Therapeutic microRNA delivery suppresses tumorigenesis in a murine liver cancer model.

Therapeutic strategies based on modulation of microRNA (miRNA) activity hold great promise due to the ability of these small RNAs to potently influence cellular behavior. In this study, we investigated the efficacy of a miRNA replacement therapy for liver cancer. We demonstrate that hepatocellular carcinoma (HCC) cells exhibit reduced expression of miR-26a, a miRNA that is normally expressed at high levels in diverse tissues. Expression of this miRNA in liver cancer cells in vitro induces cell-cycle arrest associated with direct targeting of cyclins D2 and E2. Systemic administration of this miRNA in a mouse model of HCC using adeno-associated virus (AAV) results in inhibition of cancer cell proliferation, induction of tumor-specific apoptosis, and dramatic protection from disease progression without toxicity. These findings suggest that delivery of miRNAs that are highly expressed and therefore tolerated in normal tissues but lost in disease cells may provide a general strategy for miRNA replacement therapies.

Micro-dystrophin and follistatin co-delivery restores muscle function in aged DMD model.

Pharmacologic strategies have provided modest improvement in the devastating muscle-wasting disease, Duchenne muscular dystrophy (DMD). Pre-clinical gene therapy studies have shown promise in the mdx mouse model; however, studies conducted after disease onset fall short of fully correcting muscle strength or protecting against contraction-induced injury. Here we examine the treatment effect on muscle physiology in aged dystrophic mice with significant disease pathology by combining two promising therapies: micro-dystrophin gene replacement and muscle enhancement with follistatin, a potent myostatin inhibitor. Individual treatments with micro-dystrophin and follistatin demonstrated marked improvement in mdx mice but were insufficient to fully restore muscle strength and response to injury to wild-type levels. Strikingly, when combined, micro-dystrophin/follistatin treatment restored force generation and conferred resistance to contraction-induced injury in aged mdx mice. Pre-clinical studies with miniature dystrophins have failed to demonstrate full correction of the physiological defects seen in mdx mice. Importantly, the addition of a muscle enhancement strategy with delivery of follistatin in combination with micro-dystrophin gene therapy completely restored resistance to eccentric contraction-induced injury and improved force. Eccentric contraction-induced injury is a pre-clinical parameter relevant to the exercise induced injury that occurs in DMD patients, and herein, we demonstrate compelling evidence for the therapeutic potential of micro-dystrophin/follistatin combinatorial therapy.

Retinal gene therapy in patients with choroideremia: initial findings from a phase 1/2 clinical trial.

BACKGROUND: Choroideremia is an X-linked recessive disease that leads to blindness due to mutations in the CHM gene, which encodes the Rab escort protein 1 (REP1). We assessed the effects of retinal gene therapy with an adeno-associated viral (AAV) vector encoding REP1 (AAV.REP1) in patients with this disease. METHODS: In a multicentre clinical trial, six male patients (aged 35-63 years) with choroideremia were administered AAV.REP1 (0·6-1·0×10(10) genome particles, subfoveal injection). Visual function tests included best corrected visual acuity, microperimetry, and retinal sensitivity tests for comparison of baseline values with 6 months after surgery. This study is registered with ClinicalTrials.gov, number NCT01461213. FINDINGS: Despite undergoing retinal detachment, which normally reduces vision, two patients with advanced choroideremia who had low baseline best corrected visual acuity gained 21 letters and 11 letters (more than two and four lines of vision). Four other patients with near normal best corrected visual acuity at baseline recovered to within one to three letters. Mean gain in visual acuity overall was 3·8 letters (SE 4·1). Maximal sensitivity measured with dark-adapted microperimetry increased in the treated eyes from 23·0 dB (SE 1·1) at baseline to 25·3 dB (1·3) after treatment (increase 2·3 dB [95% CI 0·8-3·8]). In all patients, over the 6 months, the increase in retinal sensitivity in the treated eyes (mean 1·7 [SE 1·0]) was correlated with the vector dose administered per mm(2) of surviving retina (r=0·82, p=0·04). By contrast, small non-significant reductions (p>0·05) were noted in the control eyes in both maximal sensitivity (-0·8 dB [1·5]) and mean sensitivity (-1·6 dB [0·9]). One patient in whom the vector was not administered to the fovea re-established variable eccentric fixation that included the ectopic island of surviving retinal pigment epithelium that had been exposed to vector. INTERPRETATION: The initial results of this retinal gene therapy trial are consistent with improved rod and cone function that overcome any negative effects of retinal detachment. These findings lend support to further assessment of gene therapy in the treatment of choroideremia and other diseases, such as age-related macular degeneration, for which intervention should ideally be applied before the onset of retinal thinning. FUNDING: UK Department of Health and Wellcome Trust.

