Genetic modification of scAAV-equine-BMP-2 transduced bone-marrow-derived mesenchymal stem cells before and after cryopreservation: An "off-the-shelf" option for fracture repair.

Optimizing the environment of complex bone healing and improving treatment of catastrophic bone fractures and segmental bone defects remains an unmet clinical need both human and equine veterinary medical orthopaedics. The objective of this study was to determine whether scAAV-equine-BMP-2 transduced cells would induce osteogenesis in equine bone marrow derived mesenchymal stem cells (BMDMSCs) in vitro, and if these cells could be cryopreserved in an effort to osteogenically prime them as an "off-the-shelf" gene therapeutic approach for fracture repair. Our study found that transgene expression is altered by cell expansion, as would be expected by a transduction resulting in episomal transgene expression, and that osteoinductive levels could still be achieved 5 days after recovery, and protein expression would continue up to 14 days after transduction. This is the first evidence that cryopreservation of genetically modified BMDMSCs would not alter the osteoinductive potential or clinical use of allogeneic donor cells in cases of equine fracture repair. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1310-1317, 2019.

Inhibition of antigen presentation during AAV gene therapy using virus peptides.

The clinical trial using adeno-associated virus (AAV) vector delivery of mini-dystrophin in patients with Duchenne Muscular Dystrophy (DMD) demonstrated a cytotoxic lymphocyte (CTL) response targeting the transgene product. These mini-dystrophin-specific T-cells have the potential to clear all transduced muscle, presenting the general gene therapy concern of overcoming the CTL response to foreign proteins that provide therapeutic benefit. In this study, we exploited a natural immunosuppression strategy employed by some viruses that results in CTL evasion only in transduced cells. After transfection of the plasmids encoding viral peptides and ovalbumin, which includes the immune-domain epitope SIINFEKL, several viral small peptides (ICP47 and US6) inhibited the SIINFEKL peptide presentation. A single AAV vector genome that consisted of either transgene AAT fused with SIINFEKL epitope and, separately, ICP47 expressed from different promoters or a single fusion protein with ICP47 linked by a furin cleavage peptide (AATOVA-ICP47) decreased antigen presentation. Compared with AAV/AATOVA in which decreased AAT expression was observed at late time points, persistent transgene expression was obtained after systemic administration of AAV/AATOVA-ICP47 vectors in mice. We extended this strategy to DMD gene therapy. After administration of AAV vector encoding human mini-dystrophin fusion protein with ICP47 into mdx mice, a lower mini-dystrophin-specific CTL response was induced. Importantly, the ICP47 fusion to mini-dystrophin inhibited CTLs mediated cytotoxicity. Although demonstrated herein using AAT and mini-dystrophin transgenes in an AAV context, the collective results have implications for all gene therapy applications resulting in foreign peptides by immune suppression in only genetically modified cells.

Gene Delivery of Activated Factor VII Using Alternative Adeno-Associated Virus Serotype Improves Hemostasis in Hemophiliac Mice with FVIII Inhibitors and Adeno-Associated Virus Neutralizing Antibodies.

