Adeno-associated Virus Type Research Articles

Direct Comparison of Administration Routes for AAV8-mediated Ocular Gene Therapy

Purpose: We recently demonstrated that direct subretinal (SR) injection of adeno-associated virus (AAV) type 8 (AAV8) into photoreceptor cells and retinal pigment epithelium (RPE) is a highly efficient model of gene delivery. The current study compared transduction efficiency and expression patterns associated with various routes of vector administration.Method: The efficacy of intravitreal (VT), SR and subconjunctival (SC) injections for delivery of AAV8-derived vectors, i.e. those expressing luciferase (Luc) and enhanced green fluorescent protein (GFP) – AAV8/Luc and AAV8/GFP, respectively – were compared in an animal (mouse) model (n = 8 mice/group). Transduction efficiency and expression patterns were examined at post-injection weeks 1 and 2, and months 1, 3, 6 and 12 via in vivo imaging.Results: One year after AAV injection, AAV8/Luc-treated mice exhibited stable and sustained high expression of vector in the VT and SR groups, but not in the SC group (VT:SR:SC = 3,218:2,923:115; 1 × 105 photons/s). Histological analysis showed that GFP expression was observed in the inner retina of VT group mice, and in photoreceptor cells and RPE of SR group mice, whereas no GFP expression was noted in the SC group. Electroretinography (ERG) revealed adverse effects following SR delivery.Conclusions: Results suggest that both SR and VT injections of AAV8 vectors are useful routes for administering ocular gene therapy, and stress the importance of selecting an appropriate administration route, i.e. one that targets specific cells, for treating ocular disorders.

Preclinical Potency and Safety Studies of an AAV2-Mediated Gene Therapy Vector for the Treatment of MERTK Associated Retinitis Pigmentosa

Abstract Proof of concept for MERTK gene replacement therapy has been demonstrated using different viral vectors in the Royal College of Surgeon (RCS) rat, a well characterized model of recessive retinitis pigmentosa that contains a mutation in the Mertk gene. MERTK plays a key role in renewal of photoreceptor outer segments (OS) by phagocytosis of shed OS tips. Mutations in MERTK cause impaired phagocytic activity and accumulation of OS debris in the interphotoreceptor space that ultimately leads to photoreceptor cell death. In the present study, we conducted a series of preclinical potency and GLP-compliant safety evaluations of an adeno-associated virus type 2 (AAV2) vector expressing human MERTK cDNA driven by the retinal pigment epithelium-specific, VMD2 promoter. We demonstrate the potency of the vector in RCS rats by improved electroretinogram (ERG) responses in treated eyes compared with contralateral untreated controls. Toxicology and biodistribution studies were performed in Sprague-Dawley (SD) rats injected with two different doses of AAV vectors and buffer control. Delivery of vector in SD rats did not result in a change in ERG amplitudes of rod and cone responses relative to balanced salt solution control-injected eyes, indicating that administration of AAV vector did not adversely affect normal retinal function. In vivo fundoscopic analysis and postmortem retinal morphology of the vector-injected eyes were normal compared with controls. Evaluation of blood smears showed the lack of transformed cells in the treated eyes. All injected eyes and day 1 blood samples were positive for vector genomes, and all peripheral tissues were negative. Our results demonstrate the potency and safety of the AAV2-VMD2-hMERTK vector in animal models tested. A GMP vector has been manufactured and is presently in clinical trial.

Restoration of Cytoskeleton Homeostasis After Gigaxonin Gene Transfer for Giant Axonal Neuropathy

Giant axonal neuropathy (GAN) is caused by loss of function of the gigaxonin protein. On a cellular level GAN is characterized by intermediate filament (IF) aggregation, leading to a progressive and fatal peripheral neuropathy in humans. This study sought to determine if re-introduction of the GAN gene into GAN-deficient cells and mice would restore proper cytoskeleton IF homeostasis. Treatment of primary skin fibroblast cultures from three different GAN patients with an adeno-associated virus type 2 (AAV2) vector containing a normal human GAN transgene significantly reduced the number of cells displaying vimentin IF aggregates. A proteomic analysis of these treated cells was also performed, wherein the abundance of 32 of 780 identified proteins significantly changed in response to gigaxonin gene transfer. While 29 of these responding proteins have not been directly described in association with gigaxonin, three were previously identified as being disregulated in GAN and were now shifted toward normal levels. To assess the potential application of this approach in vivo and eventually in humans, GAN mice received an intracisternal injection of an AAV9/GAN vector to globally deliver the GAN gene to the brainstem and spinal cord. The treated mice showed a nearly complete clearance of peripherin IF accumulations at 3 weeks post-injection. These studies demonstrate that gigaxonin gene transfer can reverse the cellular IF aggregate pathology associated with GAN.