Vascular delivery of rAAVrh74.MCK.GALGT2 to the gastrocnemius muscle of the rhesus macaque stimulates the expression of dystrophin and laminin α2 surrogates.

Overexpression of GALGT2 in skeletal muscle can stimulate the glycosylation of α dystroglycan and the upregulation of normally synaptic dystroglycan-binding proteins, some of which are dystrophin and laminin α2 surrogates known to be therapeutic for several forms of muscular dystrophy. This article describes the vascular delivery of GALGT2 gene therapy in a large animal model, the rhesus macaque. Recombinant adeno-associated virus, rhesus serotype 74 (rAAVrh74), was used to deliver GALGT2 via the femoral artery to the gastrocnemius muscle using an isolated focal limb perfusion method. GALGT2 expression averaged 44 ± 4% of myofibers after treatment in macaques with low preexisting anti-rAAVrh74 serum antibodies, and expression was reduced to 9 ± 4% of myofibers in macaques with high preexisting rAAVrh74 immunity (P < 0.001; n = 12 per group). This was the case regardless of the addition of immunosuppressants, including prednisolone, tacrolimus, and mycophenolate mofetil. GALGT2-treated macaque muscles showed increased glycosylation of α dystroglycan and increased expression of dystrophin and laminin α2 surrogate proteins, including utrophin, plectin1, agrin, and laminin α5. These experiments demonstrate successful transduction of rhesus macaque muscle with rAAVrh74.MCK.GALGT2 after vascular delivery and induction of molecular changes thought to be therapeutic in several forms of muscular dystrophy.

AAV-mediated overexpression of human α7 integrin leads to histological and functional improvement in dystrophic mice.

Duchenne muscular dystrophy (DMD) is a severe muscle disease caused by mutations in the DMD gene, with loss of its gene product, dystrophin. Dystrophin helps link integral membrane proteins to the actin cytoskeleton and stabilizes the sarcolemma during muscle activity. We investigated an alternative therapeutic approach to dystrophin replacement by overexpressing human α7 integrin (ITGA7) using adeno-associated virus (AAV) delivery. ITGA7 is a laminin receptor in skeletal and cardiac muscle that links the extracellular matrix (ECM) to the actin skeleton. It is modestly upregulated in DMD muscle and has been proposed to be an important modifier of dystrophic symptoms. We delivered rAAV8.MCK.ITGA7 to the lower limb of mdx mice through isolated limb perfusion (ILP) of the femoral artery. We demonstrated ~50% of fibers in the tibialis anterior (TA) and extensor digitorum longus (EDL) overexpressing α7 integrin at the sarcolemma following AAV gene transfer. The increase in ITGA7 in skeletal muscle significantly protected against loss of force following eccentric contraction-induced injury compared with untreated (contralateral) muscles while specific force following tetanic contraction was unchanged. Reversal of additional dystrophic features included reduced Evans blue dye (EBD) uptake and increased muscle fiber diameter. Taken together, this data shows that rAAV8.MCK.ITGA7 gene transfer stabilizes the sarcolemma potentially preserving mdx muscle from further damage. This therapeutic approach demonstrates promise as a viable treatment for DMD with further implications for other forms of muscular dystrophy.

Dystrophin immunity in Duchenne's muscular dystrophy.

