineage.

500

MSCV

Medium-strength promoter; drives gene expression in most murine or human ES
cell lines and other mammalian cell lines.

515

SV40

Medium-strength promoter

344

mPGK

Medium-strength promoter

511

hPGK

Medium-strength promoter

505

UBC

Weak promoter

1178

Mammalian Tissue-Specific Promoters
In addition to selecting a specific AAV serotype, the use of tissue specific promoters is key to achieving efficient expressionof transgenes in target tissues. Unlike constitutive promoters, tissue-specific promoters only drive the expression of target
genes in specific organs and tissues, which can greatly reduce the non-specific expression of the AAV transgene. The
tissue-specific promoters provided by VectorBuilder cover more than ten tissues and organs including nervous system,
cardiovascular system, muscle, liver, lung, bone tissue, and retina. Our tissue-specific promoters are listed in the tablebelow:
Cardiovascular system

7

Name

TissueCells

Size (bp)

cTnT

Heart

Cardiomyocytes413

αMHC(short)Heart

Cardiomyocytes2833

αMHC(long)Heart

Cardiomyocytes

5464

Hcn4

Embryonic heart

Cardiomyocytes2780

CD68(short)

Blood

Monocytes and macrophages659

CD68(long)

Blood

Monocytes and macrophages

3079

Tie1

Blood vessel

Vascular endothelial cells

1142

CD144

Lung, heart, ovary, spleen, and kidney
glomeruli

Vascular endothelial cells

2510

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Neural system
Name

TissueCells

Size (bp)Hb9

Spinal cord

Motor neurons

4003

Camk2a(short)

Brain

Pyramidal neurons

1337

Th

Brain

CA1 pyramidal cells of the hippocampus

3015

GFAP(short)

Brain

Astrocytes

681

GFAP(long)

Brain

Astrocytes

2178

Iba1

Brain

Microglia

1889

SYN1

Brain

Mature neurons

469

NSE

Nervous system

Various neurons

1807

Nes

Nervous system at embryonic stage

Neural stem/progenitor cells

1566

Cnp

Nervous systems, testis, and thymus

Oligodendrocytes and Schwann cells

3046

Tuba1aNervous systems at embryonic andearly postnatal stages

Developing neurons but not astrocytes or1121
oligodendrocytes

Name

TissueCells

Size (bp)

MHCK7

Skeletal and heart muscles

Differentiated postmitotic striated muscle
cells

771

Myog

MuscleMyoblasts

1139

ACTA1

MuscleMyocytes

2162

SM22a

Adult smooth muscle

Vascular smooth muscle cells

2960

EnSM22a

Adult smooth muscle

Vascular smooth muscle cells

594

Name

TissueCells

Size (bp)

Afp

Liver

Hepatocytes

2810

Alb

Liver

Mature hepatocytes

2336

TBG

Liver

Hepatocytes

460

Name

TissueCells

Size (bp)

ProA1

RetinaCone photoreceptor

2000

hRHO

RetinaRod photoreceptor

839

hBEST1RetinaRPE

623

MuscleLiver

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Bone Tissue
Name

TissueCells

Size (bp)Col2a1

Cartilage

Chondrocytes

1033

Runx2

Bone

Early osteoblasts

2514

CD11b

Bone marrow

Mature myeloid cell lines (neutrophils,
monocytes and macrophages)

1795

Col1a1Bone, tooth and tendon

Osteoblasts and fibroblasts

3632

OC

Bone and cartilage

Osteoblasts, osteocytes and hypertrophic
chondrocytes

2141

Name

TissueCells

Size (bp)

SPB

Lung

AT II cells (alveolar type II epithelial cells) andClara cells (bronchiolar epithelial cells)

635

Name

TissueCells

Size (bp)

NPHS2

KidneyPodocytes

2589

Name

TissueCells

Size (bp)

HIP

Pancreas

β cells

1867

Ins2

Pancreas

β cells705

Pdx1

Embryonic developing pancreas and Pancreatic progenitor cells and pancreatic β
adult pancreatic islets
cells

2930

Name

TissueCells

Size (bp)

aP2

Fat

Adipocytes

3708

AdipoqFat

Adipocytes

5411

Name

TissueCells

Size (bp)

MMTV

Mammary gland

Ductal cells of the salivary gland, mammary
epithelial cells

1315

Wap

Mammary gland

Alveolar epithelial cells of mammary tissue

2444

Name

TissueCells

Size (bp)

K14

Epidermis

Keratinocytes

2268

mTyr

Epidermis

Melanocytes and melanoma cells

560

BK5

Epidermis

Keratinocytes

5200

Lung

KidneyPancreas

Adipose

Mammary gland

Epidermal tissue

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AAV Offerings for Research
AAV Virus Packaging
Recombinant adeno-associated virus (AAV) is a versatile and popular viral vector used for in vitro and in vivo genedelivery. VectorBuilder offers superior quality AAV packaging services to support your AAV-based gene therapy
experiments. We have developed a series of proprietary technologies and reagents that have greatly improved
recombinant AAV production protocols in terms of titer, purity, potency, and consistency, especially for the AAV vector
systems used in our vector cloning services.
Types of AAV Offered
Single-stranded AAV (ssAAV) and self-complementary AAV (scAAV)
18 serotypes: 1, 2, 3, 4, 5, 6, 6.2, 7, 8, 9, rh10, DJ, DJ/8, PHP.eB, PHP.S, AAV2-retro, AAV2-QuadYF and
AAV2.7m8
AAV empty capsids or virus-like particles (VLPs)

AAV Production and QC
Triple Transfection-Based Approach
For our recombinant AAV manufacturing, the transfer plasmid carrying the gene of interest (GOI) is co-transfected with
our proprietary Rep-Cap plasmid and helper plasmid encoding adenovirus genes (E4, E2A, and VA) that mediate AAV
replication in HEK293T packaging cells. After a short incubation period, viral particles are harvested from cell lysates orsupernatants dependent on serotype and concentrated by PEG precipitation. For ultra-purified AAV (in vivo grade), viralparticles are further purified and concentrated by cesium chloride (CsCl) gradient ultracentrifugation. We use a qPCRbased approach to measure AAV titer.