Adeno-associated virus Rep78 restricts adenovirus E1B55K-mediated p53 nuclear exportation

Inactivation of p53 is needed during adenovirus type 5 DNA replication. E1B55K, an adenovirus early protein, has been reported to interact with p53 and inhibit p53 transactivation. Previous studies have shown that adeno-associated virus (AAV) type 2 could reduce the transforming potential of adenovirus by rescuing p53 from adenovirus-mediated degradation, but the details are not clear yet. We detected the Rep78-p53 interaction by co-immunoprecipitation assay. The co-localization assay revealed that Rep78 inhibits E1B55K-mediated p53 nuclear exportation. However, Rep78 did not detectably influence p53 stability and could not relieve the transcriptional inactivation of p53, as E1B55K could not be replaced from the p53-E1B55K complex by Rep78. Our results reveal a new possible mechanism that AAV-2 Rep78 inhibits adenovirus 5 by relocalizing p53 in the nucleus, which may shed some light on the regulatory mechanism of AAV-2 on its helper virus, adenovirus.

Nucleus of the Solitary Tract (Pro)Renin Receptor-Mediated Antihypertensive Effect Involves Nuclear Factor-κB-Cytokine Signaling in the Spontaneously Hypertensive Rat

The importance of the (pro)renin receptor (PRR) in the function of the central nervous system is increasingly evident because PRR seems to play a role in neuronal control of cardiovascular function. PRR expression is elevated in the nucleus of the solitary tract (NTS) of spontaneously hypertensive rats (SHR). In this study, we tested the hypothesis that altered activity of PRR in the NTS is linked to hypertension. Eight weeks of chronic knockdown of the NTS PRR, using recombinant adeno-associated virus type 2 (AAV2)-PRR-small hairpain RNA (shRNA)-mediated gene transduction, caused a significant increase in mean arterial pressure (MAP) in the SHR (shRNA, 173±5; Control, 151±6 mm Hg) but not in Wistar Kyoto rats (shRNA, 108±7; Control, 106±6 mm Hg). The MAP elevation in the SHR was associated with decreased inflammatory markers tumor necrosis factor-α, interleukin-6, C-C motif ligand 5, and their transcription factor, nuclear factor-κB. Consistent with the pressor effects of the PRR knockdown, acute bilateral NTS injection of human renin (2 pmol/side) decreased MAP and heart rate (HR) in SHR (ΔMAP, -38±4 mm Hg; Δheart rate, -40±10 bpm), with negligible responses in Wistar Kyoto rats (ΔMAP, -4±3 mm Hg; Δheart rate, -12±7 bpm). These effects in SHR were attenuated (80%) by prorenin handle region peptide but were not affected by angiotensin II type 1 or angiotensin II type 2 receptor blockers. Finally, PRR activation in SHR neuronal cultures by prorenin activated nuclear factor-κB and increased mRNA levels of interleukin-1β (250-fold), tumor necrosis factor-α (32-fold), interleukin-6 (35-fold), C-C motif ligand 5 (12-fold), and interleukin-10 (7-fold) in a nuclear factor-κB-dependent but angiotensin II type 1 receptor-independent manner. Therefore, NTS PRR mediates antihypertensive effects via an angiotensin II-independent mechanism in SHR, which involves stimulation of the nuclear factor-κB-cytokine signaling pathway.

