Stress-Induced Mouse Model of the Cardiac Manifestations of Friedreich's Ataxia Corrected by AAV-mediated Gene Therapy.

Friedreich's ataxia (FA), an autosomal recessive disorder caused by a deficiency in the expression of frataxin (FXN), is characterized by progressive ataxia and hypertrophic cardiomyopathy. Although cardiac dysfunction is the most common cause of mortality in FA, the cardiac disease remains subclinical for most of the clinical course because the neurologic disease limits muscle oxygen demands. Previous FXN knockout mouse models exhibit fatal cardiomyopathy similar to human FA, but in contrast to the human condition, untreated mice become moribund by 2 months of age, unlike humans where the cardiac disease often does not manifest until the third decade. The study was designed to create a mouse model for early FA disease relevant to the time for which a gene therapy would likely be most effective. To generate a cardiac-specific mouse model of FA cardiomyopathy similar to the human disease, we used a cardiac promoter (αMyhc) driving CRE recombinase cardiac-specific excision of FXN exon 4 to generate a mild, cardiac-specific FA model that is normal at rest, but exhibits the cardiac phenotype with stress. The hearts of αMyhc mice had decreased levels of FXN and activity of the mitochondrial complex II/complex IV respiratory chain. At rest, αMyhc mice exhibited normal cardiac function as assessed by echocardiographic assessment of ejection fraction and fractional shortening, but when the heart was stressed chemically with dobutamine, αMyhc mice compared with littermate control mice had a 62% reduction in the stress ejection fraction (p < 2 × 10-4) and 71% reduction in stress-related fractional shortening (p < 10-5). When assessing functional cardiac performance using running on an inclined treadmill, αMyhc mice stayed above the midline threefold less than littermate controls (p < 0.02). A one-time intravenous administration of 1011 genome copies of AAVrh.10hFXN, an adeno-associated virus (AAV) serotype rh10 gene transfer vector expressing human FXN, corrected the stress-induced ejection fraction and fractional shortening phenotypes. Treated αMyhc mice exhibited exercise performance on a treadmill indistinguishable from littermate controls (p > 0.07). These αMyhc mice provide an ideal model to study long-term cardiac complications due to FA and AAV-mediated gene therapy correction of stress-induced cardiac phenotypes typical of human FA.

Gene therapy for C1 esterase inhibitor deficiency in a Murine Model of Hereditary angioedema.

BACKGROUND: Hereditary angioedema (HAE) is a life-threatening, autosomal dominant disorder characterized by unpredictable, episodic swelling of the face, upper airway, oropharynx, extremities, genitalia, and gastrointestinal tract. Almost all cases of HAE are caused by mutations in the SERPING1 gene resulting in a deficiency in functional plasma C1 esterase inhibitor (C1EI), a serine protease inhibitor that normally inhibits proteases in the contact, complement, and fibrinolytic systems. Current treatment of HAE includes long-term prophylaxis with attenuated androgens or human plasma-derived C1EI and management of acute attacks with human plasma-derived or recombinant C1EI, bradykinin, and kallikrein inhibitors, each of which requires repeated administration. As an approach to effectively treat HAE with a single treatment, we hypothesized that a one-time intravenous administration of an adeno-associated virus (AAV) gene transfer vector expressing the genetic sequence of the normal human C1 esterase inhibitor (AAVrh.10hC1EI) would provide sustained circulating C1EI levels sufficient to prevent angioedema episodes. METHODS: To study the efficacy of AAVrh.10hC1EI, we used CRISPR/Cas9 technology to create a heterozygote C1EI-deficient mouse model (S63±) that shares characteristics associated with HAE in humans including decreased plasma C1EI and C4 levels. Phenotypically, these mice have increased vascular permeability of skin and internal organs. RESULTS: Systemic administration of AAVrh.10hC1EI to the S63± mice resulted in sustained human C1EI activity levels above the predicted therapeutic levels and correction of the vascular leak in skin and internal organs. CONCLUSION: A single treatment with AAVrh.10hC1EI has the potential to provide long-term protection from angioedema attacks in affected individuals.