Figure 4. Typical workflow of triple transfection-based AAV packaging.

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For each AAV produced by VectorBuilder, quality control includes titer measurement, sterility testing for bacteria and fungi,
and mycoplasma detection. If the transfer vector encodes a fluorescent protein, we also perform a transduction test to
detect corresponding fluorescence. Additionally, for ultra-purified AAV, we routinely sample virus quality by SDS-PAGE
analysis and endotoxin assay.
Price and turnaround
Scale

Applications Typical Titer

Pilot
MediumLarge

Cell culture

Minimum Titer Volume>1012 GC/ml>2x1011 GC/ml>5x1012 GC/ml>2x1012 GC/ml

Ultra-purified pilot
Ultra-purified medium

Cell culture &>2x1013 GC/ml
in vivo

Ultra-purified large>1013 GC/ml

Price (USD) Turnaround250 ul (10x25 ul)$399

1 ml (10x100 ul)$599

1 ml (10x100 ul)$999

100 ul (4x25 ul)$1,299500 ul (10x50 ul)$1,8991 ml (10x100 ul)$2,99910-18 days

16-26 days

Baculovirus-Based Approach
Our baculovirus-based AAV packaging workflow consists of two main steps as shown in Figure 5 below. Step 1 involves
the generation of two recombinant baculoviruses, one expressing the gene of interest (GOI) flanked by the AAV inverted
terminal repeats (ITRs) and a second helper baculovirus expressing the AAV Rep and Cap genes. In step 2 recombinant
AAV particles are generated by co-infecting insect cells with the two recombinant baculoviruses produced in step 1.

Figure 5. Typical workflow of baculovirus-based AAV packaging.

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For generating the two recombinant baculoviruses (one expressing the GOI flanked by AAV ITRs and the other expressing
the AAV Rep/Cap genes), the expression cassette for each baculovirus is first cloned into a baculovirus transfer vector.
The baculovirus transfer plasmid is then co-transformed with a helper plasmid expressing the Tn7 transposase into
the bacterial host harboring the empty baculovirus shuttle vector (a.k.a. bacmid) to generate recombinant bacmid. The
recombinant bacmid is then transfected into insect Sf9 cells. After a short incubation period, viral particles are harvested
from the media and further concentrated by sucrose cushion centrifugation. We use a qPCR-based approach to measurebaculovirus titer. Both recombinant baculoviruses are then co-infected into insect cells. After an incubation period of 72-96
hours, AAV particles are harvested from the cell lysate as well as the supernatant and concentrated by PEG precipitation.
For ultra-purified AAV (in vivo grade), viral particles are further purified and concentrated by cesium chloride (CsCl)
gradient ultracentrifugation. We use a qPCR-based approach to measure AAV titer.
Price and turnaround
Scale

Applications

Minimum Titer

Ultra-purified pilot
Ultra-purified medium

Cell culture & in
>5x1013 GC/ml
vivo

Ultra-purified large

VolumePrice (USD) Turnaround

1 ml (10x100 ul)$5,59942-56 days

5 ml (25x200 ul)$20,199

49-63 days

10 ml (50x200 ul)$38,199

56-70 days

Note: Baculovirus-based approach is available for the following serotypes: 1, 2, 5, 6, 8, 9.

AAV Serotype Testing Panel
Adeno-associated viruses (AAVs) have emerged as the mosteffective viral vectors for gene therapy due to their ability to transduce
a wide variety of mammalian cell types and their low immunogenicity
in host organisms. VectorBuilder offers the AAV serotype testing
panel to enable users to select the optimal AAV serotype for specificapplications by systematic comparison of a variety of serotypes incells or in animals.

Ordering Information
Product

Catalog No.

In vitro grade AAV serotype testing panel (CMV-EGFP)PANEL-AAVS01

In vitro grade AAV serotype testing panel (CAG-EGFP)PANEL-AAVS02

In vivo grade AAV serotype testing panel (CMV-EGFP)PANEL-AAVSP01

In vivo grade AAV serotype testing panel (CAG-EGFP)PANEL-AAVSP02

Titer & Volume

Unit Price (USD)>1012 GC/ml, 25 ul$59 per aliquot>1013 GC/ml, 25 ul$109 per aliquot

Experimental Validation
We have developed a number of proprietary techniques to optimize our triple transfection-based AAV packaging protocol
and our virus has been validated to exhibit high transduction efficiency in mammalian cells.

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Figure 6. HEK293T cells were transduced with 18 serotypes of recombinant AAV packaged from the same CMV>EGFP
vector (VB010000-9394npt). Representative EGFP expression at 48 h post transduction in different cell lines is shown as
indicated. Scale bars: 100 um.
A

BC

Figure 7. HeLa cells were transduced with 18 serotypes of recombinant AAV packaged from the same CMV>EGFP
vector (VB010000-9394npt). EGFP (A) Mean Fluorescence Intensity (MFI) and (B) positivity were quantified using flow
cytometry 48 h post transduction. (C) Representative EGFP expression in different cell lines is shown as indicated. Scalebars: 100 um.
A

BC

Figure 8. Huh-7 cells were transduced with 18 serotypes of recombinant AAV packaged from the same CMV>EGFP
vector (VB010000-9394npt). EGFP (A) Mean Fluorescence Intensity (MFI) and (B) positivity were quantified using flow
cytometry 48 h post transduction. (C) Representative EGFP expression in different cell lines is shown as indicated. Scalebars: 100 um.