While therapeutic expression of coagulation factors from adeno-associated virus (AAV) vectors has been successfully achieved in patients with hemophilia, neutralizing antibodies to the vector and inhibitory antibodies to the transgene severely limit efficacy. Indeed, approximately 40% of mice transduced with human factor VIII using the AAV8 serotype developed inhibitory antibodies to factor VIII (FVIII inhibitor), as well as extremely high titers (≥1:500) of neutralizing antibodies to AAV8. To correct hemophilia in these mice, AAV9, a serotype with low in vitro cross-reactivity (≤1:5) to anti-AAV8, was used to deliver mouse-activated factor VII (mFVIIa). It was found that within 6 weeks of systemic administration of 2 × 1013 particles/kg of AAV9/mFVIIa, hemophiliac mice with FVIII inhibitors and neutralizing antibodies (NAb) to AAV8 achieved hemostasis comparable to that in wild-type mice, as measured by rotational thromboelastometry. A level of 737 ng/mL mFVIIa was achieved after AAV9/mFVIIa adminstration compared to around 150 ng/mL without vector treatment, and concomitantly prothrombin time was shortened. Tissues collected after intra-articular hemorrhage from FVIII-deficient mice and mice with FVIII inhibitors were scored 4.7 and 5.5, respectively, on a scale of 0-10, indicating significant pathological damage. However, transduction with AAV9/mFVIIa decreased pathology scores to 3.6 and eliminated hemosiderin iron deposition in the synovium in most mice. Collectively, these results suggest that application of alternative serotypes of AAV vector to deliver bypassing reagents has the potential to correct hemophilia and prevent hemoarthrosis, even in the presence of FVIII inhibitor and neutralizing antibodies to AAV.

Biology of adeno-associated viral vectors in the central nervous system.

Gene therapy is a promising approach for treating a spectrum of neurological and neurodegenerative disorders by delivering corrective genes to the central nervous system (CNS). In particular, adeno-associated viruses (AAVs) have emerged as promising tools for clinical gene transfer in a broad range of genetic disorders with neurological manifestations. In the current review, we have attempted to bridge our understanding of the biology of different AAV strains with their transduction profiles, cellular tropisms, and transport mechanisms within the CNS. Continued efforts to dissect AAV-host interactions within the brain are likely to aid in the development of improved vectors for CNS-directed gene transfer applications in the clinic.

Engraftment of a galactose receptor footprint onto adeno-associated viral capsids improves transduction efficiency.

New viral strains can be evolved to recognize different host glycans through mutagenesis and experimental adaptation. However, such mutants generally harbor amino acid changes that affect viral binding to a single class of carbohydrate receptors. We describe the rational design and synthesis of novel, chimeric adeno-associated virus (AAV) strains that exploit an orthogonal glycan receptor for transduction. A dual glycan-binding AAV strain was first engineered as proof of concept by grafting a galactose (Gal)-binding footprint from AAV serotype 9 onto the heparan sulfate-binding AAV serotype 2. The resulting chimera, AAV2G9, continues to bind heparin affinity columns but interchangeably exploits Gal and heparan sulfate receptors for infection, as evidenced by competitive inhibition assays with lectins, glycans, and parental AAV strains. Although remaining hepatotropic like AAV2, the AAV2G9 chimera mediates rapid onset and higher transgene expression in mice. Similarly, engraftment of the Gal footprint onto the laboratory-derived strain AAV2i8 yielded an enhanced AAV2i8G9 chimera. This new strain remains liver-detargeted like AAV2i8 while selectively transducing muscle tissues at high efficiency, comparable with AAV9. The AAV2i8G9 chimera is a promising vector candidate for targeted gene therapy of cardiac and musculoskeletal diseases. In addition to demonstrating the modularity of glycan receptor footprints on viral capsids, our approach provides design strategies to expand the AAV vector toolkit.

Comparison of fetal mesencephalic grafts, AAV-delivered GDNF, and both combined in an MPTP-induced nonhuman primate Parkinson's model.

We combined viral vector delivery of human glial-derived neurotrophic factor (GDNF) with the grafting of dopamine (DA) precursor cells from fetal ventral mesencephalon (VM) to determine whether these strategies would improve the anti-Parkinson's effects in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated monkeys, an animal model for Parkinson's disease (PD). Both strategies have been reported as individually beneficial in animal models of PD, leading to clinical studies. GDNF delivery has also been reported to augment VM tissue implants, but no combined studies have been done in monkeys. Monkeys were treated with MPTP and placed into four balanced treatment groups receiving only recombinant adeno-associated virus serotype 5 (rAAV5)/hu-GDNF, only fetal DA precursor cells, both together, or a buffered saline solution (control). The combination of fetal precursors with rAAV5/hu-GDNF showed significantly higher striatal DA concentrations compared with the other treatments, but did not lead to greater functional improvement in this study. For the first time under identical conditions in primates, we show that all three treatments lead to improvement compared with control animals.

