Gene Delivery to the Nervous System
Authors: Manfred Schubert, Xandra Breakefield, Howard Federoff, Robert M Frederickson, Pedro R Lowenstein
Journal: Molecular Therapy (2008)

The National Institute of Neurological Disorders and Stroke (NINDS) sponsored a workshop on gene delivery to the nervous system, which took place on 12–13 November 2007 in Washington, DC. The purpose of the workshop was to convene neuroscientists, molecular virologists/vectorologists, and surgical neurologists to assess the state of the art of gene therapy for neurologic diseases and brain tumors and to address the challenges for advancing promising preclinical studies to the clinic. Since the last NINDS workshop on gene therapy, in 2000 (ref. 1Finkelstein R Baughman RW Steele FR Harvesting the neural gene therapy fruit.Mol Ther. 2001; 3: 3-7Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar), which focused on Parkinson's disease (PD) and lysosomal storage diseases (LSDs)—perceived to be the lowest-hanging neural gene therapy fruits—much progress has been achieved on several fronts. Several phase I and II clinical trials have been completed or are currently in progress, including trials for PD, LSDs, Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), epilepsy, peripheral neuropathy and pain, multiple sclerosis, and muscular dystrophy. Since the first clinical gene therapy trial, in 1989, more than 1,300 clinical trials have been carried out or are in progress worldwide. Of these, approximately 100 trials were directed either against the neurologic diseases listed above (http://www.wiley.co.uk/genetherapy/clinical)2Lowenstein PR Kroeger KM Castro MG Friedman TF et al.Gene therapy for central nervous system disorders.in: Rosenburg RN DiMauro S Paulson HL Ptácek L Nestler EJ The Molecular and Genetic Basis of Neurologic and Psychiatric Disease. 4th edn. Lippincott Williams & Wilkins, Philadelphia2007: 87-103Google Scholar or brain tumors.3Pulkkanen KJ Yla-Herttuala S Gene therapy for malignant glioma: current clinical status.Mol Ther. 2005; 12: 585-598Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar These encouraging numbers are evidence of a growing and maturing field. The shared vision of permanently correcting a neurologic defect by the delivery of a gene attracted most of the workshop participants to this research. This quest requires a detailed understanding of the dynamics of the disease, the therapeutic alternatives and strategies and their potential side effects, and the mechanisms and routes for safe in vivo gene delivery, all of which represent enormous and unprecedented challenges. During the workshop there was great excitement about the promise for gene delivery to the nervous system on several counts. First, there was a collective realization that a variety of gene delivery methods now provide strong scientific underpinnings for the design and implementation of powerful therapeutic strategies that can tackle focal as well as global disease; further, as a result of the field's maturity, the enthusiasm is now combined with a realistic assessment of the challenges to be overcome. Second, based on many gene therapy trials for a wide range of diseases, it appears that the nervous system may offer practical advantages for intervention, including a reduced immune response to vectors and transgenes in the brain parenchyma as compared with other peripheral sites of injection, focal targets that require limited gene delivery within the brain (e.g., in PD), the presence of only a few normal replicating cells that might be transformed by vector integration into their genome, and the reduced ability of vectors injected into the brain to access germline tissues as compared with peripheral injections. Third, multiple strategies and clinical trials are being implemented for several diseases of the adult nervous system, including PD4Alexander BL Ali RR Alton EW Bainbridge JW Braun S Cheng SH et al.Progress and prospects. 