We report on delivery of a functional dystrophin transgene to skeletal muscle in six patients with Duchenne's muscular dystrophy. Dystrophin-specific T cells were detected after treatment, providing evidence of transgene expression even when the functional protein was not visualized in skeletal muscle. Circulating dystrophin-specific T cells were unexpectedly detected in two patients before vector treatment. Revertant dystrophin fibers, which expressed functional, truncated dystrophin from the deleted endogenous gene after spontaneous in-frame splicing, contained epitopes targeted by the autoreactive T cells. The potential for T-cell immunity to self and nonself dystrophin epitopes should be considered in designing and monitoring experimental therapies for this disease. (Funded by the Muscular Dystrophy Association and others; ClinicalTrials.gov number, NCT00428935.).

Rapid and efficient clearance of blood-borne virus by liver sinusoidal endothelium.

The liver removes quickly the great bulk of virus circulating in blood, leaving only a small fraction to infect the host, in a manner characteristic of each virus. The scavenger cells of the liver sinusoids are implicated, but the mechanism is entirely unknown. Here we show, borrowing a mouse model of adenovirus clearance, that nearly all infused adenovirus is cleared by the liver sinusoidal endothelial cell (LSEC). Using refined immunofluorescence microscopy techniques for distinguishing macrophages and endothelial cells in fixed liver, and identifying virus by two distinct physicochemical methods, we localized adenovirus 1 minute after infusion mainly to the LSEC (∼90%), finding ∼10% with Kupffer cells (KC) and none with hepatocytes. Electron microscopy confirmed our results. In contrast with much prior work claiming the main scavenger to be the KC, our results locate the clearance mechanism to the LSEC and identify this cell as a key site of antiviral activity.

Impaired regeneration in LGMD2A supported by increased PAX7-positive satellite cell content and muscle-specific microrna dysregulation.

INTRODUCTION: Recent in vitro studies suggest that CAPN3 deficiency leads initially to accelerated myofiber formation followed by depletion of satellite cells (SC). In normal muscle, up-regulation of miR-1 and miR-206 facilitates transition from proliferating SCs to differentiating myogenic progenitors. METHODS: We examined the histopathological stages, Pax7 SC content, and muscle-specific microRNA expression in biopsy specimens from well-characterized LGMD 2A patients to gain insight into disease pathogenesis. RESULTS: Three distinct stages of pathological changes were identified that represented the continuum of the dystrophic process from prominent inflammation with necrosis and regeneration to prominent fibrosis, which correlated with age and disease duration. Pax7-positive SCs were highest in the fibrotic group and correlated with down-regulation of miR-1, miR-133a, and miR-206. CONCLUSIONS: These observations, and other published reports, are consistent with microRNA dysregulation leading to inability of Pax7-positive SCs to transit from proliferation to differentiation. This results in impaired regeneration and fibrosis.

Knee extensor strength exhibits potential to predict function in sporadic inclusion-body myositis.

INTRODUCTION: In this study we address the challenging issue of potential use of muscle strength to predict function in clinical trials. This has immediate relevance to translational studies that attempt to improve quadriceps strength in sporadic inclusion-body myositis (sIBM). METHODS: Maximum voluntary isometric contraction testing as a measure of muscle strength and a battery of functional outcomes were tested in 85 ambulatory subjects with sIBM. RESULTS: Marked quadriceps weakness was noted in all patients. Strength was correlated with distance walked at 2 and 6 minutes. Additional correlations were found with time to get up from a chair, climb stairs, and step up on curbs. CONCLUSIONS: Quadriceps (knee extensor) strength correlated with performance in this large cohort of sIBM subjects, which demonstrated its potential to predict function in this disease. These data provide initial support for use of muscle strength as a surrogate for function, although validation in a clinical trial is required.

Sustained alpha-sarcoglycan gene expression after gene transfer in limb-girdle muscular dystrophy, type 2D.