AAV9.I-1c Delivered via Direct Coronary Infusion in a Porcine Model of Heart Failure Improves Contractility and Mitigates Adverse Remodeling

Heart failure is characterized by impaired function and disturbed Ca2+ homeostasis. Transgenic increases in inhibitor-1 activity have been shown to improve Ca2 cycling and preserve cardiac performance in the failing heart. The aim of this study was to evaluate the effect of activating the inhibitor (I-1c) of protein phosphatase 1 (I-1) through gene transfer on cardiac function in a porcine model of heart failure induced by myocardial infarction. Myocardial infarction was created by a percutaneous, permanent left anterior descending artery occlusion in Yorkshire Landrace swine (n=16). One month after myocardial infarction, pigs underwent intracoronary delivery of either recombinant adeno-associated virus type 9 carrying I-1c (n=8) or saline (n=6) as control. One month after myocardial infarction was created, animals exhibited severe heart failure demonstrated by decreased ejection fraction (46.4±7.0% versus sham 69.7±8.5%) and impaired (dP/dt)max and (dP/dt)min. Intracoronary injection of AAV9.I-1c prevented further deterioration of cardiac function and led to a decrease in scar size. In this preclinical model of heart failure, overexpression of I-1c by intracoronary in vivo gene transfer preserved cardiac function and reduced the scar size.

Recombinant adeno-associated virus type 8 mediated dual-luciferase gene expression in mouse

Recombinant adeno-associated virus type 8 (rAAV8) mediating transgene expression in mice was investigated using co-expressed report gene of secreted Gaussia princeps luciferase (Gluc) and non-secreted firefly luciferase(Fluc). rAAV8-Gluc/Fluc was prepared and infected HEK293 cells to test its performance in vitro. BALB/c mice were received rAAV8-Gluc/Fluc at different doses by intravenous injection (iv) or intramuscular injection (im). Then Glue activities in blood were measured,the whole-body images for Flue activities were performed and Flue activities of tissue lysate were also detected. rAAV8-Gluc/Fluc was successfully prepared and could infected HEK293 cells. The Glue was mainly detected in the culture media while the Flue was mainly detected within cells. The blood Glue activities of mice received rAAV8-Gluc/Fluc by iv or im peaked at 10-20 d post injection and persisted for at least 120d. The blood Gluc activities increased at the rAAV8-Gluc/Fluc dose-dependent manner. For mice received rAAV8 by iv, Fluc mainly expressed in liver and minor Fluc expression was also found in cardiac muscle and skeletal muscle. For mice received rAAV8-Gluc/Fluc by im, Fluc mainly expressed in local skeletal muscle and secondly in liver. rAAV8-Gluc/Fluc has been prepared successfully and its mediating transgene expression in mice has been investigated. This research will facilitate preclinical studies for rAAV8-mediated gene therapy.

Hsp40 Gene Therapy Exerts Therapeutic Effects on Polyglutamine Disease Mice via a Non-Cell Autonomous Mechanism

The polyglutamine (polyQ) diseases such as Huntington’s disease (HD), are neurodegenerative diseases caused by proteins with an expanded polyQ stretch, which misfold and aggregate, and eventually accumulate as inclusion bodies within neurons. Molecules that inhibit polyQ protein misfolding/aggregation, such as Polyglutamine Binding Peptide 1 (QBP1) and molecular chaperones, have been shown to exert therapeutic effects in vivo by crossing of transgenic animals. Towards developing a therapy using these aggregation inhibitors, we here investigated the effect of viral vector-mediated gene therapy using QBP1 and molecular chaperones on polyQ disease model mice. We found that injection of adeno-associated virus type 5 (AAV5) expressing QBP1 or Hsp40 into the striatum both dramatically suppresses inclusion body formation in the HD mouse R6/2. AAV5-Hsp40 injection also ameliorated the motor impairment and extended the lifespan of R6/2 mice. Unexpectedly, we found even in virus non-infected cells that AAV5-Hsp40 appreciably suppresses inclusion body formation, suggesting a non-cell autonomous therapeutic effect. We further show that Hsp40 inhibits secretion of the polyQ protein from cultured cells, implying that it inhibits the recently suggested cell-cell transmission of the polyQ protein. Our results demonstrate for the first time the therapeutic effect of Hsp40 gene therapy on the neurological phenotypes of polyQ disease mice.