1. Gene therapy clinical trials.Gene Ther. 2007; 14: 1439-1447Crossref PubMed Scopus (86) Google Scholar and glioblastoma multiforme,3Pulkkanen KJ Yla-Herttuala S Gene therapy for malignant glioma: current clinical status.Mol Ther. 2005; 12: 585-598Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar with the hope that at least one will provide therapeutic benefit in these high-profile and relatively common conditions. Many studies have been published regarding delivery of genes to the nervous system. Strength of transgene expression and distribution throughout the brain are dependent on vector backbone, promoters used, and site, volume, and rate of injection.5Thomas CE Schiedner G Kochanek S Castro MG Lowenstein PR Peripheral infection with adenovirus causes unexpected long-term brain inflammation in animals injected intracranially with first-generation, but not with high-capacity, adenovirus vectors: toward realistic long-term neurological gene therapy for chronic diseases.Proc Natl Acad Sci USA. 2000; 97: 7482-7487Crossref PubMed Scopus (234) Google Scholar,6Lowenstein PR Mandel RJ Xiong WD Kroeger K Castro MG Immune responses to adenovirus and adeno-associated vectors used for gene therapy of brain diseases: the role of immunological synapses in understanding the cell biology of neuroimmune interactions.Curr Gene Ther. 2007; 7: 347-360Crossref PubMed Scopus (130) Google Scholar Different vectors, as well as delivery in various stages of brain development, have been used to achieve either focal or diffuse delivery throughout larger areas of the brain. However, diffuse vector delivery covering large areas of the brain remains an important challenge. This will be especially important in the treatment of diseases such as AD, which affects the structure of most of the neocortex and hippocampus. One approach that has been proposed to homogeneously cover larger areas of the brain is convection-enhanced delivery.7Fiandaca MS Forsayeth JR Dickinson PJ Bankiewicz KS Image-guided convection-enhanced delivery platform in the treatment of neurological diseases.Neurotherapeutics. 2008; 5: 123-127Crossref PubMed Scopus (114) Google Scholar This method, using programmable pumps and specifically designed injection catheters, has been used to make adeno-associated virus type 2 (AAV-2) vector delivery to the nonhuman primate striatum much more even, efficient, and reproducible than single injections of smaller volumes (as reported at the workshop by Drs. Bankiewicz, Starr, and Lonser—see accompanying list of participants). The convection-enhanced-delivery approach can achieve comprehensive delivery to the striatum/globus pallidus/subthalamic nuclei using AAV-2 vectors. Gene delivery can also be imaged in real time using co-injections of liposomes containing gadolinium (Bankiewicz) or gadolinium bound to albumin (Lonser). An alternative approach to accomplishing widespread delivery throughout the nervous system was described by Dr. Wolfe, who achieved essentially global gene product delivery expressed from AAV vectors in animal models of LSDs. Upon viral entry, the vector cores are retrogradely transported within neurons through their extensive processes depending on the vector serotype and the injection site. This delivery strategy consists of injecting vectors into nuclei from which originate long diffuse neuronal tracts throughout much of the forebrain (e.g., the locus coeruleus). In addition, the vector-encoded gene product moves within neurons through axonal transport, is released at synaptic terminals, and is then taken up by cells located at the target areas. In this way the replacement enzyme ends up being transferred from cell to cell. One microliter of vector suspension was sufficient for nearly global delivery to mouse brain, representing 1–2 ml on the scale of the human brain. This strategy replaces the invasiveness of multiple vector injections by using the neuronal network itself for delivery of the transgene and its encoded product.8Cearley CN Wolfe JH A single injection of an adeno-associated virus vector into nuclei with divergent connections results in widespread vector distribution in the brain and global correction of a neurogenetic disease.J Neurosci. 