Overexpression of SOD in retina: need for increase in H2O2-detoxifying enzyme in same cellular compartment.

In retinitis pigmentosa (RP), various mutations cause rod photoreceptor cell death leading to increased oxygen levels in the outer retina, progressive oxidative damage to cones, and gradual loss of cone cell function. We have been exploring the potential of overexpressing components of the endogenous antioxidant defense system to preserve cone cell function in rd10(+/+) mice, a model of RP. rd10(+/+) mice deficient in superoxide dismutase 1 (SOD1) showed increased levels of superoxide radicals and carbonyl adducts (a marker of oxidative damage) in the retina and more rapid loss of cone function than rd10(+/+) mice with normal levels of SOD1. This suggests that SOD1 is an important component of the antioxidant defense system of cones, but increased expression of SOD1 in rd10(+/+) mice increased oxidative damage and accelerated the loss of cone function. Coexpression of SOD1 with glutathione peroxidase 4 (Gpx4), which like SOD1 is localized in the cytoplasm, but not with catalase targeted to the mitochondria, reduced oxidative damage in the retina and significantly slowed the loss of cone cell function in rd10(+/+) mice. Gene transfer resulting in increased expression of SOD2, but not coexpression of SOD2 and mitochondrial Gpx4, resulted in high levels of H(2)O(2) in the retina. These data suggest that to provide benefit in RP, overexpression of an SOD must be combined with expression of a peroxide-detoxifying enzyme in the same cellular compartment.

Reduced IL-2 expression in NOD mice leads to a temporal increase in CD62Llo FoxP3+ CD4+ T cells with limited suppressor activity.

IL-2 plays a critical role in the induction and maintenance of FoxP3-expressing regulatory T cells (FoxP3(+) Tregs). Reduced expression of IL-2 is linked to T-cell-mediated autoimmune diseases such as type 1 diabetes (T1D), in which an imbalance between FoxP3(+) Tregs and pathogenic T effectors exists. We investigated the contribution of IL-2 to dysregulation of FoxP3(+) Tregs by comparing wildtype NOD mice with animals congenic for a C57BL/6-derived disease-resistant Il2 allele and in which T-cell secretion of IL-2 is increased (NOD.B6Idd3). Although NOD mice exhibited a progressive decline in the frequency of CD62L(hi) FoxP3(+) Tregs due to an increase in CD62L(lo) FoxP3(+) Tregs, CD62L(hi) FoxP3(+) Tregs were maintained in the pancreatic lymph nodes and islets of NOD.B6Idd3 mice. Notably, the frequency of proliferating CD62L(hi) FoxP3(+) Tregs was elevated in the islets of NOD.B6Idd3 versus NOD mice. Increasing levels of IL-2 in vivo also resulted in larger numbers of CD62L(hi) FoxP3(+) Tregs in NOD mice. These results demonstrate that IL-2 influences the suppressor activity of the FoxP3(+) Tregs pool by regulating the balance between CD62L(lo) and CD62L(hi) FoxP3(+) Tregs. In NOD mice, reduced IL-2 expression leads to an increase in nonsuppressive CD62L(lo) FoxP3(+) Tregs, which in turn correlates with a pool of CD62L(hi) FoxP3(+) Tregs with limited proliferation.

Transient demyelination increases the efficiency of retrograde AAV transduction.