OBJECTIVE: The aim of this study was to attain long-lasting alpha-sarcoglycan gene expression in limb-girdle muscular dystrophy, type 2D (LGMD2D) subjects mediated by adeno-associated virus (AAV) gene transfer under control of a muscle specific promoter (tMCK). METHODS: rAAV1.tMCK.hSGCA (3.25 × 10¹¹ vector genomes) was delivered to the extensor digitorum brevis muscle of 3 subjects with documented SGCA mutations via a double-blind, randomized, placebo controlled trial. Control sides received saline. The blind was not broken until the study was completed at 6 months and all results were reported to the oversight committee. RESULTS: Persistent alpha-sarcoglycan gene expression was achieved for 6 months in 2 of 3 LGMD2D subjects. Markers for muscle fiber transduction other than alpha-sarcoglycan included expression of major histocompatibility complex I, increase in muscle fiber size, and restoration of the full sarcoglycan complex. Mononuclear inflammatory cells recruited to the site of gene transfer appeared to undergo programmed cell death, demonstrated by terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate nick-end labeling and caspase-3 staining. A patient failing gene transfer demonstrated an early rise in neutralizing antibody titers and T-cell immunity to AAV, validated by enzyme-linked immunospot on the second day after gene injection. This was in clear distinction to other participants with satisfactory gene expression. INTERPRETATION: The findings of this gene replacement study in LGMD2D subjects have important implications not previously demonstrated in muscular dystrophy. Long-term, sustainable gene expression of alpha-sarcoglycan was observed following gene transfer mediated by AAV. The merit of a muscle-specific tMCK promoter, not previously used in a clinical trial, was evident, and the potential for reversal of disease was displayed.

Persistent expression of FLAG-tagged micro dystrophin in nonhuman primates following intramuscular and vascular delivery.

Animal models for Duchenne muscular dystrophy (DMD) have species limitations related to assessing function, immune response, and distribution of micro- or mini-dystrophins. Nonhuman primates (NHPs) provide the ideal model to optimize vector delivery across a vascular barrier and provide accurate dose estimates for widespread transduction. To address vascular delivery and dosing in rhesus macaques, we have generated a fusion construct that encodes an eight amino-acid FLAG epitope at the C-terminus of micro-dystrophin to facilitate translational studies targeting DMD. Intramuscular (IM) injection of AAV8.MCK.micro-dys.FLAG in the tibialis anterior (TA) of macaques demonstrated robust gene expression, with muscle transduction (50-79%) persisting for up to 5 months. Success by IM injection was followed by targeted vascular delivery studies using a fluoroscopy-guided catheter threaded through the femoral artery. Three months after gene transfer, >80% of muscle fibers showed gene expression in the targeted muscle. No cellular immune response to AAV8 capsid, micro-dystrophin, or the FLAG tag was detected by interferon-gamma (IFN-gamma) enzyme-linked immunosorbent spot (ELISpot) at any time point with either route. In summary, an epitope-tagged micro-dystrophin cassette enhances the ability to evaluate site-specific localization and distribution of gene expression in the NHP in preparation for vascular delivery clinical trials.

Infectious molecular clones of adeno-associated virus isolated directly from human tissues.

Adeno-associated virus (AAV) replication and biology have been extensively studied using cell culture systems, but there is precious little known about AAV biology in natural hosts. As part of our ongoing interest in the in vivo biology of AAV, we previously described the existence of extrachromosomal proviral AAV genomes in human tissues. In the current work, we describe the molecular structure of infectious DNA clones derived directly from these tissues. Sequence-specific linear rolling-circle amplification was utilized to isolate clones of native circular AAV DNA. Several molecular clones containing unit-length viral genomes directed the production of infectious wild-type AAV upon DNA transfection in the presence of adenovirus help. DNA sequence analysis of the molecular clones revealed the ubiquitous presence of a double-D inverted terminal repeat (ITR) structure, which implied a mechanism by which the virus is able to maintain ITR sequence continuity and persist in the absence of host chromosome integration. These data suggest that the natural life cycle of AAV, unlike that of retroviruses, might not have genome integration as an obligatory component.

Limb-girdle muscular dystrophy type 2D gene therapy restores alpha-sarcoglycan and associated proteins.