2007; 27: 9928-9940Crossref PubMed Scopus (158) Google Scholar Direct delivery of virus vectors into the brain ventricles has also been attempted in order to achieve diffuse distribution of transgene products. Although this may play a role during in utero gene delivery or very early postnatally (when the immune system is still immature), once the immune system has developed, this is unlikely to be of clinical utility. Data presented by Dr. Lowenstein indicate that intraventricular injection of adenoviral vectors can elicit a strong T-cell response, similar to those stimulated by direct injection of adenoviral vectors systemically. Such responses can block transgene expression in the central nervous system (CNS); some of the loss of transgene expression is due to the immune-mediated killing of vector-transduced brain cells following local cell recruitment induced by inflammatory responses. In contrast, intraparenchymal injections of viral vectors do not stimulate a systemic immune response, most likely because of the absence of viral vector diffusion and a lack of immune-cell recruitment. This finding has important implications for the selection of target sites for vector injection because it can affect the safety and efficiency of gene delivery to the CNS.9Barcia C Thomas CE Curtin JF King GD Wawrowsky K Candolfi M et al.In vivo mature immunological synapses forming SMACs mediate clearance of virally infected astrocytes from the brain.J Exp Med. 2006; 203: 2095-2107Crossref PubMed Scopus (80) Google Scholar Delivery of therapeutic transgenes or even vectors from peripheral tissues into neurons remains an important step toward delivery to areas of the brain such as the spinal cord.8Cearley CN Wolfe JH A single injection of an adeno-associated virus vector into nuclei with divergent connections results in widespread vector distribution in the brain and global correction of a neurogenetic disease.J Neurosci. 2007; 27: 9928-9940Crossref PubMed Scopus (158) Google Scholar Some success was reported using monoclonal antibodies to the transferrin receptor or insulin receptor conjugated to liposomes to cross the blood–brain barrier (Boado), via uptake through the neuromuscular junctions using the tetanus toxin C (TTC) fragment conjugated to either superoxide dismutase (SOD) (TTC:SOD; Fishman) or β-glucuronidase (GUSB:TTC; Wolfe), and by intraventricular injection of glial cell line–derived neurotrophic factor (GDNF:TTC; Fishman). Nevertheless, there are serious immunologic considerations to take into account when using TTC, because tetanus toxin is highly immunogenic and the majority of the US population has been immunized against it. To increase targeted delivery from the periphery using viral vectors, Dr. Boulis finds increased neuronal uptake of AAV-1 by inserting into the viral capsid protein a short tet1 peptide, specifically selected from a phage display library to recognize the tetanus toxin receptor. Peptide insertion resulted in retrograde transport, but peptide presentation still needs to be optimized. Neurotropic lentiviral vectors—equine infectious anemia virus (EIAV) and HIV-1—pseudotyped with rabies glycoprotein were considered potential alternatives for targeting neurons,10Jakobsson J Lundberg C Lentiviral vectors for use in the central nervous system.Mol Ther. 2006; 13: 484-493Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar,11Wong LF Goodhead L Prat C Mitrophanous KA Kingsman SM Mazarakis ND Lentivirus-mediated gene transfer to the central nervous system: therapeutic and research applications.Hum Gene Ther. 2006; 17: 1-9Crossref PubMed Scopus (156) Google Scholar including motor neurons following intramuscular injections12Mazarakis ND Azzouz M Rohll JB Ellard FM Wilkes FJ Olsen AL et al.Rabies virus glycoprotein pseudotyping of lentiviral vectors enables retrograde axonal transport and access to the nervous system after peripheral delivery.Hum Mol Genet. 2001; 10: 2109-2121Crossref PubMed Scopus (378) Google Scholar,13Mentis GZ Gravell M Hamilton R Shneider NA O'Donovan MJ Schubert M Transduction of motor neurons and muscle fibers by intramuscular injection of HIV-1-based vectors pseudotyped with select rabies virus glycoproteins.J Neurosci Methods. 2006; 157: 208-217Crossref PubMed Scopus (47) Google Scholar (Mazarakis and Schubert). AAV vectors expressing insulin growth factor-1 for the potential treatment of ALS have also been delivered to the spinal cord via muscles, presumably through transport by way of motor and sensory neurons.14Kaspar BK Lladó J Sherkat N Rothstein JD Gage FH Retrograde viral delivery of IGF-1 prolongs survival in a mouse ALS model.Science. 