Tin Oxide Nanowires Suppress Herpes Simplex Virus-1 Entry and Cell-to-Cell Membrane Fusion

The advent of nanotechnology has ushered in the use of modified nanoparticles as potential antiviral agents against diseases such as herpes simplex virus 1 and 2 (HSV-1) (HSV-2), human immunodeficiency virus (HIV), monkeypox virus, and hepatitis B virus. Here we describe the application of tin oxide (SnO2) nanowires as an effective treatment against HSV-1 infection. SnO2 nanowires work as a carrier of negatively charged structures that compete with HSV-1 attachment to cell bound heparan sulfate (HS), therefore inhibiting entry and subsequent cell-to-cell spread. This promising new approach can be developed into a novel form of broad-spectrum antiviral therapy especially since HS has been shown to serve as a cellular co-receptor for a number of other viruses as well, including the respiratory syncytial virus, adeno-associated virus type 2, and human papilloma virus.

The Adeno-Associated Virus Type 5 Small Rep Proteins Expressed via Internal Translation Initiation Are Functional

Although precluded from using splicing to produce multiple small Rep proteins, adeno-associated virus type 5 (AAV5) generates a Rep40-like protein by alternative translation initiation at an internal AUG. A defined region upstream of the internal AUG was both required and sufficient to program internal initiation within AAV5 and may act similarly in heterologous contexts. The internally initiated AAV5 Rep40-like protein was functional and had helicase activity similar to that of AAV2 Rep40. Surprisingly, both the AAV5 Rep40-like protein and Rep52 were able to be translated from the AAV5 upstream P7-generated RNAs; however, the relative level of small to large Rep proteins was reduced compared to that of the wild type. A P19 mutant AAV5 infectious clone generated near-wild-type levels of the double-stranded monomer replicative form (mRF) replicative intermediate but reduced levels of virus, consistent with the previously defined role of Rep40-like proteins in genome encapsidation. Levels of mutant virus were dramatically reduced upon amplification.

Site‐Specific Modification of Adeno‐Associated Viruses via a Genetically Engineered Aldehyde Tag

As a consequence of their well-defined nanostructure and intrinsic bioactive functionality, virus-based nanoparticles have shown promise for mediating gene delivery. Adeno-associated virus (AAV) nanoparticles, which possess an excellent safety profile and therapeutic potential, hold potential for use in human gene therapy. However, because of their native tropisms, the applicability of AAV nanoparticles is often limited to restricted ranges of cells or tissues. Thus, retargeting AAV particles to the desired cell populations has continued to be a major research focus in many gene therapy applications. In this study, a general strategy is reported for nanoparticle targeting. This involves the site-specific modification of AAV type 2 (AAV2) by genetically incorporating a short peptide, in this case an aldehyde tag, in the viral capsid. Such a tag can be exploited for site-specific attachment of targeting molecules and allows for further introduction of targeting antibodies or ligands. It is shown that this modification neither affects the level of infectious viral titer nor intracellular trafficking properties. Furthermore, the site-specific conjugation of targeting antibodies could significantly enhance viral transduction to those target cells that have otherwise exhibited very low permissiveness to AAV2 infection. This method also allows the functional incorporation of RGD peptides onto AAV2 for enhanced delivery with implications for cancer gene therapy.

AAV2/IL-12 gene delivery into dendritic cells (DC) enhances CTL stimulation above other IL-12 applications

Adoptive transfer of antigen-specific cytotoxic T lymphocytes (CTL) holds significant promise in treating cancer and Th1 response cytokines are critical for their stimulation. Recently we reported that interleukin 7-(IL-7) and interferongamma-(IFNγ) autocrine/T cell gene delivery resulted in superior ex vivo CTL stimulation over paracrine/DC delivery. IL-12 is yet another important Th1 cytokine which affects both DC and T cells. Here, using adeno-associated virus Type 2 (AAV2) gene delivery, IL-12-paracrine/DC gene delivery gave significantly superior stimulation of carcinoembryonic antigen (CEA)-specific CTL killing over that induced by autocrine gene delivery (or exogenous IL-12 addition). This is surprising as both AAV2/IL-12-treated T cells and DC secreted approximately the same level of IL-12. Paracrine IL-12 gene delivery also resulted in highest IL-12/IL-10 secretion ratio by DC and highest CD40, CD80, CD83 and CD86 expression. Moreover, AAV2/IL-12-DC stimulated the highest T-cell IFNγ production, highest T cell proliferation, highest CD69+/CD8+ levels, and lowest level of CD25+/CD4+ Treg. These data strongly suggest that the primary activity of IL-12 during CTL generation is upon the DC. These data are also consistent with there being novel activity for IL-12 within the DC itself, not involving its surface receptor; an “intracrine” activity. Given the plethora of IL-12 studies, these data also suggest that this gene delivery comparison approach could be useful for uncovering new cytokine activities and mechanism(s) of action gone unrecognized by conventional immunologic assays. Finally, these data further suggest AAV2/IL-12 intracrine gene delivery into DC may have utility in immunotherapy protocols involving antigen-specific CTL.