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shRNA (3+1) AAV Packaging
The knockdown effects of empirically designed shRNAs are often
limited by variations in specificity and efficiency observed fromone shRNA to another. Therefore, it is important to test multiple
shRNAs to find the most potent shRNA for knocking down your GOI.
VectorBuilder’s shRNA (3+1) virus packaging services enable you
to select optimal shRNAs for your target genes at highly affordable
prices. This offering includes cloning and packaging three custom
shRNA viruses targeting your GOI and one scramble control virus.

Ordering Information
Scale

Applications

Pilot
MediumPrice (USD)*$1,248Cell culture$1,748Large$2,748Ultra-purified pilot$4,148Ultra-purified medium

Turnaround**Cell culture & in vivo

Ultra-purified large$5,64817-30 days

23-38 days

$8,748* Price includes both vector cloning and virus packaging.
** Turnaround includes the production time for both vector cloning and virus packaging. It does not include transit time forshipping final deliverables to the customer.

Control AAV Virus
AAV control virus is designed to match the biological application of the custom virus and to be used for testing AAV
transduction. For example, if the custom virus overexpresses a gene, then the control virus provided will be EGFP control
AAV (AAV overexpressing EGFP), and if the custom virus expresses an shRNA against a gene, then the control virus
provided will express a scramble shRNA. Detailed information on the control virus is shown below:
Vector System

Control Virus Vector NameControl Virus Vector ID

ssAAV gene expression system

pAAV[Exp]-CMV>EGFP:WPRE

VB010000-9394npt

scAAV gene expression system

pscAAV[Exp]-CMV>EGFP

VB010000-9304aud

ssAAV U6-based shRNA
knockdown system

pAAV[shRNA]-EGFP-U6>Scramble_shRNA

VB010000-0023jze

scAAV U6-based shRNA
knockdown system

pscAAV[shRNA]-EGFP-U6>Scramble_shRNA

VB010000-9343nhh

ssAAV miR30-based shRNA
knockdown system

pAAV[miR30]-CMV>EGFP:Scramble_miR30shRNA:WPRE

VB010000-9397wgw

ssAAV CRISPR system

pAAV[Exp]-CMV>EGFP:WPRE

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AAV Virus-Like Particles (VLPs)
VectorBuilder offers premade as well as custom AAV virus-like particles that can be used for a variety of applications
during the development of AAV gene therapy vectors, including optimization of analytical assays, biodistribution
assessment, and evaluation of in vivo immune responses to specific serotypes of AAV capsids. Our AAV virus-likeparticles are extensively characterized and can therefore, serve as reliable standards for evaluating the quality of
AAV virus-like particles generated in house. Moreover, they can be used as negative controls in studies intended todemonstrate that an observed effect is caused by the expression cassette harbored in an AAV vector rather than its
capsid.
Highlights
Ready-to-ship in vivo grade AAV virus-like particles of serotypes 1, 2, 5, 8, and 9
Custom services available for AAV virus-like particles produced using any desired serotype, production system,
purification method, or QC requirement
Thorough characterization using various methods including SDS-PAGE, BCA, endotoxin testing, mycoplasma
detection, and full/empty capsid ratio analysis

Ordering Information
Scale
12

10 ug (1.6x10 VPs)
13

100 ug (1.6x10 VPs)

VolumePrice (USD)

25 ul

$379

0.1 ml (1x100 ul)$2,19913

0.5 ml (5x100 ul)$8,29914

1 ml (10x100 ul)$12,399

500 ug (8x10 VPs)
1 mg (1.6x10 VPs)

Experimental ValidationACB

AAV VLPs

Conventional AAV

99.4%

72.5%
15.7%
0.0%

Figure 9. (A) Silver stained SDS-PAGE of ready-to-ship AAV virus-like particles of serotypes 1, 2, 5, 8, and 9.
(B) Transmission electron microscopy of serotype 9 AAV virus-like particles showing empty (black-filled spheres) and filled(white-filled spheres) capsids. (C) Charge detection mass spectrometry analysis indicating percentages of empty (black)and filled (green) capsids for AAV virus-like particles and conventional AAV preparations of serotype 8.

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AAV GMP Manufacturing
Virus Manufacturing
VectorBuilder has extensive expertise in producing many different types of viruses. We can provide viruses at different
scales and with different quality control requirements to meet the full range of demands along the gene therapy drug
development pipeline. We have established and validated a platform of technologies for large-scale GMP manufacturingof adeno-associated virus (AAV).

Different Grades of AAV Offered
AAV for Research
Research-grade AAV is suitable for basic research and drug discovery studies. It is produced in conventional cell
culture facilities. Stringent QC assays are performed to ensure the quality of the virus meets the standards requested
by customers.

GMP-like AAV
GMP-like AAV is suitable for pre-clinical studies including drug safety and metabolism testing in animals. The
manufacturing of GMP-like virus follows key features of GMP guidelines, such that the production process and quality
attributes are comparable to GMP with document control and traceability. GMP-like grade is therefore intended to bea small-scale mimic of the final GMP product, but with significantly lower cost and a much faster timeline. Productrelease is accompanied by a certificate of analysis (COA). TSE/BSE statement can be provided upon request.

GMP AAV
GMP-grade AAV is produced in our certified GMP cell culture suite following strict GMP guidelines. A comprehensive
quality assurance system is embedded throughout the manufacturing process, including an array of in-process andrelease QC assays to ensure that the virus meets customer specifications and regulatory standards for quality andsafety. At product release, a COA and a batch release report fully documenting the production process are provided.

Workflow for GMP AAV Production▶▷

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Platform Technologies
We package AAV in HEK293T cells under either adherent conditions (Cell Factory or fixed-bed bioreactors) or serum-freesuspension conditions (up to 200 L single-use bioreactors). We also package AAV in suspension Sf9 insect cells. We canachieve a scale of up to 1017 GC AAV per batch.