Adeno-associated virus (AAV) is capable of mediating retrograde viral transduction of central and peripheral neurons. This occurs at a relatively low efficiency, which we previously found to be dependent upon capsid serotype. We sought to augment retrograde transduction by providing increased axonal access to peripherally delivered AAV. Others have described utilizing full transection of peripheral nerves to mediate retrograde viral transduction of motor neurons. Here, we examined the ability of a transient demyelinating event to modulate levels of retrograde AAV transduction. Transient demyelination does not cause lasting functional deficits. Ethidium bromide (EtBr)-induced transient demyelination of the sciatic nerve resulted in significant elevation of retrograde transduction of both motor and sensory neurons. Retrograde transduction levels of motor neurons and heavily myelinated, large-diameter sensory neurons increased at least sixfold following peripheral delivery of self-complementary AAV serotype 1 (scAAV1) and serotype 2 (scAAV2), when preceded by demyelination. These findings identify a means of significantly enhancing retrograde vector transport for use in experimental paradigms requiring either retrograde neuronal identification and gene expression, or translational treatment paradigms.

Self-complementary AAV virus (scAAV) safe and long-term gene transfer in the trabecular meshwork of living rats and monkeys.

PURPOSE: AAV vectors produce stable transgene expression and elicit low immune response in many tissues. AAVs have been the vectors of choice for gene therapy for the eye, in particular the retina. scAAVs are modified AAVs that bypass the required second-strand DNA synthesis to achieve transcription of the transgene. The goal was to investigate the ability of AAV vectors to induce long-term, safe delivery of transgenes to the trabecular meshwork of living animals. METHODS: Single doses of AAV2.GFP and AAV2.RGD.GFP/Ad5.LacZ were injected intracamerally (IC) into rats (n = 28 eyes). A single dose of scAAV.GFP was IC-injected into rats (n = 72 eyes) and cynomolgus monkeys (n = 3). GFP expression was evaluated by fluorescence, immunohistochemistry, and noninvasive gonioscopy. Intraocular pressure (IOP) was measured with calibrated tonometer (rats) and Goldmann tonometer (monkeys). Differential expression of scAAV-infected human trabecular meshwork cells (HTM) was determined by microarrays. Humoral and cell-mediated immune responses were evaluated by ELISA and peripheral blood proliferation assays. RESULTS: No GFP transduction was observed on the anterior segment tissues of AAV-injected rats up to 27 days after injection. In contrast, scAAV2 transduced the trabecular meshwork very efficiently, with a fast onset (4 days). Eyes remained clear and no adverse effects were observed. Transgene expression lasted >3.5 months in rats and >2.35 years in monkeys. CONCLUSIONS: The scAAV viral vector provides prolonged and safe transduction in the trabecular meshwork of rats and monkeys. The stable expression and safe properties of this vector could facilitate the development of trabecular meshwork drugs for gene therapy for glaucoma.

Engineering and selection of shuffled AAV genomes: a new strategy for producing targeted biological nanoparticles.

We report a DNA shuffling-based approach for developing cell type-specific vectors through directed evolution. Capsid genomes of adeno-associated virus (AAV) serotypes 1-9 were randomly fragmented and reassembled using PCR to generate a chimeric capsid library. A single infectious clone (chimeric-1829) containing genome fragments from AAV1, 2, 8, and 9 was isolated from an integrin minus hamster melanoma cell line previously shown to have low permissiveness to AAV. Molecular modeling studies suggest that AAV2 contributes to surface loops at the icosahedral threefold axis of symmetry, while AAV1 and 9 contribute to two- and fivefold symmetry interactions, respectively. The C-terminal domain (AAV9) was identified as a critical structural determinant of melanoma tropism through rational mutagenesis. Chimeric-1829 utilizes heparan sulfate as a primary receptor and transduces melanoma cells more efficiently than all serotypes. Further, chimeric-1829 demonstrates altered tropism in rodent skeletal muscle, liver, and brain including nonhuman primates. We determined a unique immunological profile based on neutralizing antibody (NAb) titer and crossreactivity studies strongly supporting isolation of a synthetic laboratory-derived capsid variant. Application of this technology to alternative cell/tissue types using AAV or other viral capsid sequences is likely to yield a new class of biological nanoparticles as vectors for human gene transfer.