2003; 301: 839-842Crossref PubMed Scopus (750) Google Scholar Perceived limitations in the use of viral vectors in terms of potential toxicity have stimulated the development of “synthetic” or “nonviral” vectors. Nonviral vectors have come of age, with various clinical trials either finished or in preparation.15Herweijer H Wolff JA Gene therapy progress and prospects: hydrodynamic gene delivery.Gene Ther. 2007; 14: 99-107Crossref PubMed Scopus (160) Google Scholar However, none of these has yet been used in the CNS or for the clinical treatment of brain diseases. Data presented by Dr. Wolff support the advantage of rational design in generating synthetic vectors for nucleic acid delivery. Chemical components (polyethylene glycol) were used to achieve serum stability and receptor targeting, joined to a polymer with pH-cleavable bonds, which allow release from the endosomal compartment for delivery of genetic information—e.g., short interfering RNAs—to the cytoplasm. This strategy is currently very effective at delivery to the liver; studies in the brain are ongoing. Dr. Pun described ways to increase gene delivery to neurons, including targeting using the tet1 peptide (developed by Dr. Boulis), which enters via the tetanus toxin receptor. Her strategy combines peptides that target retrograde motors with peptides that are lytic so as to achieve nucleic acid release from endosomes into the cytoplasm, which is critical for activity. An issue not directly addressed at the workshop was that, as compared with viral vectors, the relatively short duration of transgene expression by most synthetic vectors must be improved if they are to be useful for many therapeutic applications—especially if they require invasive delivery procedures. For clinical indications that require only short-term expression (e.g., during early post-stroke interventions), transitory gene expression may be less of a limitation. Relative to other diseases, brain tumors are rapidly fatal, killing patients in less than 2 years after diagnosis. Although the main tumor mass is usually localized at diagnosis, even complete resection of adult glioblastoma multiforme fails to cure affected patients because of invasive microfoci. Thus, novel alternatives are being discussed. Despite recent improvement in the treatment of such tumors using novel chemotherapeutic approaches, none of these is likely to work in all patients. At the workshop, three very promising and potentially complementary strategies for gene therapy of brain tumors were outlined. Dr. Castro described an enhancement of immune rejection of tumors by using the adenovirus–thymidine kinase/ganciclovir (Ad-TK/GCV) vector system for delivery, combined with Ad-Flt3L. Flt3L has been shown to attract dendritic cells to the brain; importantly, those immune cells stimulate a systemic, long-term, CD8+ T-cell-mediated immune response directly from the brain itself.16Ali S King GD Curtin JR Candolfi M Xiong W Liu C et al.Combined immunostimulation and conditional cytotoxic gene therapy provide long-term survival in a large glioma model.Cancer Res. 2005; 65: 7194-7204Crossref PubMed Scopus (100) Google Scholar The idea is that this strategy may prime the immune system to recognize glioma cells that have undergone new mutations, one of the main challenges in the treatment of glioblastoma multiforme. In another strategy, oncolytic adenoviral vectors with delta-24 mutation in E1A were used because they proliferate preferentially in Rb(–) tumors. Infection can be targeted to integrins, preferentially found on glioma cells via the RGD tripeptide. Subsequent death of the tumor cells apparently occurs through an autophagic pathway17Jiang H Gomez-Manzano C Aoki H Alonso MM Kondo S McCormick F et al.Examination of the therapeutic potential of Delta-24-RGD in brain tumor stem cells: role of autophagic cell death.J Natl Cancer Inst. 2007; 99: 1410-1414Crossref PubMed Scopus (239) Google Scholar (Fueyo). Dr. Chiocca also demonstrated that innate immune responses can reduce the efficiency of viral vectors in the brain. The therapeutic effect of oncolytic herpes simplex virus (HSV) vectors can be enhanced by co-administration of cyclophosphamide, which decreases on-site production of γ-interferon that would otherwise stimulate CD68+ and CD163+ lymphocyte infiltration, result in a curtailment of the spread of the oncolytic virus, and cause an enhanced toxic inflammatory response in the brain.18Fulci G Breymann L Gianni D Kurozomi K Rhee SS Yu J et al.Cyclophosphamide enhances glioma virotherapy by inhibiting innate immune responses.Proc Natl Acad Sci USA. 2006; 103: 12873-12878Crossref PubMed Scopus (289) Google Scholar Importantly, in the United States there have been phase I trials of Ad-TK and replication-competent adenoviruses, and others are expected to begin soon; moreover, the results of a large multicenter double-blind phase III trial recently completed in Europe are expected this summer. A phase I trial combining Ad-TK and Ad-Flt3L is expected to commence in late 2008 or early 2009. Late infantile neuronal ceroid lipofuscinosis (Batten disease). Treatment for the LSD known as late infantile neuronal ceroid lipofuscinosis using AAV-2-mediated gene delivery is in early-phase clinical stages; the field will need to await the results of longer follow-up of patients and larger clinical trials to deduce indices of therapeutic benefit. Current protocol modifications include switches to AAV-10 for increased time of transgene expression and earlier intervention in the course of the disease (Crystal).19Hackett NR Redmond DE Sondhi D Giannaris EL Vassallo E Stratton J et al.Safety of direct administration of AAV2(CU)hCLN2, a candidate treatment for the central nervous system manifestations of late infantile neuronal ceroid lipofuscinosis, to the brain of rats and nonhuman primates.Hum Gene Ther. 2005; 16: 1484-1503Crossref PubMed Scopus (41) Google Scholar Pain. Chronic neuropathic pain is the consequence of microglia and astrocyte activation in the spinal cord with release of proinflammatory cytokines. This response can be alleviated by the delivery of interleukin 10 using adenovirus, AAV, and nonviral vectors. The duration of interleukin 10 expression should be extended (Milligan). The groups of David Fink (University of Michigan) and Joe Glorioso (University of Pittsburgh) are also very close to implementing clinical trials of gene therapy for pain using HSV-1 vectors20Glorioso JC Fink DJ Herpes vector-mediated gene transfer in treatment of diseases of the nervous system.Annu Rev Microbiol. 2004; 58: 253-271Crossref PubMed Scopus (94) Google Scholar to deliver the endogenous morphine-like peptide precursor proenkephalin and/or other proteins such as interleukin 4, glutamic acid decarboxylase, tumor necrosis factor-α@ or GDNF, which can reduce pain. Neurodegenerations: ALS, AD, PD, and prion diseases. Delivery of insulin growth factor-1 in a mutant SOD model of motor neuron degeneration acts by reducing activation of astrocytes and microglia and prevents death of motor neurons in mouse models of ALS through activation of the Akt pathway (Kaspar).14Kaspar BK Lladó J Sherkat N Rothstein JD Gage FH Retrograde viral delivery of IGF-1 prolongs survival in a mouse ALS model.Science. 2003; 301: 839-842Crossref PubMed Scopus (750) Google Scholar Initial clinical trials using fibroblasts and AAV to deliver nerve growth factor in AD brains showed no adverse events with positive signs of increased glucose uptake and neuronal sprouting, which are signs of reversal of the metabolic decrease after gene delivery. A phase II clinical trial is being planned on AAV-mediated gene delivery of nerve growth factor in AD (Tuszynski).21Tuszynski MH Nerve growth factor gene therapy in Alzheimer disease.Alzheimer Dis Assoc Disord. 2007; 21: 179-189Crossref PubMed Scopus (97) Google Scholar Another strategic approach for AD is active vaccination against the amyloid-β (Aβ) peptide, which accumulates in amyloid plaques, initially using HSV-derived amplicon vectors. This approach allows for immune shaping in which a T-helper 2 (Th2) response driving therapeutic anti-Aβ antibodies was achieved instead of the Th1-type response, which results in cytotoxic T-cell response; the latter is believed to have caused aseptic meningoencephalitis in the Elan Pharmaceuticals AD peptide vaccination trial. This approach was followed by a prion model of neuronal degeneration using another immunologic strategy involving intracranial (thalamus/striatum) injections of AAV-2 vectors expressing a single-chain antibody, which blocks the conversion of the cellular prion protein (PrP) to scrapie PrPSc amyloids. This strategy conferred increased survival, reduced disease burden, and improved neurobehavioral functions in mice that were challenged with PrPSc (Federoff).22Federoff HJ Bowers WJ Immune shaping and the development of Alzheimer's disease vaccines.Sci Aging Knowledge Environ. 