AAV Provides an Alternative for Gene Therapy of the Peripheral Sensory Nervous System
Authors: Andreas S Beutler
Journal: Molecular Therapy (2010)

Recombinant adeno-associated virus (rAAV) is a promising vector for applications in the central nervous system (CNS). Clinical phase I/II studies have demonstrated safety of rAAV-expressing therapeutic transgenes for a variety of benign (i.e., noncancer) CNS disorders, namely, Canavan disease,1Janson C McPhee S Bilaniuk L Haselgrove J Testaiuti M Freese A et al.Clinical protocol. Gene therapy of Canavan disease: AAV-2 vector for neurosurgical delivery of aspartoacylase gene (ASPA) to the human brain.Hum Gene Ther. 2002; 13: 1391-1412Crossref PubMed Scopus (205) Google Scholar Parkinson's disease,2Kaplitt 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 (841) Google Scholar,3Eberling JL Jagust WJ Christine CW Starr P Larson P Bankiewicz KS et al.Results from a phase I safety trial of hAADC gene therapy for Parkinson disease.Neurology. 2008; 70: 1980-1983Crossref PubMed Scopus (307) Google Scholar,4Marks Jr, WJ Ostrem JL Verhagen L Starr PA Larson PS Bakay RA et al.Safety and tolerability of intraputaminal delivery of CERE-120 (adeno-associated virus serotype 2-neurturin) to patients with idiopathic Parkinson's disease: an open-label, phase I trial.Lancet Neurol. 2008; 7: 400-408Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar and Batten disease.5Worgall S Sondhi D Hackett NR Kosofsky B Kekatpure MV Neyzi N et al.Treatment of late infantile neuronal ceroid lipofuscinosis by CNS administration of a serotype 2 adeno-associated virus expressing CLN2 cDNA.Hum Gene Ther. 2008; 19: 463-474Crossref PubMed Scopus (329) Google Scholar Human studies were also recently reported targeting the retina.6Bainbridge JW Smith AJ Barker SS Robbie S Henderson R Balaggan K et al.Effect of gene therapy on visual function in Leber's congenital amaurosis.N Engl J Med. 2008; 358: 2231-2239Crossref PubMed Scopus (1623) Google Scholar, 7Maguire AM Simonelli F Pierce EA Pugh EN Jr Mingozzi F Bennicelli J et al.Safety and efficacy of gene transfer for Leber's congenital amaurosis.N Engl J Med. 2008; 358: 2240-2248Crossref PubMed Scopus (1719) Google Scholar,8Cideciyan AV Aleman TS Boye SL Schwartz SB Kaushal S Roman AJ et al.Human gene therapy for RPE65 isomerase deficiency activates the retinoid cycle of vision but with slow rod kinetics.Proc Natl Acad Sci USA. 2008; 105: 15112-15117Crossref PubMed Scopus (590) Google Scholar,9Hauswirth W Aleman TS Kaushal S Cideciyan AV Schwartz SB Wang L et al.Phase I trial of leber congenital amaurosis due to RPE65 mutations by ocular subretinal injection of adeno-associated virus gene vector: short-term results.Hum Gene Ther. 2008; 19: 979-990Crossref PubMed Scopus (806) Google Scholar,9Hauswirth W Aleman TS Kaushal S Cideciyan AV Schwartz SB Wang L et al.Phase I trial of leber congenital amaurosis due to RPE65 mutations by ocular subretinal injection of adeno-associated virus gene vector: short-term results.Hum Gene Ther. 2008; 19: 979-990Crossref PubMed Scopus (806) Google Scholar From the early stages of preclinical development, rAAV was appealing because it was found to be neurotrophic and capable of long-term activity without immune interference or toxicity when injected into the CNS.10Kaplitt MG Leone P Samulski RJ Xiao X Pfaff DW O'Malley KL et al.Long-term gene expression and phenotypic correction using adeno- associated virus vectors in the mammalian brain.Nat Genet. 1994; 8: 148-154Crossref PubMed Scopus (915) Google Scholar But what about the potential of rAAV for the peripheral nervous system (PNS)? The PNS has a major role in a large number of neurological disorders, including a common one, chronic pain. The PNS is also, in principle, more easily accessible without major surgery, a potential advantage for any clinical gene therapy application. The PNS consists of three groups of neurons: primary sensory neurons, primary motor neurons, and autonomic neurons. Primary sensory neurons reside in the dorsal root ganglia (DRG)—hence their frequent designation as DRG neurons—and in the trigeminal ganglion. Primary sensory neurons are of clinical interest because of their role in sensory neuropathies and a wide range of chronic pain states. Testing of rAAV in the PNS was initially reported as a sidekick of an adenovirus marker gene study,11Glatzel M Flechsig E Navarro B Klein MA Paterna JC Büeler H et