Efficient retrograde neuronal transduction utilizing self-complementary AAV1.

Adeno-associated virus (AAV) is frequently used for gene transfer into the central nervous system (CNS). Similar to adenovirus and rabies virus, AAV can be taken up by axons and retrogradely transported, resulting in neuronal gene expression distant from the injection site. We investigated the retrograde transport properties of self-complementary AAV (scAAV) serotypes 1-6 following peripheral injection. Injection of scAAV into either rat extensor carpi muscle or sciatic nerve resulted in detectable retrograde vector transport and reporter gene expression in spinal cord motor neurons (MNs). Serotype 1 resulted in the highest level of retrograde transport, with 4.1 +/- 0.3% of cervical MNs projecting to the extensor carpi transduced following intramuscular injection, and 7.5 +/- 3.1% of lumbar MNs transduced after sciatic nerve injection. In contrast to scAAV1, retrograde transduction with scAAV2 was undetectable following intramuscular injection, and was detected in only 0.81 +/- 0.15% of MNs projecting to the sciatic nerve following intranerve injection. Furthermore, sciatic injection of single-stranded AAV1 required injection of tenfold higher numbers of viral particles for detectable transgene expression compared to scAAV1, and then only 0.91 +/- 0.24% of lumbar MNs were transduced. Our data provide the basis for increased retrograde transduction efficiency using peripheral injections of scAAV1 vectors for therapeutic gene delivery to the spinal cord.

Engineering and Selection of Shuffled AAV Genomes: A New Strategy for Producing Targeted Biological Nanoparticles.

We report a DNA shuffling-based approach for developing cell type-specific vectors through directed evolution. Capsid genomes of adeno-associated virus (AAV) serotypes 1-9 were randomly fragmented and reassembled using PCR to generate a chimeric capsid library. A single infectious clone (chimeric-1829) containing genome fragments from AAV1, 2, 8, and 9 was isolated from an integrin minus hamster melanoma cell line previously shown to have low permissiveness to AAV. Molecular modeling studies suggest that AAV2 contributes to surface loops at the icosahedral threefold axis of symmetry, while AAV1 and 9 contribute to two- and fivefold symmetry interactions, respectively. The C-terminal domain (AAV9) was identified as a critical structural determinant of melanoma tropism through rational mutagenesis. Chimeric-1829 utilizes heparan sulfate as a primary receptor and transduces melanoma cells more efficiently than all serotypes. Further, chimeric-1829 demonstrates altered tropism in rodent skeletal muscle, liver, and brain including nonhuman primates. We determined a unique immunological profile based on neutralizing antibody (NAb) titer and crossreactivity studies strongly supporting isolation of a synthetic laboratory-derived capsid variant. Application of this technology to alternative cell/tissue types using AAV or other viral capsid sequences is likely to yield a new class of biological nanoparticles as vectors for human gene transfer.

Efficient Retrograde Neuronal Transduction Utilizing Self-complementary AAV1.

Adeno-associated virus (AAV) is frequently used for gene transfer into the central nervous system (CNS). Similar to adenovirus and rabies virus, AAV can be taken up by axons and retrogradely transported, resulting in neuronal gene expression distant from the injection site. We investigated the retrograde transport properties of self-complementary AAV (scAAV) serotypes 1-6 following peripheral injection. Injection of scAAV into either rat extensor carpi muscle or sciatic nerve resulted in detectable retrograde vector transport and reporter gene expression in spinal cord motor neurons (MNs). Serotype 1 resulted in the highest level of retrograde transport, with 4.1 ± 0.3% of cervical MNs projecting to the extensor carpi transduced following intramuscular injection, and 7.5 ± 3.1% of lumbar MNs transduced after sciatic nerve injection. In contrast to scAAV1, retrograde transduction with scAAV2 was undetectable following intramuscular injection, and was detected in only 0.81 ± 0.15% of MNs projecting to the sciatic nerve following intranerve injection. Furthermore, sciatic injection of single-stranded AAV1 required injection of tenfold higher numbers of viral particles for detectable transgene expression compared to scAAV1, and then only 0.91 ± 0.24% of lumbar MNs were transduced. Our data provide the basis for increased retrograde transduction efficiency using peripheral injections of scAAV1 vectors for therapeutic gene delivery to the spinal cord.