OBJECTIVE: alpha-Sarcoglycan deficiency results in a severe form of muscular dystrophy (limb-girdle muscular dystrophy type 2D [LGMD2D]) without treatment. Gene replacement represents a strategy for correcting the underlying defect. Questions related to this approach were addressed in this clinical trial, particularly the need for immunotherapy and persistence of gene expression. METHODS: A double-blind, randomized controlled trial using rAAV1.tMCK.hSGCA injected into the extensor digitorum brevis muscle was conducted. Control sides received saline. A 3-day course of methylprednisolone accompanied gene transfer without further immune suppression. RESULTS: No adverse events were encountered. SGCA gene expression increased 4-5-fold over control sides when examined at 6 weeks (2 subjects) and 3 months (1 subject). The full sarcoglycan complex was restored in all subjects, and muscle fiber size was increased in the 3-month subject. Adeno-associated virus serotype 1 (AAV1)-neutralizing antibodies were seen as early as 2 weeks. Neither CD4+ nor CD8+ cells were increased over contralateral sides. Scattered foci of inflammation could be found, but showed features of programmed cell death. Enzyme-linked immunospot (ELISpot) showed no interferon-gamma response to alpha-SG or AAV1 capsid peptide pools, with the exception of a minimal capsid response in 1 subject. Restimulation to detect low-frequency capsid-specific T cells by ELISpot assays was negative. Results of the first 3 subjects successfully achieved study aims, precluding the need for additional enrollment. INTERPRETATION: The finding of this gene replacement study in LGMD2D has important implications for muscular dystrophy. Sustained gene expression was seen, but studies over longer time periods without immunotherapy will be required for design of vascular delivery gene therapy trials.

Physiological properties of hypothalamic MCH neurons identified with selective expression of reporter gene after recombinant virus infection.

Neurons that synthesize melanin-concentrating hormone (MCH) may modulate arousal and energy homeostasis. The scattered MCH neurons have been difficult to study, as they have no defining morphological characteristics. We have developed a viral approach with AAV for selective long-term reporter gene (GFP) expression in MCH neurons, allowing the study of their cellular physiology in hypothalamic slices. MCH neurons showed distinct membrane properties compared to other neurons infected with the same virus with a cytomegalovirus promoter. Transmitters of extrahypothalamic arousal systems, including norepinephrine, serotonin, and the acetylcholine agonist muscarine, evoked direct inhibitory actions. Orexigenic neuropeptide Y was inhibitory by pre- and postsynaptic mechanisms; an anorexigenic melanocortin agonist had no effect. In contrast, the hypothalamic arousal peptide hypocretin/orexin evoked a direct inward current and increased excitatory synaptic activity and spike frequency in the normally silent MCH neurons. Together, these data support the view that MCH neurons may integrate information within the arousal system in favor of energy conservation.

Safety and Efficacy of AAV Retrograde Pancreatic Ductal Gene Delivery in Normal and Pancreatic Cancer Mice.

Recombinant adeno-associated virus (rAAV)-mediated gene delivery shows promise to transduce the pancreas, but safety/efficacy in a neoplastic context is not well established. To identify an ideal AAV serotype, route, and vector dose and assess safety, we have investigated the use of three AAV serotypes (6, 8, and 9) expressing GFP in a self-complementary (sc) AAV vector under an EF1α promoter (scAAV.GFP) following systemic or retrograde pancreatic intraductal delivery. Systemic delivery of scAAV9.GFP transduced the pancreas with high efficiency, but gene expression did not exceed >45% with the highest dose, 5 × 1012 viral genomes (vg). Intraductal delivery of 1 × 1011 vg scAAV6.GFP transduced acini, ductal cells, and islet cells with >50%, ∼48%, and >80% efficiency, respectively, and >80% pancreatic transduction was achieved with 5 × 1011 vg. In a KrasG12D-driven pancreatic cancer mouse model, intraductal delivery of scAAV6.GFP targeted acini, epithelial, and stromal cells and exhibited persistent gene expression 5 months post-delivery. In normal mice, intraductal delivery induced a transient increase in serum amylase/lipase that resolved within a day of infusion with no sustained pancreatic inflammation or fibrosis. Similarly, in PDAC mice, intraductal delivery did not increase pancreatic intraepithelial neoplasia progression/fibrosis. Our study demonstrates that scAAV6 targets the pancreas/neoplasm efficiently and safely via retrograde pancreatic intraductal delivery.