2005; 46: pe35Google Scholar Several strategies are being tested in phase I trials for PD. In one trial an effort was directed toward reducing neuronal loss by AAV delivery of the neurotrophic factor neurturin, which reacts with the same receptors that GDNF does and leads to an increase in 18F-dopa uptake in the brains of both aged monkeys and monkeys treated with MPTP (Kordower). There is no evidence of toxicity at the maximal tolerable dose in different animal models in phase II trials being conducted by Ceregene Inc. (Bartus). Another promising trophic effort is the delivery of the heat shock protein HSP104 and the PD gene protein parkin in mice overexpressing α-synuclein using a lentivirus vector (Zufferey). Interestingly, GDNF does not prevent α-synuclein toxicity. In some cases human PD is caused by elevated expression of α-synuclein. This indicates the need for multiple animal models to parallel the multiple known causes of PD in humans. Given that L-dopa is the common therapy for PD but loses its effectiveness over time, efforts are also underway to increase L-dopa conversion to dopamine by AAV delivery of aromatic amino acid decarboxylase to facilitate the conversion of L-dopa to dopamine (Bankiewicz)23Bankiewicz KS Forsayeth J Eberling JL Sanchez-Pernaute R Pivirotto P Bringas J et al.Long-term clinical improvement in MPTP-lesioned primates after gene therapy with AAV-hAADC.Mol Ther. 2006; 14: 564-570Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar or to increase de novo synthesis of dopamine using a multicistronic lentivirus vector based on EIAV that encodes the biosynthetic enzymes tyrosine hydroxylase, aromatic amino acid decarboxylase, and guanosine triphosphate cyclohydrolase (Mazarakis).11Wong LF Goodhead L Prat C Mitrophanous KA Kingsman SM Mazarakis ND Lentivirus-mediated gene transfer to the central nervous system: therapeutic and research applications.Hum Gene Ther. 2006; 17: 1-9Crossref PubMed Scopus (156) Google Scholar Phase I–II trials have been approved and are commencing in France. Another promising PD therapeutic strategy is AAV-2-mediated delivery of glutamic acid decarboxylase in the subthalamic nucleus to increase levels of limited γ-aminobutyric acid production and thereby inhibit excessive firing of neurons in the substantia nigra, which contributes to compromised motor function in PD. A phase I safety trial was completed without adverse effects. The clinical protocol approved by the FDA limited the treatment to one hemisphere. A phase II trial is planned to verify the potential benefits that were recorded on the patients' contralateral side, as had been anticipated (Kaplitt and During).4Alexander BL Ali RR Alton EW Bainbridge JW Braun S Cheng SH et al.Progress and prospects. 1. Gene therapy clinical trials.Gene Ther. 2007; 14: 1439-1447Crossref PubMed Scopus (86) Google Scholar,24Kaplitt MG Feigin A Tang C Fitzsimons HL Mattis P Lawlor PA et al.Safety and tolerability of gene therapy with an adeno-associated virus (AAV) borne GAD gene for Parkinson's disease: an open label, phase I trial.Lancet. 2007; 369: 2097-2105Abstract Full Text Full Text PDF PubMed Scopus (849) Google Scholar For many years, gene therapy's strength was in the overexpression of therapeutic genes. In a number of applications—foremost the dominantly inherited neurologic disorders, as exemplified by Huntington's disease—inhibiting the expression of the disease-causing alleles would be the ideal therapeutic goal. The use of RNA interference is now fulfilling this promise, and a number of experimental approaches have achieved major breakthroughs in this area. It is likely that clinical trials using RNAi for the treatment of dominant diseases will be implemented in the very near future.25Davidson BL Boudreau RL RNA interference: a tool for querying nervous system function and an emerging therapy.Neuron. 