Optimization of self-complementary AAV vectors for liver-directed expression results in sustained correction of hemophilia B at low vector dose.

Self-complementary adeno-associated virus (scAAV) vectors can significantly minimize the vector load required to achieve sustained transgene expression. In this study, transcriptional regulatory elements were systematically screened to produce constitutive and liver-specific scAAV factor IX (FIX) expression cassettes. In addition, optimization of GC content, cis- regulatory elements, and codon usage in the human FIX (hFIX) transgene increased expression 4-20-fold. A vector was developed that was capable of expressing high FIX levels in comparison with the single-stranded (ss) AAV vector used in a recent clinical trial. The ssAAV and scAAV vectors display different transgene expression and genome stability patterns in the liver, as determined by immunohistochemical staining, in situ messenger RNA (mRNA) hybridization and vector genome quantitation. The ssAAV2 vector promoted strong FIX expression in only a subset of hepatocytes. The scAAV2-hFIX vector showed widespread ( approximately 80% of hepatocytes), moderate FIX expression levels similar to normal livers with correction of coagulation function in FIX-deficient mice. The ability of low dose scAAV-FIX vectors to achieve near-physiological expression may circumvent inflammatory responses in the liver. In addition to providing an improved scAAV vector for potential application in future hemophilia B clinical trials and liver-directed gene delivery, these studies underscore the need for rigorous analysis and optimization of vector genome cassettes.

Adeno-associated virus type 2 contains an integrin alpha5beta1 binding domain essential for viral cell entry.

Integrins have been implicated as coreceptors in the infectious pathways of several nonenveloped viruses. For example, adenoviruses are known to interact with alphaV integrins by virtue of a high-affinity arginine-glycine-aspartate (RGD) domain present in the penton bases of the capsids. In the case of adeno-associated virus type 2 (AAV2), which lacks this RGD motif, integrin alphaVbeta5 has been identified as a coreceptor for cellular entry. However, the molecular determinants of AAV2 capsid-integrin interactions and the potential exploitation of alternative integrins as coreceptors by AAV2 have not been established thus far. In this report, we demonstrate that integrin alpha5beta1 serves as an alternative coreceptor for AAV2 infection in human embryonic kidney 293 cells. Such interactions appear to be mediated by a highly conserved domain that contains an asparagine-glycine-arginine (NGR) motif known to bind alpha5beta1 integrin with moderate affinity. The mutation of this domain reduces transduction efficiency by an order of magnitude relative to that of wild-type AAV2 vectors in vitro and in vivo. Further characterization of mutant and wild-type AAV2 capsids through transduction assays in cell lines lacking specific integrins, cell adhesion studies, and cell surface/solid-phase binding assays confirmed the role of the NGR domain in promoting AAV2-integrin interactions. Molecular modeling studies suggest that NGR residues form a surface loop close to the threefold axis of symmetry adjacent to residues previously implicated in binding heparan sulfate, the primary receptor for AAV2. The aforementioned results suggest that the internalization of AAV2 in 293 cells might follow a "click-to-fit" mechanism that involves the cooperative binding of heparan sulfate and alpha5beta1 integrin by the AAV2 capsids.

Separate basic region motifs within the adeno-associated virus capsid proteins are essential for infectivity and assembly.