A translational approach for limb vascular delivery of the micro-dystrophin gene without high volume or high pressure for treatment of Duchenne muscular dystrophy.

BACKGROUND: Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder with monogenic mutations setting the stage for successful gene therapy treatment. We have completed a study that directly deals with the following key issues that can be directly adapted to a gene therapy clinical trial using rAAV considering the following criteria: 1) A regional vascular delivery approach that will protect the patient from widespread dissemination of virus; 2) an approach to potentially facilitate safe passage of the virus for efficient skeletal muscle transduction; 3) the use of viral doses to accommodate current limitations imposed by vector production methods; 4) and at the same time, achieve a clinically meaningful outcome by transducing multiple muscles in the lower limb to prolong ambulation. METHODS: The capacity of AAV1, AAV6 or AAV8 to cross the vascular endothelial barrier carrying a micro-dystrophin cDNA was compared under identical conditions with delivery through a catheter placed in the femoral artery of the mdx mouse. Transduction efficiency was assessed by immuno-staining using an antibody (Manex1a) that recognizes the N-terminus of micro-dystrophin. The degree of physiologic correction was assessed by measuring tetanic force and protection from eccentric contraction in the extensor digitorum longus muscle (EDL). The vascular delivery paradigm found successful in the mouse was carried to the non-human primate to test its potential translation to boys with DMD. RESULTS: Regional vascular delivery resulted in transduction by rAAV8.micro-dystrophin reaching 94.5 +/- 0.9 (1 month), 91.3 +/- 3.1 (2 months), and 89.6 +/- 1.6% (3 months). rAAV6.micro-dystrophin treated animals demonstrated 87.7 +/- 6.8 (1 month), 78.9 +/- 7.4 (2 months), and 81.2 +/- 6.2% (3 months) transduction. In striking contrast, rAAV1 demonstrated very low transduction efficiency [0.9 +/- 0.3 (1 month), 2.1 +/- 0.8 (2 months), and 2.1 +/- 0.7% (3 months)] by vascular delivery. Micro-dystrophin delivered by rAAV8 and rAAV6 through the femoral artery significantly improved tetanic force and protected against eccentric contraction. Mouse studies translated to the hindlimb of cynamologous macaques using a similar vascular delivery paradigm. rAAV8 carrying eGFP in doses proportional to the mouse (5 x 1012 vg/kg in mouse vs 2 x 1012 vg/kg in monkey) demonstrated widespread gene expression [medial gastrocnemius - 63.8 +/- 4.9%, lateral gastrocnemius - 66.0 +/- 4.5%, EDL - 80.2 +/- 3.1%, soleus - 86.4 +/- 1.9%, TA - 72.2 +/- 4.0%. CONCLUSION: These studies demonstrate regional vascular gene delivery with AAV serotype(s) in mouse and non-human primate at doses, pressures and volumes applicable for clinical trials in children with DMD.

Characterization of Adeno-Associated Viral Vector-Mediated Human Factor VIII Gene Therapy in Hemophilia A Mice.