GMP Facilities
VectorBuilder currently has about 100,000 sq ft of modern GMP
facilities with advanced designs and state-of-the-art equipment.
All our facilities are designed to meet GMP regulations andguidelines of the US, EU, Japan, China, and PIC/S. They
are suitable for Phase I/II/III clinical trials and commercial
manufacturing. Our facilities currently include 10 GMP
manufacturing suites, multiple fill/finish suites, QC laboratories
totaling 9,500 sq ft, and multiple GMP suites totaling 8,000 sq ft
for PD/pilot runs.
Additionally, our Stage 3 GMP facilities (~500,000 sq ft),capable of large-scale commercial gene and cell therapy drug
manufacturing, are under construction and will be operational inearly 2024.

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AAV CRO Services
Library Construction
VectorBuilder can help you build custom pooled libraries to perform large-scale functional screens. We have considerable
experience in constructing high-quality pooled libraries utilizing our optimized vectors and proprietary technologies. We
can deliver your library as an E. coli stock, DNA, or recombinant virus, depending on your needs. Our custom libraries are
fully validated by next-generation sequencing (NGS) to ensure uniform clone representation and low error rates.

Libraries Offered
CRISPR libraries (CRISPR KO, CRISPRa/i,
CROP-seq, Perturb-seq, etc.)
shRNA libraries
Barcode libraries
Enhancer/promoter screening libraries

DNA shuffling libraries
Two-hybrid libraries
Capsid screening libraries
And many more

Peptide expression libraries

AAV Capsid Evolution
Directed evolution is a high-throughput approach widely used for engineering novel AAV capsids. It mimics the process ofnatural selection through repeated genetic diversification and selection. Directed evolution of AAV capsids is performedby mutating the wildtype AAV capsid gene to generate highly diverse AAV capsid libraries, which are then screened to
identify novel capsid variants with improved properties.
Capsid Evolution Services Offered
Full-service platform to fulfill all your needs along the workflow of generating high diversity AAV capsid libraries
High-complexity capsid libraies construction via any mutagenesis or combinatorial approach, including errorprone PCR, random peptide display, DNA family shuffling, and in silico design
High-titer packaging of viral capsid libraries by either one-step or two-step approaches
In vivo screening in multiple species including mice, rats, and NHPs
Full technical support covering every aspect of your AAV capsid project from library design and construction toin vivo screening and NGS analysis▶▷

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Error-prone
PCR

Random peptide
display

DNA family
shuffling

In silicodesign

AAV capsid library (plasmid)

AAV capsid library (virus)

Recovery ofenriched
chimeric AAVs

In vitro or in vivo screening

Repeatedscreening

Smaller library withenriched variants

Characterization and validationof novel AAV variants withdesired property

Figure 10. Above is the typical workflow for screening novel AAV capsids by directed evolution.The first and most criticalstep in the entire workflow of AAV capsid evolution is the generation of a highly diverse AAV capsid library in which eachplasmid carries a chimeric AAV genome consisting of a Rep gene and a capsid gene variant. The capsid gene variants
can be efficiently generated using various approaches, such as error-prone PCR, random peptide display, DNA family
shuffling, or in silico design. The capsid library is then packaged into viral particles, and each viral particle harbors a
corresponding capsid variant in its genome. The viral library is then subsequently subjected to a screening process. Viral
genomes that pass screening are recovered from target cells and made into a smaller library for the second round ofscreening. Multiple rounds of screening are usually performed to enrich high-confidence hits. The resulting hits are thenvalidated and characterized to identify novel AAV capsid variants with enhanced properties.

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AAV Biodistribution Profiling
Assessing the distribution and persistence of AAV vectors in various body tissues and organs at the developmental andpreclinical stages is critical to ensure the success of AAV-based gene therapy. AAV biodistribution studies have beenhighly instrumental in identifying off-target effects, thereby playing a significant role in the safety assessment of AAV
vectors.
Service Highlights
Full-service platform to fulfill all your needs along the workflow of biodistribution assessment for your AAV
vectors
Services available for multiple species, including mice, rats, and NHPs
Multiple analytical assays, including fluorescence imaging, flow cytometry analysis, luciferase assay, qPCR,
and RT-qPCR
Multiplexing analysis using barcodes and NGS for assessing the biodistribution of different vectors within thesame animal
Multiple routes of AAV administration by highly trained experts, including tail vein injection, facial vein injection(for neonatal mice and rats), intracerebroventricular injection, intrathecal injection, subretinal injection,
intravitreal injection, intratympanic injection, and intramuscular injection
Full technical support that covers every aspect of your AAV biodistribution project

Experimental Validation

Figure 11. AAV9 carrying CAG promoter driving EGFP was administered to mice by various routes. EGFP and DAPI
fluorescence was analyzed in the following organs: (A) inner ear, images were taken 13 days after vector delivery byintratympanic injection to the left ear; (B) hippocampus and spinal cord anterior horn, images were taken 10 days after
vector delivery by facial vein injection; (C) retina, liver, pancreas, and skeletal muscle, images were taken 12 days after
vector delivery by tail vain injection.

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Figure 12. AAV1 carrying CMV promoter driving EGFP was administered to mice by tail vein injection. EGFP and DAPI
fluorescence was analyzed in lung, liver, pancreas, and skeletal muscle. Images were taken 12 days after injection.
A

B

Figure 13. (A) AAV8 carrying ProA1 promoter driving mCherry or (B) AAV8 carrying hRHO promoter driving EGFP was
administered to mice by subretinal injection. mCherry, EGFP, and DAPI fluorescence was analyzed in retina. Images weretaken 20 days after injection.