Adeno-associated virus (AAV) is gaining momentum as a gene therapy vector for human applications. However, there remain impediments to the development of this virus as a vector. One of these is the incomplete understanding of the biology of the virus, including nuclear targeting of the incoming virion during initial infection, as well as assembly of progeny virions from structural components in the nucleus. Toward this end, we have identified four basic regions (BR) on the AAV2 capsid that represent possible nuclear localization sequence (NLS) motifs. Mutagenesis of BR1 ((120)QAKKRVL(126)) and BR2 ((140)PGKKRPV(146)) had minor effects on viral infectivity ( approximately 4- and approximately 10-fold, respectively), whereas BR3 ((166)PARKRLN(172)) and BR4 ((307)RPKRLN(312)) were found to be essential for infectivity and virion assembly, respectively. Mutagenesis of BR3, which is located in Vp1 and Vp2 capsid proteins, does not interfere with viral production or trafficking of intact AAV capsids to the nuclear periphery but does inhibit transfer of encapsidated DNA into the nucleus. Substitution of the canine parvovirus NLS rescued the BR3 mutant to wild-type (wt) levels, supporting the role of an AAV NLS motif. In addition, rAAV2 containing a mutant form of BR3 in Vp1 and a wt BR3 in Vp2 was found to be infectious, suggesting that the function of BR3 is redundant between Vp1 and Vp2 and that Vp2 may play a role in infectivity. Mutagenesis of BR4 was found to inhibit virion assembly in the nucleus of transfected cells. This affect was not completely due to the inefficient nuclear import of capsid subunits based on Western blot analysis. In fact, aberrant capsid foci were observed in the cytoplasm of transfected cells, compared to the wild type, suggesting a defect in early viral assembly or trafficking. Using three-dimensional structural analysis, the lysine- and arginine-to-asparagine change disrupts hydrogen bonding between these basic residues and adjacent beta strand glutamine residues that may prevent assembly of intact virions. Taken together, these data support that the BR4 domain is essential for virion assembly. Each BR was also found to be conserved in serotypes 1 to 11, suggesting that these regions are significant and function similarly in each serotype. This study establishes the importance of two BR motifs on the AAV2 capsid that are essential for infectivity and virion assembly.

Mechanisms of AAV transduction in glaucoma-associated human trabecular meshwork cells.

BACKGROUND: Glaucoma is a chronic eye disease which leads to irreversible blindness. The trabecular meshwork tissue controls intraocular pressure (IOP), which is the major risk factor for glaucoma. Gene therapy treatment of chronic diseases requires the use of long-term expression, low toxicity and lack of immune response vectors. Adeno-associated viruses (AAV) possess these characteristics but have been unable to transduce the trabecular meshwork. Because of the importance of regulating elevated IOP by long-term gene therapy, we investigated mechanisms of AAV transduction to the human trabecular meshwork (TM). METHODS: Primary human trabecular meshwork cells (HTM) and perfused organ cultures were infected with rAAV2-GFP, RGD-pseudotyped rAAV2-GFP alone, or combined with recombinant DeltaE1/E3 adenoviruses. Intracellular rAAV2 DNA and RNA were measured by relative quantitative and real-time TaqMan polymerase chain reaction (PCR). Host transcriptome was analyzed using high-density oligonucleotide microarrays. One transduction mechanism was tested using self-complementary AAV (scAAV). RESULTS: The dramatic transduction enhancement obtained upon co-infection of rAAV2 with DeltaE1/E3 adenoviruses provides insights into transduction mechanisms in the HTM. Even if not transduced, rAAV2 enters TM cells. GeneChip analysis showed significant changes in host genes involved in cell cycle and DNA replication. Consequently, scAAV-GFP transduction was highly efficient. Other transduction-enhancement genes included coxsackie adenovirus receptor (CAR) and genes relevant to trabecular meshwork function. CONCLUSIONS: The rate-limiting step of AAV transduction was not viral entry failure but, at least in part, host downregulation of DNA replication. Additional specific host genes might be involved. Our study revealed genes and mechanisms which led for the first time to efficient AAV transduction of the HTM.