Exome sequencing-based identification of novel type 2 diabetes risk allele loci in the Qatari population.

BACKGROUND: Type 2 diabetes (T2D) susceptibility is influenced by genetic and lifestyle factors. To date, the majority of genetic studies of T2D have been in populations of European and Asian descent. The focus of this study is on genetic variations underlying T2D in Qataris, a population with one of the highest incidences of T2D worldwide. RESULTS: Illumina HiSeq exome sequencing was performed on 864 Qatari subjects (574 T2D cases, 290 controls). Sequence kernel association test (SKAT) gene-based analysis identified an association for low frequency potentially deleterious variants in 6 genes. However, these findings were not replicated by SKAT analysis in an independent cohort of 12,699 exomes, primarly due to the absence of low frequency potentially deleterious variants in 5 of the 6 genes. Interestingly one of the genes identified, catenin beta 1 (CTNNB1, ß-catenin), is the key effector of the Wnt pathway and interacts with the nuclear receptor transcription factor 7-like 2 (TCF7L2), variants which are the most strongly associated with risk of developing T2D worldwide. Single variant analysis did not identify any associated variants, suggesting the SKAT association signal was not driven by individual variants. None of the 6 associated genes were among 634 previously described T2D genes. CONCLUSIONS: The observation that genes not previously linked to T2D in prior studies of European and Asian populations are associated with T2D in Qatar provides new insights into the complexity of T2D pathogenesis and emphasizes the importance of understudied populations when assessing genetic variation in the pathogenesis of common disorders.

In situ reprogramming to transdifferentiate fibroblasts into cardiomyocytes using adenoviral vectors: Implications for clinical myocardial regeneration.

OBJECTIVE: The reprogramming of cardiac fibroblasts into induced cardiomyocyte-like cells improves ventricular function in myocardial infarction models. Only integrating persistent expression vectors have thus far been used to induce reprogramming, potentially limiting its clinical applicability. We therefore tested the reprogramming potential of nonintegrating, acute expression adenoviral (Ad) vectors. METHODS: Ad or lentivirus vectors encoding Gata4 (G), Mef2c (M), and Tbx5 (T) were validated in vitro. Sprague-Dawley rats then underwent coronary ligation and Ad-mediated administration of vascular endothelial growth factor to generate infarct prevascularization. Three weeks later, animals received Ad or lentivirus encoding G, M, or T (AdGMT or LentiGMT) or an equivalent dose of a null vector (n = 11, 10, and 10, respectively). Outcomes were analyzed by echocardiography, magnetic resonance imaging, and histology. RESULTS: Ad and lentivirus vectors provided equivalent G, M, and T expression in vitro. AdGMT and LentiGMT both likewise induced expression of the cardiomyocyte marker cardiac troponin T in approximately 6% of cardiac fibroblasts versus <1% cardiac troponin T expression in AdNull (adenoviral vector that does not encode a transgene)-treated cells. Infarcted myocardium that had been treated with AdGMT likewise demonstrated greater density of cells expressing the cardiomyocyte marker beta myosin heavy chain 7 compared with AdNull-treated animals. Echocardiography demonstrated that AdGMT and LentiGMT both increased ejection fraction compared with AdNull (AdGMT: 21% ± 3%, LentiGMT: 14% ± 5%, AdNull: -0.4% ± 2%; P < .05). CONCLUSIONS: Ad vectors are at least as effective as lentiviral vectors in inducing cardiac fibroblast transdifferentiation into induced cardiomyocyte-like cells and improving cardiac function in postinfarct rat hearts. Short-term expression Ad vectors may represent an important means to induce cardiac cellular reprogramming in humans.

Vectored Intracerebral Immunization with the Anti-Tau Monoclonal Antibody PHF1 Markedly Reduces Tau Pathology in Mutant Tau Transgenic Mice.