Figure 14. Recombinant AAV9 particles (~1.5X1010 GC/mouse) were intravenously administered to three neonatal mice
(two male and one female) within 48h after birth. AAV9 biodistribution in several organs was quantified using qPCR six
weeks after the injection.
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AAV Knowledge BaseAAV Serotypes and Tissue Tropism
Many strains of AAV have been identified in nature. They are divided into different serotypes based on different
antigenicity of the capsid protein on the viral surface. Different serotypes can render the virus with different tissue tropism(i.e. tissue specificity of infection). When our AAV vectors are packaged into the virus, different serotypes can be conferredto the virus by using different capsid proteins for the packaging. The table below lists the tissue specificity conferred bydifferent AAV serotypes.
Tissue type

Recommended AAV serotypes

Smooth muscle

AAV1, AAV2, AAV3, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10

Skeletal muscle

AAV1, AAV9CNS

AAV1, AAV2, AAV4, AAV5, AAV7, AAV8, AAV9, AAVrh10, AAV-PHP.eB

PNS

AAV-PHP.SBrain

AAV1, AAV2, AAV5, AAV7, AAV8, AAV-DJ/8

RetinaAAV1, AAV2, AAV4, AAV5, AAV7, AAV8, AAV9, AAVrh10, AAV2-QuadYF, AAV2.7m8

Inner ear

AAV1, AAV2, AAV6.2, AAV8, AAV9, AAV2.7m8

Lung

AAV1, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV9, AAVrh10

Liver

AAV1, AAV2, AAV3, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAVrh10, AAV-DJ, AAV-DJ/8

Pancreas

AAV1, AAV2, AAV6, AAV8, AAV9, AAVrh10

Heart

AAV1,AAV4, AAV5, AAV6, AAV8, AAV9, AAVrh10, AAV-DJ

KidneyAAV2, AAV4, AAV8, AAV9, AAVrh10, AAV-DJ, AAV-DJ/8

Adipose

AAV6, AAV8, AAV9Testes

AAV2, AAV9

SpleenAAV-DJ, AAV-DJ/8

Spinal nerves

AAV2-retro

Endothelial cells

AAV2-QuadYF▶▷

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Protocols for AAV In Vitro and In Vivo Applications
Storage and Handling
1. VectorBuilder's non-ultra-purified AAV is recommended for in vitro applications. Our non-ultra-purified AAV is stored ina Tris-based buffer. VectorBuilder’s ultra-purified AAV is recommended for in vivo applications. Our ultra-purified AAV
is stored in a PBS-based buffer.
2. After thawing, AAV can be dispensed into smaller aliquots according to the quantity used in your experiment and thenrefrozen. If you need to dilute the virus, you may use PBS, but do so ONLY immediately prior to use.
3. Upon receiving, AAV should be stored at -80°C for long term storage (stable for at least 1 year), or -20°C for short term
storage (stable for 2~3 weeks).
4. Thaw the vial of AAV on ice prior to use and keep it on ice during the experiment. Thawed AAV can be stored at 4°C
for 1-2 weeks without significant loss of biological activity.
Note: Do not freeze and thaw your AAV sample multiple times. AAV can be frozen and thawed several
times with minimal loss of activity, but it is better to avoid this.

AAV In Vitro Transduction
Transduction of Targeted Cells
AAV transduction is cell type dependent. Some cell types exhibit low transduction efficiency, while others transduce very
readily. When designing AAV transduction experiments, it is recommended to use different serotypes with a reporter
vector such an AAV expressing EGFP (e.g. VectroBuilder's Serotype Testing Panel) to determine the optimal serotype
for transduction of your tissue or cell culture. Start transducing the cells at a multiplicity of infection (MOI) between 1x104
and 1x106 genome copies (GC) per cell if the cells are readily transducible. With some cell lines a higher MOI might beneeded, look for the highest transduction with minimal cell death. For some cell lines, high transduction levels cannot be
achieved.

Protocol for Transducing Mammalian Cell Lines
1. Day before transduction (Day 0)
Plate target cells in appropriate medium so that they will be 30-50% confluent at the time of transduction. Incubate 1820 hours at 37°C in a humidified 5% CO2 incubator. For example, when using HEK293T cells, we usually plate
3x105 cells per well in a 6-well plate.
2. Day of transduction (Day 1)
Thaw virus on ice. Take the appropriate amount of virus as needed to achieve the desired MOI, place in an
appropriate amount of medium, and mix gently (but do not vortex). To maximize transduction efficiency, use theminimum amount of medium necessary to cover the surface of the plate. For example, when infection is performedin 6-well plates, we use 1 ml of medium per well.
Aspirate old medium from target cells, then add the virus-containing medium onto the cells.
Swirl the plate gently to mix and cover the cells. Incubate at 37°C in a humidified 5% CO2 incubator overnight.
Note: If you are concerned that exposure to the viral supernatant may adversely affect the target cells,
limit the transduction to 6-8 hours.
3. Day 2
Remove the virus-containing medium and replace with fresh complete culture medium. Incubate at 37°C in ahumidified 5% CO2 incubator overnight.

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4. Day 3 and onward
Analyze gene expression at desired time points following viral infection. In general, detectable levels of your geneproduct should be evident 24-48 hours after transduction.
Note: In actively dividing cells (i.e. doubling time of approximately 24 hours), transgene expression is
generally detectable within 24 hours of transduction, with maximal expression observed at 48-96 hours (2-4days) post transduction. Expression levels generally start to decline 5 days post-transduction. In cell linesthat exhibit longer doubling times or non-dividing cell lines, high levels of transgene expression normallypersists for a longer time. If you are transducing AAV into your mammalian cell line for the first time, we
recommend performing a time course study to determine the optimal temporal conditions for expressionof your transgene.
Anticipated Results
An example of successful transduction is shown below:

Figure 15. EGFP expressing AAV2 was used to transduce HEK293T cells at MOI 10,000 using the protocol in thisdocument. Images were taken at 100X. Left: bright field; right: EGFP.

AAV In Vivo Transduction
Protocol for Mouse Tail Vein Injection
Intravenous (IV) injection via the lateral tail vein is an efficient means of transducing mouse tissues with AAV. Tail vein
injections are preferred for their simplicity, as they do not require surgery or anesthesia. Other injection sites include
the portal and jugular veins, but these require surgery. Tail vein injections are more efficient at delivering AAV to tissues
compared to portal vein injections.