2007; 53: 781-788Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar Vectors that seem most promising for clinical applications in neurologic diseases are nonreplicating AAV and gutless adenovirus vectors for neurodegenerative disorders and replication-defective and conditionally replicating (oncolytic) adenovirus and HSV vectors for brain tumors. The first clinical trial with HIV-1-derived lentiviral vectors targeted CD4+ T lymphocytes in AIDS patients. The cells were stably transduced by the vector and expanded ex vivo. The autologous HIV-1-resistant cells were reinfused into the patient with no apparent adverse effects (Dropulic).26Levine BL Humeau LM Boyer J MacGregor RR Rebello T Lu X et al.Gene transfer in humans using a conditionally replicating lentiviral vector.Proc Natl Acad Sci USA. 2006; 103: 17372-17377Crossref PubMed Scopus (418) Google Scholar Integrating lentiviral vectors are currently used in clinical trials to target hematopoietic cells to treat mucopolysaccharidosis type VII. The facility with which lentiviral vectors transduce bone marrow ex vivo is likely to be harnessed to treat several diseases, including metachromatic leukodystrophy.27Biffi A Capotondo A Fasano S del Carro U Marchesini S Azuma H et al.Gene therapy of metachromatic leukodystrophy reverses neurological damage and deficits in mice.J Clin Invest. 2006; 116: 3070-3082Crossref PubMed Scopus (185) Google Scholar Safety and efficacy studies of lentiviral vectors in the nervous system have been carried out in nonhuman primate models of PD (Mazarakis). There is a lack of evidence that HIV-1 integration can cause tumors in AIDS patients, and long-term follow-up will be necessary to evaluate the potential risk of integration by HIV-1-based vectors. The potential use of these vectors for the nervous system should be further explored. Results of the recently approved phase I–II clinical trials of an EIAV-based lentiviral vector against PD will be very important, although the vector safety may not be known for many years. In the case of AAV, cataloguing of the many new serotypes derived from humans and rhesus macaque monkeys with respect to infection of cells in the nervous system must be undertaken. Differences in vector-uptake efficiency among the various neural cell types, along with differences in the cellular transport mechanisms for the vector core, could greatly help in the targeting of transgene expression to specific regions within the nervous system (Mandel). For all vector types, standards will need to be established. Ideally, this should be done in concert with the US Food and Drug Administration (FDA). Resources will need to be made available for the construction of Good Manufacturing Practice–grade vectors and for carrying out clinical trials. A systematic functional mapping of vector distribution and expression might highlight new vector delivery routes that could potentially reduce the total number of injections and thereby increase vector efficacy and safety. Vector loss during purification and injections (e.g., binding to tubes) must be minimized to lower the cost of highly purified vector preparations and reduce toxic contaminants. The need for transgene regulation continues to be debated within the community. There was disagreement about whether transgene regulation should be required for clinical trials. For some therapeutic purposes, drug regulation of transgene expression may prove important, but the current regulatory strategies have complications of leakiness, added side effects of the drugs used to induce or inhibit transgene expression, and the potentially unknown immunogenicity of the regulatory proteins, most of which are derived from prokaryotic organisms. Further, from a regulatory standpoint, the use of currently available inducible systems in clinical trials substantially raises the financial bar in testing the safety and efficacy of the inducible systems themselves. Innovative and potentially simpler approaches to transgene regulation should be explored and are likely to have a major impact on the future of gene therapy. Gene therapy researchers have worked for decades to achieve long-term expression vectors. Added to the particular immune privilege of the brain, the long-term effects of expressing powerful growth factors in the brains of pat