Adeno-associated viral (AAV) vectors are promising vehicles for hemophilia gene therapy, with favorable clinical trial data seen in the treatment of hemophilia B. In an effort to optimize the expression of human coagulation factor VIII (hFVIII) for the treatment of hemophilia A, an extensive study was performed with numerous combinations of liver-specific promoter and enhancer elements with a codon-optimized hFVIII transgene. After generating 42 variants of three reduced-size promoters and three small enhancers, transgene cassettes were packaged within a single AAV capsid, AAVrh10, to eliminate performance differences due to the capsid type. Each hFVIII vector was administered to FVIII knockout (KO) mice at a dose of 1010 genome copies (GC) per mouse. Criteria for distinguishing the performance of the different enhancer/promoter combinations were established prior to the initiation of the studies. These criteria included prominently the level of hFVIII activity (0.12-2.12 IU/mL) and the pattern of development of anti-hFVIII antibodies. In order to evaluate the impact of capsid on hFVIII expression and antibody formation, one of the enhancer and promoter combinations that exhibited high hFVIII immunogenicity was evaluated using AAV8, AAV9, AAVrh10, AAVhu37, and AAVrh64R1 capsids. The capsids subdivided into two groups: those that generated anti-hFVIII antibodies in ≤20% of mice (AAV8 and AAV9), and those that generated anti-hFVIII antibodies in >20% of mice (AAVrh10, AAVhu37, and AAVrh64R1). The results of this study, which entailed extensive vector optimization and in vivo testing, demonstrate the significant impact that transcriptional control elements and capsid can have on vector performance.

Adeno-Associated Virus Gene Therapy for Liver Disease.

The field of adeno-associated virus (AAV) gene therapy has progressed rapidly over the past decade, with the advent of novel capsid serotype and organ-specific promoters, and an increasing understanding of the immune response to AAV administration. In particular, liver-directed therapy has made remarkable strides, with a number of clinical trials currently planned and ongoing in hemophilia A and B, as well as other liver disorders. This review focuses on liver-directed AAV gene therapy, including historic context, current challenges, and future developments.

Rational design and engineering of a modified adeno-associated virus (AAV1)-based vector system for enhanced retrograde gene delivery.

BACKGROUND: After injection into muscle and peripheral nerves, a variety of viral vectors undergo retrograde transport to lower motor neurons. However, because of its attractive safety profile and durable gene expression, adeno-associated virus (AAV) remains the only vector to have been applied to the human nervous system for the treatment of neurodegenerative disease. Nonetheless, only a very small fraction of intramuscularly injected AAV vector arrives at the spinal cord. OBJECTIVE: To engineer a novel AAV vector by inserting a neuronal targeting peptide (Tet1), with binding properties similar to those of tetanus toxin, into the AAV1 capsid. METHODS: Integral to this approach was the use of structure-based design to increase the effectiveness of functional capsid engineering. This approach allowed the optimization of scaffolding regions for effective display of the foreign epitope while minimizing disruption of the native capsid structure. We also validated an approach by which low-titer tropism-modified AAV vectors can be rescued by particle mosaicism with unmodified capsid proteins. RESULTS: Importantly, our rationally engineered AAV1-based vectors exhibited markedly enhanced transduction of cultured motor neurons, diminished transduction of nontarget cells, and markedly superior retrograde delivery compared with unmodified AAV1 vector. CONCLUSION: This approach promises a significant advancement in the rational engineering of AAV vectors for diseases of the nervous system and other organs.

Human α7 Integrin Gene (ITGA7) Delivered by Adeno-Associated Virus Extends Survival of Severely Affected Dystrophin/Utrophin-Deficient Mice.

Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene. It is the most common, severe childhood form of muscular dystrophy. We investigated an alternative to dystrophin replacement by overexpressing ITGA7 using adeno-associated virus (AAV) delivery. ITGA7 is a laminin receptor in skeletal muscle that, like the dystrophin-glycoprotein complex, links the extracellular matrix to the internal actin cytoskeleton. ITGA7 is expressed in DMD patients and overexpression does not elicit an immune response to the transgene. We delivered rAAVrh.74.MCK.ITGA7 systemically at 5-7 days of age to the mdx/utrn(-/-) mouse deficient for dystrophin and utrophin, a severe mouse model of DMD. At 8 weeks postinjection, widespread expression of ITGA7 was observed at the sarcolemma of multiple muscle groups following gene transfer. The increased expression of ITGA7 significantly extended longevity and reduced common features of the mdx/utrn(-/-) mouse, including kyphosis. Overexpression of α7 expression protected against loss of force following contraction-induced damage and increased specific force in the diaphragm and EDL muscles 8 weeks after gene transfer. Taken together, these results further support the use of α7 integrin as a potential therapy for DMD.