Lateral vein

Vertebra
Ventral artery

Figure 16. Diagram of a transverse sectional view of mouse tail lateral veins and ventral artery.

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1. Physical restraint
Mice can be physically restrained using a commercially available restraining device. Gently introduce the mouse to be
injected into restrainer making sure the tail is exposed for further manipulations.
2. Vasodilation
To induce peripheral vasodilation, warm the mouse by either dipping the tail in warm water (43°C) or placing theanimal near a heat lamp for 5 to 10 minutes prior to injection.
Note: When using a heat lamp, place the lamp about 15 to 25 cm above the cage and observe the mice for5 minutes. Stop heating once the mice huddle together in one corner of the cage.
3. Injection
Making sure the mouse is placed in the restraining device in a stern position, slightly rotate the tail to visualize thelateral vein. Disinfect the injection site with 70% ethanol and wipe off excess alcohol with gauze pad.
Using a 0.5 ml syringe with a 27 G needle, draw up at least 0.1 ml of ultra-purified AAV particles. Carefully remove
air from the needle.
Grasp the distal part of the tail and twist it slightly to the lateral vein’s side (Figure 16). Insert the needle at a shallow
angle (as the tail vein is relatively close to the surface) and inject 0.1 ml of diluted AAV into the vein. The injection
should proceed smoothly without any resistance if the needle is correctly positioned, and the vein will transiently
shift to a clear color. Stop injecting if any local swelling is observed, as in that case the virus is being injected into
the tail tissue, not the vein.
Note: Start your injection from the lower portion of the tail about 1/3 from the tip. This allows you to moveup the tail if the previous injection was unsuccessful.
Remove the needle and apply pressure with a clean gauze until bleeding stops.
Return mouse to original cage.
4. Validation
AAV particles are predominantly sequestered by tissues following intravenous injection. To determine the efficiency
of in vivo transduction, mice can be sacrificed at day 7 after virus infusion and target tissue(s) can be processed foranalysis.
Anticipated Results
An example of successful in vivo transduction is shown below:

Figure 17. Mice were injected with the ultra-purified
EGFP expressing AAV9 at a dose of 1x1013 GC/kg using
the protocol of IV injection in this document. 10 days
after the injection, the livers from injected animals weredissected for analysis. Images were taken by a confocal
microscope. Green: EGFP. Blue: DAPI.

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Protocol for Mouse Intramuscular (IM) Injection
1.

Restrain the animal. Assistance for restraint or use of anesthesia is helpful.

2.

Secure the rear foot nearest you.

3.

Injections can be made into the thigh muscles caudal to (behind) the femur (semitendinosus - semimembranosus) or
those cranial to (in front of) the femur (quadriceps).

4.

Isolate the muscle mass to be used with your fingers.

5.

Insert the needle to avoid the sciatic nerve. Direct the needle caudally (toward tail) if using the caudal thigh muscles
or cranially (toward head) if using the quadriceps muscle. 25-30 G needle is needed.

6.

Aspirate to ensure that you have not entered a blood vessel. If blood is aspirated, redirect the needle and aspirate
again. For mouse, the injection volume generally is 25 ul. If no blood is aspirated, slowly inject the material.

7.

Withdraw needle. If there is any bleeding at the injection site, apply pressure to the site for approximately 1 minuteuntil bleeding stops.

8.

Return the animal to its cage and observe the animal for any signs of pain or distress.

Protocol for Mouse Intraperitoneal (IP) Injection
Intraperitoneal injection is a simple, rapid and non-invasive AAV delivery method. Glybera, a commercial AAV genetherapy drug for lipoprotein esterase deficiency, was administered by intraperitoneal injection. The protocol of
intraperitoneal injection is as follows:
1. Draw up the amount of AAV virus to be administered into the syringe and needle (the maximum volume per
intraperitoneal injection is 10 ml/kg).
2. Restrain the mouse in an appropriate manner: “scruff” the mouse (gather the skin) with your non-dominant hand andhold the animal with its head slightly lower than the abdomen.
3. Identify anatomical landmarks to locate the appropriate IP injection site. Typically, the injection site is the animal’s
lower right quadrant of the abdomen to reduce the chances of damage to the urinary bladder, cecum and otherabdominal organs.
4. Disinfect the injection site with 70% ethanol. Insert needle at a 30-40ᵒ angle to the horizontal plane of the animal withthe bevel facing up.
5. Direct the needle towards the head of the animal. With a 25-27 G needle, approximately 1/2 the needle length isinserted.
6. When the needle inserts into the abdomen, the resistance from tissue dissipates. Pull back on the plunger to ensure
negative pressure and that nothing is aspirated before injecting.
Note: If aspirate is green material, likely the bowel has been punctured. If aspirate is a yellow liquid, likely
the bladder has been punctured. If aspirate contains blood, urine or gastrointestinal contents, the syringe
and needle must be discarded. Restart the injection using new syringe and needle. Record any possible
complications for the animal.
7. Proceed with the injection. The injection can be completed in 1-2 seconds. Do not allow the needle to move further inor out of the abdomen.
8. When the injection is completed, remove needle from the abdomen at the same angle as it was inserted.
9. Return the animal to its cage and observe for any complications.