al.Adenoviral and adeno-associated viral transfer of genes to the peripheral nervous system.Proc Natl Acad Sci USA. 2000; 97: 442-447Crossref PubMed Scopus (106) Google Scholar followed by a report of its usefulness for analgesic treatments.12Xu Y Gu Y Xu GY Wu P Li GW Huang LY Adeno-associated viral transfer of opioid receptor gene to primary sensory neurons: a strategy to increase opioid antinociception.Proc Natl Acad Sci USA. 2003; 100: 6204-6209Crossref PubMed Scopus (78) Google Scholar Both studies used rAAV consisting of inverted terminal repeats and capsid proteins derived from AAV serotype 2 (the original rAAV vectors developed in the mid-1990s) and administered the vectors directly into the DRG. In this issue of Molecular Therapy, Mason et al. rekindle this approach, reporting on the targeting of primary sensory neurons by injection of rAAV into the DRG.13Mason MRJ Ehlert EME Eggers R Pool CW Hermening S Huseinovic A et al.Comparison of AAV serotypes for gene delivery to dorsal root ganglion neurons.Mol Ther. 2010; 18: 715-724Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar The authors packaged AAV2 recombinant genomes into seven different capsids—the original serotype 2 as well as the newer types 1, 3, 4, 5, 6, and 8—and compared their efficacy for expressing the marker gene eGFP. Their best-performing vector, AAV5, transduced >90% of DRG neurons—it can't get better than that. So is it prime time yet for a clinical trial targeting the PNS by direct rAAV injection into the DRG? A critical set of studies may help to light the way. First, it would be of interest to repeat the study in other animal species to evaluate whether the marked performance difference among the various capsid serotypes reported by Mason et al.13Mason MRJ Ehlert EME Eggers R Pool CW Hermening S Huseinovic A et al.Comparison of AAV serotypes for gene delivery to dorsal root ganglion neurons.Mol Ther. 2010; 18: 715-724Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar seems generalizable or is rodent-specific. The original reports on capsid pseudotyping of rAAV showed very marked differences between rodent muscle14Chao H Liu Y Rabinowitz J Li C Samulski RJ Walsh CE Several log increase in therapeutic transgene delivery by distinct adeno-associated viral serotype vectors.Mol Ther. 2000; 2: 619-623Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar and rodent brain15Davidson BL Stein CS Heth JA Martins I Korin RM Derksen TA et al.Recombinant adeno-associated virus type 2, 4, and 5 vectors: transduction of variant cell types and regions in the mammalian central nervous system.Proc Natl Acad Sci USA. 2000; 97: 3428-3432Crossref PubMed Scopus (572) Google Scholar resembling or exceeding the differences between serotypes demonstrated by Mason et al. in the rodent DRG (e.g., in the muscle the difference was several log10 orders of magnitude14Chao H Liu Y Rabinowitz J Li C Samulski RJ Walsh CE Several log increase in therapeutic transgene delivery by distinct adeno-associated viral serotype vectors.Mol Ther. 2000; 2: 619-623Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar). Unfortunately, in the case of muscle- or brain-directed rAAV gene transfer, similarly systematic serotype comparisons seem not to have been performed in nonhuman primates or other large animals (dogs or pigs could be considered), but the "word on the street" (evaluating single-serotype studies and anecdotal findings in only a few animals) implies differences that are less than overwhelming. In the case of DRG gene transfer, any firmer guidance would certainly help clinical researchers to choose the best rAAV serotype when planning human trials in the PNS. Second, any piece of evidence supporting the lack of immunogenicity of rAAV vectors would be reassuring. rAAV is notable for its inability to induce cellular immunity in rodents. However, administration of AAV2 into the portal vein in humans led to an apparent delayed immune response that ended a clinical trial prematurely.16Manno CS Pierce GF Arruda VR Glader B Ragni M Rasko JJ et al.Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response.Nat Med. 2006; 12: 342-347Crossref PubMed Scopus (1578) Google Scholar,17Mingozzi F Maus MV Hui DJ Sabatino DE Murphy SL Rasko JE et al.CD8(+) T-cell responses to adeno-associated virus capsid in humans.Nat Med. 2007; 13: 419-422Crossref PubMed Scopus (532) Google Scholar,18Li H Murphy SL Giles-Davis W Edmonson S Xiang Z Li Y et al.Pre-existing AAV capsid-specific CD8+ T cells are unable to eliminate AAV-transduced hepatocytes.Mol Ther. 2007; 15: 792-800Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar,19Herzog RW Immune responses to AAV capsid: are mice not humans after all?.Mol Ther. 2007; 15: 649-650Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar rAAV has also been used as a platform for vaccine development, suggesting that it can induce strong immunity under some circumstances.20Lin J Calcedo R Vandenberghe LH Bell P Somanathan S Wilson JM A new genetic vaccine platform based on an adeno-associated virus isolated from a rhesus macaque.J Virol. 2009; 83: 12738-12750Crossref PubMed Scopus (47) Google Scholar Perhaps the most interesting observation reported by Mason et al. relates to this problem of rAAV immunogenicity—expression of AAV6 (but not any of the other serotypes) declined 12 weeks after DRG injection and was accompanied by neuropathological tissue damage at the injection site. Interestingly, the authors found that only AAV6 among the vectors tested was able to transduce nonneuronal cells in the DRG, namely, satellite cells. Accordingly, the authors speculate that the less discriminate transduction profile of AAV6 may have led to AAV6 uptake by antigen-presenting cells triggering immunity, which consequently led to tissue damage and loss of transgene expression.13Mason MRJ Ehlert EME Eggers R Pool CW Hermening S Huseinovic A et al.Comparison of AAV serotypes for gene delivery to dorsal root ganglion neurons.Mol Ther. 2010; 18: 715-724Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar Although these findings are intriguing, the present study leaves open many questions. For example, what happens to expression at later time points (loss of expression is only partial at 12 weeks)? Would neuropathological toxicity hold up in a blinded experiment (random variation in injection technique and tissue procurement remain a possibility)? Finally, what is the nature of the immune response (presumably cytotoxic), and is it directed against the transgene (eGFP), the virus capsid, or both? A careful follow-up study would be most welcome and certainly highly informative for future clinical research. Third, a lingering concern with rAAV is whether it might be tumorigenic. Wild-type AAV has not been associated with carcinogenesis. But rAAV causes an increased incidence of liver tumors when administered to newborn mice of certain susceptible strains,21Donsante A Vogler C Muzyczka N Crawford JM Barker J Flotte T et al.Observed incidence of tumorigenesis in long-term rodent studies of rAAV vectors.Gene Ther. 2001; 8: 1343-1346Crossref PubMed Scopus (221) Google Scholar in which vector integration was demonstrated in tumor tissues. In at least some of the cases, integration occurred in a micro-RNA locus on mouse chromosome 12. This led to regional transcriptional activation of many small RNA genes with known growth-regulatory properties, a plausible mechanism of carcinogenesis.22Donsante A Miller DG Li Y Vogler C Brunt EM Russell DW et al.AAV vector integration sites in mouse hepatocellular carcinoma.Science. 2007; 317: 477Crossref PubMed Scopus (474) Google Scholar Another study found an increased incidence of liver tumors only with a specific transgene but not with rAAV administration per se.23Bell P Moscioni AD McCarter RJ Wu D Gao G Hoang A et al.Analysis of tumors arising in male B6C3F1 mice with and without AAV vector delivery to liver.Mol Ther. 2006; 14: 34-44Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar Such findings seem to be limited to rAAV administration in newborn (as opposed to adult) mice and might occur only in specific, susceptible strains (such as knockout mice with lysosomal storage disease) in which liver tumors can occur even spontaneously. They could also have been favored by relatively high vector doses (1.5 × 1011 particles per newborn mouse) and have thus far not been noted in other organs or other species. It is therefore not surprising that no such findings were noted by Mason et al.13Mason MRJ Ehlert EME Eggers R Pool CW Hermening S Huseinovic A et al.Comparison of AAV serotypes for gene delivery to dorsal root ganglion neurons.Mol Ther. 2010; 18: 715-724Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar Nevertheless, continued careful attention to even a low incidence of tumor formation will need to be an integral part of any preclinical testing. Fourth, an obvious challenge is the development of a minimally invasive DRG injection technique resembling the method practiced by Mason et al. in rodents but applicable to humans. The human DRG is approximately 50 times larger than that of a rodent, e.g., approximately 150–200 mm3 in humans24Shen J Wang HY Chen JY Liang BL Morphologic analysis of normal human lumbar dorsal root ganglion by 3D MR imaging.AJNR Am J Neuroradiol. 