Adeno-Associated Viral Vectors Based on Serotype 3b Use Components of the Fibroblast Growth Factor Receptor Signaling Complex for Efficient Transduction

Adeno-associated virus type 3b (AAV3b) has been largely ignored by gene therapists because of the inability of vectors based on this serotype to transduce target tissues efficiently. Here we describe a phenomenon unique to AAV3b in that vectors based on this serotype mediate enhanced transduction in the presence of heparin. Among the many biological functions attributed to heparin, its interaction with, and ability to regulate, several growth factors (GFs) and growth factor receptors (GFRs) has been well characterized. Using GFR-overexpressing cell lines, soluble GFs and heparins, as well as specific GFR inhibitors, we have demonstrated a requirement for fibroblast growth factor receptor-2 (FGFR2) and FGF1 in the heparin-mediated augmentation of AAV3b vector transduction. In contrast to AAV2, we establish that heparin can be used as an adjunct with AAV3b to further increase transduction in a variety of cells and target tissues, additionally suggesting that AAV3b may be an attractive viral vector for clinical use during procedures in which heparin is used. In summary, AAV3b exhibits FGFR2-dependent, markedly enhanced transduction efficiency in the presence of heparin and FGFs, which could make it a useful vector for gene therapy in a variety of human diseases.

Comparison of gene transfer to the murine liver following intraperitoneal and intraportal delivery of hepatotropic AAV pseudo-serotypes

Adeno-associated virus (AAV) vectors are highly efficient for liver-targeted gene delivery in murine models and show promise in early phase human clinical trials. This efficiency is capsid-dependent and was only achieved after discovery that the AAV2 vector genome could be trans-encapsidated into the capsids of other AAV serotypes. This confers novel host-vector biology and target tissue tropism. Optimal exploitation of the growing number of AAV vector pseudo-serotypes, however, requires detailed context-dependent characterisation of transduction performance. In this study, we compared the pattern and efficiency of gene delivery to the adult mouse liver following intraportal and intraperitoneal injection of vectors pseudo-serotyped with known hepatotropic capsids from AAV type 7, 8, 9 and rhesus 10. Vectors pseudo-serotyped with these hepatotropic capsids proved relatively efficient irrespective of administration route, with higher transgene expression in males despite equivalent vector genome delivery in females. Transgene expression was predominantly centrilobular in contrast to the AAV2 capsid, which gave a periportal pattern of expression. Most intriguingly, vector genome performance appeared to be delivery route-dependent, consistent with the possibility of in vivo capsid modification. These data not only inform the experimental use of AAV vectors, but also provide insight into novel aspects of host-vector biology requiring further focused analysis.

Limitations of encapsidation of recombinant self-complementary adeno-associated viral genomes in different serotype capsids and their quantitation.