Passive immunization with anti-tau monoclonal antibodies has been shown by several laboratories to reduce age-dependent tau pathology and neurodegeneration in mutant tau transgenic mice. These studies have used repeated high weekly doses of various tau antibodies administered systemically for several months and have reported reduced tau pathology of ∼40-50% in various brain regions. Here we show that direct intrahippocampal administration of the adeno-associated virus (AAV)-vectored anti-phospho-tau antibody PHF1 to P301S tau transgenic mice results in high and durable antibody expression, primarily in neurons. Hippocampal antibody levels achieved after AAV delivery were ∼50-fold more than those reported following repeated systemic administration. In contrast to systemic passive immunization, we observed markedly reduced (≥80-90%) hippocampal insoluble pathological tau species and neurofibrillary tangles following a single dose of AAV-vectored PHF1 compared with mice treated with an AAV-IgG control vector. Moreover, the hippocampal atrophy observed in untreated P301S mice was fully rescued by treatment with the AAV-vectored PHF1 antibody. Vectored passive immunotherapy with an anti-tau monoclonal antibody may represent a viable therapeutic strategy for treating or preventing such tauopathies as frontotemporal dementia, progressive supranuclear palsy, or Alzheimer's disease. SIGNIFICANCE STATEMENT: We have used an adeno-associated viral (AAV) vector to deliver the genes encoding an anti-phospho-tau monoclonal antibody, PHF1, directly to the brain of mice that develop neurodegeneration due to a tau mutation that causes frontotemporal dementia (FTD). When administered systemically, PHF1 has been shown to modestly reduce tau pathology and neurodegeneration. Since such antibodies do not readily cross the blood-brain barrier, we used an AAV vector to deliver antibody directly to the hippocampus and observed much higher antibody levels and a much greater reduction in tau pathology. Using AAV vectors to deliver antibodies like PHF1 directly to brain may constitute a novel approach to treating various neurodegenerative disorders, such as FTD and Alzheimer's disease.

Anti-Epidermal Growth Factor Receptor Gene Therapy for Glioblastoma.

Glioblastoma multiforme (GBM) is the most common and aggressive primary intracranial brain tumor in adults with a mean survival of 14 to 15 months. Aberrant activation of the epidermal growth factor receptor (EGFR) plays a significant role in GBM progression, with amplification or overexpression of EGFR in 60% of GBM tumors. To target EGFR expressed by GBM, we have developed a strategy to deliver the coding sequence for cetuximab, an anti-EGFR antibody, directly to the CNS using an adeno-associated virus serotype rh.10 gene transfer vector. The data demonstrates that single, local delivery of an anti-EGFR antibody by an AAVrh.10 vector coding for cetuximab (AAVrh.10Cetmab) reduces GBM tumor growth and increases survival in xenograft mouse models of a human GBM EGFR-expressing cell line and patient-derived GBM. AAVrh10.CetMab-treated mice displayed a reduction in cachexia, a significant decrease in tumor volume and a prolonged survival following therapy. Adeno-associated-directed delivery of a gene encoding a therapeutic anti-EGFR monoclonal antibody may be an effective strategy to treat GBM.

Correction: Type 2 Diabetes Risk Allele Loci in the Qatari Population.

[This corrects the article DOI: 10.1371/journal.pone.0156834.].

Gene Therapy for Alpha-1 Antitrypsin Deficiency Lung Disease.

Alpha-1 antitrypsin (AAT) deficiency, characterized by low plasma levels of the serine protease inhibitor AAT, is associated with emphysema secondary to insufficient protection of the lung from neutrophil proteases. Although AAT augmentation therapy with purified AAT protein is efficacious, it requires weekly to monthly intravenous infusion of AAT purified from pooled human plasma, has the risk of viral contamination and allergic reactions, and is costly. As an alternative, gene therapy offers the advantage of single administration, eliminating the burden of protein infusion, and reduced risks and costs. The focus of this review is to describe the various strategies for AAT gene therapy for the pulmonary manifestations of AAT deficiency and the state of the art in bringing AAT gene therapy to the bedside.