2006; 27: 2098-2103PubMed Google Scholar as compared with 4–5 mm3 for the rat.25West CA Davies KA Hart AM Wiberg M Williams SR Terenghi G Volumetric magnetic resonance imaging of dorsal root ganglia for the objective quantitative assessment of neuron death after peripheral nerve injury.Exp Neurol. 2007; 203: 22-33Crossref PubMed Scopus (25) Google Scholar As a result, single-site injections will not work. Bankiewicz et al. tackled this problem in the CNS through the development of an administration technique that they refer to as convection-enhanced delivery, which they studied in different species, including primates.26Cunningham J Oiwa Y Nagy D Podsakoff G Colosi P Bankiewicz KS Distribution of AAV-TK following intracranial convection-enhanced delivery into rats.Cell Transplant. 2000; 9: 585-594PubMed Google Scholar,27Bankiewicz KS Eberling JL Kohutnicka M Jagust W Pivirotto P Bringas J et al.Convection-enhanced delivery of AAV vector in parkinsonian monkeys; in vivo detection of gene expression and restoration of dopaminergic function using pro-drug approach.Exp Neurol. 2000; 164: 2-14Crossref PubMed Scopus (363) Google Scholar They defined the technique in terms of biomechanical details such as flow and pressure,28Cunningham J Pivirotto P Bringas J Suzuki B Vijay S Sanftner L et al.Biodistribution of adeno-associated virus type-2 in nonhuman primates after convection-enhanced delivery to brain.Mol Ther. 2008; 16: 1267-1275Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar,29Hadaczek P Yamashita Y Mirek H Tamas L Bohn MC Noble C et al.The "perivascular pump" driven by arterial pulsation is a powerful mechanism for the distribution of therapeutic molecules within the brain.Mol Ther. 2006; 14: 69-78Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar,30Krauze MT Saito R Noble C Bringas J Forsayeth J McKnight TR et al.Effects of the perivascular space on convection-enhanced delivery of liposomes in primate putamen.Exp Neurol. 2005; 196: 104-111Crossref PubMed Scopus (67) Google Scholar tested it for safety with different vector types,31Hadaczek P Forsayeth J Mirek H Munson K Bringas J Pivirotto P et al.Transduction of nonhuman primate brain with adeno-associated virus serotype 1: vector trafficking and immune response.Hum Gene Ther. 2009; 20: 225-237Crossref PubMed Scopus (79) Google Scholar,32Hadaczek P Kohutnicka M Krauze MT Bringas J Pivirotto P Cunningham J et al.Convection-enhanced delivery of adeno-associated virus type 2 (AAV2) into the striatum and transport of AAV2 within monkey brain.Hum Gene Ther. 2006; 17: 291-302Crossref PubMed Scopus (101) Google Scholar,33Krauze MT Vandenberg SR Yamashita Y Saito R Forsayeth J Noble C et al.Safety of real-time convection-enhanced delivery of liposomes to primate brain: a long-term retrospective.Exp Neurol. 2008; 210: 638-644Crossref PubMed Scopus (44) Google Scholar developed imaging for it,34Saito R Krauze MT Bringas JR Noble C McKnight TR Jackson P et al.Gadolinium-loaded liposomes allow for real-time magnetic resonance imaging of convection-enhanced delivery in the primate brain.Exp Neurol. 2005; 196: 381-389Crossref PubMed Scopus (128) Google Scholar,35Fiandaca MS Varenika V Eberling J McKnight T Bringas J Pivirotto P et al.Real-time MR imaging of adeno-associated viral vector delivery to the primate brain.Neuroimage. 2009; 47: T27-T35Crossref PubMed Scopus (92) Google Scholar,36Krauze MT McKnight TR Yamashita Y Bringas J Noble CO Saito R et al.Real-time visualization and characterization of liposomal delivery into the monkey brain by magnetic resonance imaging.Brain Res Brain Res Protoc. 2005; 16: 20-26Crossref PubMed Scopus (82) Google Scholar and refined it down to such details as the needle to be employed.37Krauze MT Saito R Noble C Tamas M Bringas J Park JW et al.Reflux-free cannula for convection-enhanced high-speed delivery of therapeutic agents.J Neurosurg. 2005; 103: 923-929Crossref PubMed Scopus (184) Google Scholar,38Yin D Forsayeth J Bankiewicz KS Optimized cannula design and placement for convection-enhanced delivery in rat striatum.J Neurosci Methods. 