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VectorBuilder
Protocol for Mouse Intra-Cerebroventricular Injection
Intra-cerebroventricular (ICV) injection is a procedure to deliver the ultra-purified AAV into the left and right lateral
ventricles, followed by its diffusion into the central nervous system (CNS). The brain ventricles and CNS containcerebrospinal fluid (CSF) which is produced in the two lateral ventricles. The CSF starts circulating from the lateral
ventricles and eventually flows into the subarachnoid space. Since the subarachnoid space covers the entire brain, spinal
cord, and sacrum, the ICV injection can be used to introduce the virus sample or other therapeutic agents to the CNS
without being obstructed by the blood-brain barrier. To determine the optimal injection dose for your study, you could
conduct pilot testing in your animals by using reporter AAV, such as EGFP-expressing AAV.
1. Preparation of operating environment
Wipe the operating table with 70% ethanol.
Cool down the brain stereotaxic device by adding 5 ml of absolute ethanol in the groove of the operating table.
Consistently add dry ice to the absolute ethanol to stabilize the temperature of the operating table at about 0°C. At
the same time, pre-warm a small heating pad.
Note: To avoid the death of mice, the temperature of the operating environment should not be below 0°C.
2. Preparation of injection syringe
Take a 33-34 G needle and sleeve it tightly with a PE tube, and then tighten the other side of the PE tube witha 10 ul syringe.
Draw up 5 ul of ultra-purified AAV into the needle, and carefully remove air from the needle.
Restrain the syringe and needle in the stereotaxic device.
3. Anesthesia
Anesthetize the mouse to unconsciousness by gently placing the mouse on ice for 4 minutes.
4. Physical restraint
After anesthesia, the head of the mouse can be physically restrained on the stereotaxic device gently.
5. Injection
5.1. Left ventricle
Disinfect the mouse head with 70% ethanol and wipe off excess alcohol with gauze pad.
Place the needle just above the lambda structure of the mouse (Figure 18). Record the indexes of x and y axis on
the vernier scale.

Figure 18. Diagram of the top view of mouse lambda structure and injection sites.

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Place the needle just above the left ventricle (x + 1.2 mm, y + 1.6 mm). Then pierce the mouse skull with theneedle by adjusting the z axis.
Immediately after piercing the skull, withdraw the needle out of the mouse slowly.
Note: The z axis position of the mouse skull can be slightly shifted when being pierced due to thepressure from the needle. Withdrawing the needle immediately after piercing the skull can help the skull
move back to the original position
Carefully put the needle back into the skull and move the needle down until the needle tip just touches the brain.
Record the index of z axis (z1) on the vernier scale.
Lower the needle slowly for 2.5~3 mm so that the needle tip penetrates the center of the left ventricle. The indexes
on the vernier scale should be x + 1.2 mm, y + 1.6 mm, z - 2.5~3 mm.
Push the syringe slowly and inject 2.5 ul virus sample into the left ventricle. To prevent the AAV leaking from thebrain, wait for 1 minute or even longer after injecting the virus sample.
Slowly adjust the z axis to withdraw the needle from the mouse. This process should take about 2 minutes.
Withdrawing the needle too fast may lead to AAV leakage.
5.2. Right ventricle
Place the needle above the right ventricle (x - 1.2 mm, y + 1.6 mm). Then pierce the mouse skull with the needle
by adjusting the z-axis.
Immediately after piercing the skull, withdraw the needle out of the mouse slowly.
Note: The z axis position of the mouse skull can be slightly shifted when being pierced due to thepressure from the needle. Withdrawing the needle immediately after piercing the skull can help the skull
move back to the original position.
Carefully put the needle back into the skull and move the needle down until the needle tip just touches the brain.
Record the index of z axis (z2) on the vernier scale.
Lower the needle slowly for 2.5~3 mm so that the needle tip penetrates the center of the right ventricle. The
indexes on the vernier scale should be x - 1.2 mm, y + 1.6 mm, z - 2.5~3 mm.
Push the syringe slowly and inject 2.5 ul virus sample to the right ventricle. To prevent the AAV leaking from thebrain, wait for 1 minute or even longer after injecting the virus sample.
Slowly adjust the z axis to withdraw the needle from the mouse. This process should take about 2 minutes.
Withdrawing the needle too fast may lead to the AAV leakage.
Note: To avoid the death of mice, mice should not be kept at low temperature for over 30 minutes.
6. Recovery
Right after the injection, place the mouse on the pre-warmed heating pad until the body temperature and movement
are restored.
7. Observation
Return the mouse to the cage with the mother mouse. Monitor the condition of the mice until starting further
experiments.

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Anticipated Results
An example of successful in vivo transduction is shown below:

Figure 19. Mice were injected with the ultra-purified
EGFP expressing AAV9 at a dose of 1x10 13 GC/kg
using the protocol of ICV in this document. 10 days
after the injection, the brains from injected animals
were dissected for analyses. Images of hippocampus
were taken by a confocal microscope. Green: EGFP.
Blue: DAPI.

Comparisons of AAV Delivery Methods In Vivo
CNS Gene Delivery
Delivery method

Advantages

Disadvantages

Direct injection

Efficiently transduce a small area

Invasive

Allows broad distribution of the vector acrossthe CNS

Limited penetration into the brain
parenchyma

Intracisternal injection
Intraventricular injection
Intrathecal injection

Distributes the vector through the CSF, but
mostly targets the spinal cord and dorsal root N/Aganglia (DRGs)

Intramuscular injection

Targets motor and sensory neurons via
retrograde transport

N/A

Intravascular administration

Widespread transduction throughout the CNS

Requires high vector doses and peripheral
exposure to the vector

Liver Gene Delivery
Delivery method

Advantages

Disadvantages

Liver lobes injection
Direct Liver
DeliveryPortal vein injection
Splenic capsuleinjection

Mouse
High Transduction
efficiency

Highly invasive, require minor to
major surgery, risk of operativeand postoperative complications incoagulation-deficient animal models

Hepatic artery injection

Peripheral
Vector Delivery

Intraperitoneal injection
Intravenous injection

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SpeciesRat, Dog,
NHP
Mouse
NHP

Less effective in large animal modeldue to the increased volume of theLess invasive, easy
peritoneal cavity
to perform
Limited injection volume compared to
intraperitoneal injection

Mouse
Mouse, Dog,
NHP, Humanwww.vectorbuilder.com

Cardiac Gene Delivery
Delivery method

Advantages

Disadvantages

Peripheral intravenous
injection• Non-invasive• Simple• Dilution issues• High expression in nontargeted organs• Low myocardial transduction rate

Antegrade coronary injection• Clinically relevant• Less invasive• Lower transduction efficiency• Limited delivery to the ischemic area