We previously reported that self-complementary adeno-associated virus (scAAV) type 2 genomes of up to 3.3 kb can be successfully encapsidated into AAV2 serotype capsids. Here we report that such oversized AAV2 genomes fail to undergo packaging in other AAV serotype capsids, such as AAV1, AAV3, AAV6, and AAV8, as determined by Southern blot analyses of the vector genomes, although hybridization signals on quantitative DNA slot-blots could still be obtained. Recently, it has been reported that quantitative real-time PCR assays may result in substantial differences in determining titers of scAAV vectors depending on the distance between the primer sets and the terminal hairpin structure in the scAAV genomes. We also observed that the vector titers determined by the standard DNA slot-blot assays were highly dependent on the specific probe being used, with probes hybridizing to the ends of viral genomes being significantly overrepresented compared with the probes hybridizing close to the middle of the viral genomes. These differences among various probes were not observed using Southern blot assays. This overestimation of titer is a systemic error during scAAV genome quantification, regardless of viral genome sequences and capsid serotypes. Furthermore, different serotypes capsid and modification of capsid sequence may affect the ability of packaging intact, full-length AAV genomes. Although the discrepancy is modest with wild-type serotype capsid and short viral genomes, the measured titer could be as much as fivefold different with capsid mutant vectors and large genomes. Thus, based on our data, we suggest that Southern blot analyses should be performed routinely to more accurately determine the titers of recombinant AAV vectors. At the very least, the use of probes/primers hybridizing close to the mutant inverted terminal repeat in scAAV genomes is recommended to avoid possible overestimation of vector titers.

Cytoplasmic Trafficking, Endosomal Escape, and Perinuclear Accumulation of Adeno-Associated Virus Type 2 Particles Are Facilitated by Microtubule Network

Understanding adeno-associated virus (AAV) trafficking is critical to advance our knowledge of AAV biology and exploit novel aspects of vector development. Similar to the case for most DNA viruses, after receptor binding and entry, AAV traverses the cytoplasm and deposits the viral genome in the cell nucleus. In this study, we examined the role of the microtubule (MT) network in productive AAV infection. Using pharmacological reagents (e.g., nocodazole), live-cell imaging, and flow cytometry analysis, we demonstrated that AAV type 2 (AAV2) transduction was reduced by at least 2-fold in the absence of the MT network. Cell surface attachment and viral internalization were not dependent on an intact MT network. In treated cells at 2 h postinfection, quantitative three-dimensional (3D) microscopy determined a reproducible difference in number of intracellular particles associated with the nuclear membrane or the nucleus compared to that for controls (6 to 7% versus 26 to 30%, respectively). Confocal microscopy analysis demonstrated a direct association of virions with MTs, further supporting a critical role in AAV infection. To investigate the underling mechanisms, we employed single-particle tracking (SPT) to monitor the viral movement in real time. Surprisingly, unlike other DNA viruses (e.g., adenovirus [Ad] and herpes simplex virus [HSV]) that display bidirectional motion on MTs, AAV2 displays only unidirectional movement on MTs toward the nuclei, with peak instantaneous velocities at 1.5 to 3.5 μm/s. This rapid and unidirectional motion on MTs lasts for about 5 to 10 s and results in AAV particles migrating more than 10 μm in the cytoplasm reaching the nucleus very efficiently. Furthermore, electron microscopy analysis determined that, unlike Ad and HSV, AAV2 particles were transported on MTs within membranous compartments, and surprisingly, the acidification of AAV2-containing endosomes was delayed by the disruption of MTs. These findings together suggest an as-yet-undescribed model in which after internalization, AAV2 exploits MTs for rapid cytoplasmic trafficking in endosomal compartments unidirectionally toward the perinuclear region, where most acidification events for viral escape take place.

Decreased expression of neuronal nitric oxide synthase in the nucleus tractus solitarii inhibits sympathetically mediated baroreflex responses in rat

Despite numerous studies it remains controversial whether nitric oxide (NO·) synthesized by neuronal NOS (nNOS) plays an excitatory or inhibitory role in transmission of baroreflex signals in the nucleus tractus solitarii (NTS). In the current studies we sought to test the hypothesis that nNOS is involved in excitation of baroreflex pathways in NTS while excluding pharmacological interventions in assessing the influence of nNOS. We therefore developed, validated and utilized a short hairpin RNA (shRNA) to reduce expression of nNOS in the NTS of rats whose baroreflex activity was then studied. We demonstrate downregulation of nNOS through transduction with adeno-associated virus type 2 (AAV2) carrying shRNA for nNOS. When injected bilaterally into NTS AAV2nNOSshRNA significantly reduced reflex tachycardic responses to acute hypotension while not affecting reflex bradycardic responses to acute increases of arterial pressure. Control animals treated with intravenous propranolol to block sympathetically mediated chronotropic responses manifested the same baroreflex responses as animals that had been treated with AAV2nNOSshRNA. Neither AAV2 eGFP nor AAV2nNOScDNA affected baroreflex responses. Blocking cardiac vagal influences with atropine similarly reduced baroreflex-mediated bradycardic responses to increases in arterial pressure both in control animals and in those treated with AAV2nNOSshRNA. We conclude that NO· synthesized by nNOS in the NTS is integral to excitation of baroreflex pathways involved in reflex tachycardia, a largely sympathetically mediated response, but not reflex bradycardia, a largely parasympathetically mediated response. We suggest that, at the basal state, nNOS is maximally engaged. Thus, its upregulation does not augment the baroreflex.