2010; 187: 46-51Crossref PubMed Scopus (65) Google Scholar From a neurosurgical perspective, the DRG is a structure that is routinely visualized in the scope of many spine operations. But spine surgery would ideally not be required for injecting rAAV. Instead, a minimally invasive technique would be desirable, particularly given that clinical syndromes involving the PNS would ultimately call for treatment of DRG at multiple spinal levels and possibly bilaterally. The absence of a clinically established procedure for injecting DRG in humans calls for some creative surgical research. For the past decade, PNS gene therapy has been the domain of herpes simplex virus, a vector that transduces primary sensory neurons effectively in animal models when injected subcutaneously.39Goss JR Mata M Goins WF Wu HH Glorioso JC Fink DJ Antinociceptive effect of a genomic herpes simplex virus-based vector expressing human proenkephalin in rat dorsal root ganglion.Gene Ther. 2001; 8: 551-556Crossref PubMed Scopus (148) Google Scholar,40Glorioso JC Fink DJ Herpes vector-mediated gene transfer in the treatment of chronic pain.Mol Ther. 2009; 17: 13-18Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar Many crucial questions, such as those asked above for rAAV, have been successfully resolved for herpes simplex virus, prompting ongoing clinical trials in patients with intractable pain.41Wolfe D Mata M Fink DJ A human trial of HSV-mediated gene transfer for the treatment of chronic pain.Gene Ther. 2009; 16: 455-460Crossref PubMed Scopus (55) Google Scholar,42Wolfe D Wechuck J Krisky D Mata M Fink DJ A clinical trial of gene therapy for chronic pain.Pain Med. 2009; 10: 1325-1330Crossref PubMed Scopus (31) Google Scholar For rAAV-based gene therapy of the PNS, it took several more years after the initial studies11Glatzel M Flechsig E Navarro B Klein MA Paterna JC Büeler H et al.Adenoviral and adeno-associated viral transfer of genes to the peripheral nervous system.Proc Natl Acad Sci USA. 2000; 97: 442-447Crossref PubMed Scopus (106) Google Scholar,12Xu Y Gu Y Xu GY Wu P Li GW Huang LY Adeno-associated viral transfer of opioid receptor gene to primary sensory neurons: a strategy to increase opioid antinociception.Proc Natl Acad Sci USA. 2003; 100: 6204-6209Crossref PubMed Scopus (78) Google Scholar for a more comprehensive picture to emerge that covered various administration techniques such as intrathecal delivery of various rAAV serotypes,43Storek B Harder NM Banck MS Wang C McCarty DM Janssen WG et al.Intrathecal long-term gene expression by self-complementary adeno-associated virus type 1 suitable for chronic pain studies in rats.Mol Pain. 2006; 2: 4Crossref PubMed Scopus (48) Google Scholar,44Beutler AS Banck MS Walsh CE Milligan ED Intrathecal gene transfer by adeno-associated virus for pain.Curr Opin Mol Ther. 2005; 7: 431-439PubMed Google Scholar intraneural and systemic targeting of DRG neurons by rAAV6 (ref. 45Towne C Pertin M Beggah AT Aebischer P Decosterd I Recombinant adeno-associated virus serotype 6 (rAAV2/6)-mediated gene transfer to nociceptive neurons through different routes of delivery.Mol Pain. 2009; 5: 52Crossref PubMed Scopus (118) Google Scholar), and long-term efficacy for the treatment of neuropathic pain.46Storek B Reinhardt M Wang C Janssen WG Harder NM Banck MS et al.Sensory neuron targeting by self-complementary AAV8 via lumbar puncture for chronic pain.Proc Natl Acad Sci USA. 2008; 105: 1055-1060Crossref PubMed Scopus (105) Google Scholar,47Beutler AS Reinhardt M AAV for pain: steps towards clinical translation.Gene Ther. 2009; 16: 461-469Crossref PubMed Scopus (32) Google Scholar The study by Mason et al.13Mason MRJ Ehlert EME Eggers R Pool CW Hermening S Huseinovic A et al.Comparison of AAV serotypes for gene delivery to dorsal root ganglion neurons.Mol Ther. 2010; 18: 715-724Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar adds seminal data refocusing on rAAV injection directly into the DRG and further supports the candidacy of rAAV as an alternative for gene therapy of the PNS. The author thanks Kendall Lee, Departments of Neurosurgery and Biomedical Engineering at the Mayo Clinic, for discussions. The author's research is supported by the National Institute of Neurological Disorders and Stroke (R01NS063022 and R21NS062271) and the Richard M. Schulze Family Foundation.