Retrograde coronary
injection

Higher transduction efficiency

Requires blockage of antegrade flow

Aortic clamping

Higher transduction efficiency

Highly invasive

Direct intramyocardial
injection• No endothelial barrier• No first pass-effect of liver and spleen• Reduced T-cell triggered inflammation• Independent of neutralizing antibodies• Endomyocardial approach being lessinvasive• Surgical delivery being the most invasive
delivery method• Damage along the needle track• Region of interest might be notcompletely covered as a result of a
restricted area of injection• Limitation of vector delivery because ofleakage at the injection site

• Minimally invasive• High concentration and long duration ofvector possible• Little systemic vector distribution• Transduction mostly in pericardial cells
and only very limited in myocardial cells• Possible leakage of injected agent
into thorax cavity with transduction of
nontargeted organs• In future clinical use, difficult toapply after pericarditis or previous
cardiovascular surgery

Pericardial injection

Muscle Gene Delivery
Delivery method
Intramuscular injection

Localized
DeliveryTransvenous limb
perfusion
Intracoronary infusion
Recirculating delivery

Advantages

Disadvantages• Easy to administer
High transduction
in muscle
Not able to transduce large• Low off-target
muscle mass
vector delivery• Low immune
response

Transendocardial injection
Intravascular injection
Systemic
deliveryIntraperitoneal injection

Diseases
A1ATD, LGMD2D,
Pompe disease, etc.
DMD
Heart failure, myocardial
infarction and LGMDHeart failure
Heart failure and DMD

Widespreadtransduction oflarge muscle mass,potentially themuscles of wholebody• Off-target vector delivery
Immune responses• Need of high doses ofvector• Limited to certain rAAV
serotypes

FSHD, myocardial
infarction
DMD, LGMD▶▷

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Sources
Wang D, Zhong L, Nahid MA, Gao G. The potential of adeno-associated viral vectors for gene delivery to muscle
tissue. Expert Opin Drug Deliv. 2014 Mar;11(3):345-364. doi: 10.1517/17425247.2014.871258.

Moscoso CG, Steer CJ. The Evolution of Gene Therapy in the Treatment of Metabolic Liver Diseases. Genes(Basel). 2020 Aug 10;11(8):915. doi: 10.3390/genes11080915.

Palaschak B, Herzog RW, Markusic DM. AAV-Mediated Gene Delivery to the Liver: Overview of Current
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User Citations
snRNA-seq reveals a subpopulation of adipocytes that regulates thermogenesis
Nature. 2021. doi: 10.1038/s41586-020-2856-x. IF: 69.054
Cholinergic neurons constitutively engage the ISR for dopamine modulation and skill learning in mice
Science. 2021. doi: 10.1126/science.abe1931. IF: 63.714
Lipolysis drives expression of the constitutively active receptor GPRR3 to induce adipose thermogenesis
Cell. 2021. doi: 10.1016/j.cell.2021.04.037. IF: 66.850
De novo formation of the biliary system by TGFβ-mediated hepatocyte transdifferentiation
Nature. 2018. doi: 10.1038/s41586-018-0075-5. IF: 69.054
Positron emission tomography reporter gene strategy for use in the central nervous system
Proc Natl Acad Sci U S A. 2019. doi: 10.1073/pnas.1901645116. IF:12.779
PIAS2-mediated blockade of IFN-β signaling: a basis for sporadic Parkinson disease dementia
Mol Psychiatry. 2021. doi: 10.1038/s41380-021-01207-w. IF:13.437
TREK-1 and TRAAK are principal K+ channels at the nodes of Ranvier for rapid action potential conduction on
mammalian myelinated afferent nerves
Neuron. 2019. doi: 10.1016/j.neuron.2019.08.042. IF: 18.688
Leveraging gene therapy to achieve long-term continuous or controllable expression of biotherapeutics
Sci Adv. 2022. doi: 10.1126/sciadv.abm1890. IF: 14.957
Loss of LAMP5 interneurons drives neuronal network dysfunction in Alzheimer’s disease
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Endoplasmic reticulum chaperone genes encode effectors of long-term memory
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Single-cell transcriptomics of suprachiasmatic nuclei reveal a Prokineticin-driven circadian network
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Olanzapine: A potent agonist at the hM4D (Gi) DREADD amenable to clinical translation of chemogenetics
Sci Adv. 2019. doi: 10.1126/sciadv.aaw1567. IF: 14.957
Cardiomyocyte homeodomain-interacting protein kinase 2 maintains basal cardiac function via extracellularsignal-regulated kinase signaling
Circulation. 2019. doi: 10.1161/CIRCULATIONAHA.119.040740. IF: 39.918▶▷

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Endocannabinoid signaling collapse mediates stress-induced amygdalo-cortical strengthening
Neuron. 2020. doi: 10.1016/j.neuron.2019.12.024. IF: 18.688
Neuronal metabolic rewiring promotes resilience to neurodegeneration caused by mitochondrial
dysfunction
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O-GlcNAcylated p53 in the liver modulates hepatic glucose production
Nat Commun. 2021. doi: 10.1038/s41467-021-25390-0. IF: 17.694
Astrocyte-targeted gene delivery of interleukin 2 specifically increases brain-resident regulatory T cell numbers
and protects against pathological neuroinflammation
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Ablation of the stress protease OMA1 protects against heart failure in mice
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Hepatic IRF3 fuels dysglycemia in obesity through direct regulation of Ppp2r1b
Sci Transl Med. 2022. doi: 10.1126/scitranslmed.abh3831. IF: 19.319
Fasting-induced FGF21 signaling activates hepatic autophagy and lipid degradation via
JMJD3 histone demethylase
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ALOX15-launhed PUFA-phospholipids peroxidation increases the sysceptibility of ferroptosis in ischemiainduced myocardial damage
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FGF1 and insulin control lipolysis by convergent pathways
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