Restoration of Hearing in the VGLUT3 Knockout Mouse Using Virally Mediated Gene Therapy

Mice lacking the vesicular glutamate transporter-3 (VGLUT3) are congenitally deaf due to loss of glutamate release at the inner hair cell afferent synapse. Cochlear delivery of VGLUT3 using adeno-associated virus type 1 (AAV1) leads to transgene expression in only inner hair cells (IHCs), despite broader viral uptake. Within 2weeks of AAV1-VGLUT3 delivery, auditory brainstem response (ABR) thresholds normalize, along with partial rescue of the startle response. Lastly, we demonstrate partial reversal of the morphologic changes seen within the afferent IHC ribbon synapse. These findings representa successful restoration of hearing by gene replacement in mice, which is a significant advance toward gene therapy of human deafness.

The Threefold Protrusions of Adeno-Associated Virus Type 8 Are Involved in Cell Surface Targeting as Well as Postattachment Processing

Adeno-associated virus (AAV) has attracted considerable interest as a vector for gene therapy owing its lack of pathogenicity and the wealth of available serotypes with distinct tissue tropisms. One of the most promising isolates for vector development, based on its superior gene transfer efficiency to the liver in small animals compared to AAV type 2 (AAV2), is AAV8. Comparison of the in vivo gene transduction of rAAV2 and rAAV8 in mice showed that single amino acid exchanges in the 3-fold protrusions of AAV8 in the surface loops comprised of residues 581 to 584 and 589 to 592 to the corresponding amino acids of AAV2 and vice versa had a strong influence on transduction efficiency and tissue tropism. Surprisingly, not only did conversion of AAV8 to AAV2 cap sequences increase the transduction efficiency and change tissue tropism but so did the reciprocal conversion of AAV2 to AAV8. Insertion of new peptide motifs at position 590 in AAV8 also enabled retargeting of AAV8 capsids to specific tissues, suggesting that these sequences can interact with receptors on the cell surface. However, a neutralizing monoclonal antibody that binds to amino acids (588)QQNTA(592) of AAV8 does not prevent cell binding and virus uptake, indicating that this region is not necessary for receptor binding but rather that the antibody interferes with an essential step of postattachment processing in which the 3-fold protrusion is also involved. This study supports a multifunctional role of the 3-fold region of AAV capsids in the infection process.

Humoral and Cellular Capsid-Specific Immune Responses to Adeno-Associated Virus Type 1 in Randomized Healthy Donors

A major impediment to the use of adeno-associated virus (AAV)-mediated gene delivery to muscle in clinical applications is the pre-existing immune responses against the vector. Pre-existing humoral response to different AAV serotypes is now well documented. In contrast, cellular responses to AAV capsid have not been analyzed in a systematic manner, despite the risk of T cell reactivation upon gene transfer. AAV1 has been widely used in humans to target muscle. In this study, we analyzed PBMCs and sera of healthy donors for the presence of AAV1 capsid-specific T cell responses and AAV1 neutralizing factors. Approximately 30% of donors presented AAV1 capsid-specific T cells, mainly effector memory CD8(+) cells. IFN-γ-producing cells were also observed among effector memory CD4(+) cells for two of these donors. Moreover, to our knowledge, this study shows for the first time on a large cohort that there was no correlation between AAV1-specific T cell and humoral responses. Indeed, most donors presenting specific Ig and neutralizing factors were negative for cellular response (and vice versa). These new data raise the question of prescreening patients not only for the humoral response, but also for the cellular response. Clearly, a better understanding of the natural immunology of AAV serotypes will allow us to improve AAV gene therapy and make it an efficient treatment for genetic disease.