Type 2 Diabetes Risk Allele Loci in the Qatari Population.

BACKGROUND: The prevalence of type 2 diabetes (T2D) is increasing in the Middle East. However, the genetic risk factors for T2D in the Middle Eastern populations are not known, as the majority of studies of genetic risk for T2D are in Europeans and Asians. METHODS: All subjects were ≥3 generation Qataris. Cases with T2D (n = 1,124) and controls (n = 590) were randomly recruited and assigned to the 3 known Qatari genetic subpopulations [Bedouin (Q1), Persian/South Asian (Q2) and African (Q3)]. Subjects underwent genotyping for 37 single nucleotide polymorphisms (SNPs) in 29 genes known to be associated with T2D in Europeans and/or Asian populations, and an additional 27 tag SNPs related to these susceptibility loci. Pre-study power analysis suggested that with the known incidence of T2D in adult Qataris (22%), the study population size would be sufficient to detect significant differences if the SNPs were risk factors among Qataris, assuming that the odds ratio (OR) for T2D SNPs in Qatari's is greater than or equal to the SNP with highest known OR in other populations. RESULTS: Haplotype analysis demonstrated that Qatari haplotypes in the region of known T2D risk alleles in Q1 and Q2 genetic subpopulations were similar to European haplotypes. After Benjamini-Hochberg adjustment for multiple testing, only two SNPs (rs7903146 and rs4506565), both associated with transcription factor 7-like 2 (TCF7L2), achieved statistical significance in the whole study population. When T2D subjects and control subjects were assigned to the known 3 Qatari subpopulations, and analyzed individually and with the Q1 and Q2 genetic subpopulations combined, one of these SNPs (rs4506565) was also significant in the admixed group. No other SNPs associated with T2D in all Qataris or individual genetic subpopulations. CONCLUSIONS: With the caveats of the power analysis, the European/Asian T2D SNPs do not contribute significantly to the high prevalence of T2D in the Qatari population, suggesting that the genetic risks for T2D are likely different in Qataris compared to Europeans and Asians.

Anti-hIgE gene therapy of peanut-induced anaphylaxis in a humanized murine model of peanut allergy.

BACKGROUND: Peanuts are the most common food to provoke fatal or near-fatal anaphylactic reactions. Treatment with an anti-hIgE mAb is efficacious but requires frequent parenteral administration. OBJECTIVE: Based on the knowledge that peanut allergy is mediated by peanut-specific IgE, we hypothesized that a single administration of an adeno-associated virus (AAV) gene transfer vector encoding for anti-hIgE would protect against repeated peanut exposure in the host with peanut allergy. METHODS: We developed a novel humanized murine model of peanut allergy that recapitulates the human anaphylactic response to peanuts in NOD-scid IL2Rgammanull mice transferred with blood mononuclear cells from donors with peanut allergy and then sensitized with peanut extract. As therapy, we constructed an adeno-associated rh.10 serotype vector coding for a full-length, high-affinity, anti-hIgE antibody derived from the Fab fragment of the anti-hIgE mAb omalizumab (AAVrh.10anti-hIgE). In the reconstituted mice peanut-specific IgE was induced by peanut sensitization and hypersensitivity, and reactions were provoked by feeding peanuts to mice with symptoms similar to those of human subjects with peanut allergy. RESULTS: A single administration of AAVrh.10anti-hIgE vector expressed persistent levels of anti-hIgE. The anti-hIgE vector, administered either before sensitization or after peanut sensitization and manifestation of the peanut-induced phenotype, blocked IgE-mediated alterations in peanut-induced histamine release, anaphylaxis scores, locomotor activity, and free IgE levels and protected animals from death caused by anaphylaxis. CONCLUSION: If this degree of persistent efficacy translates to human subjects, AAVrh.10anti-hIgE could be an effective 1-time preventative therapy for peanut allergy and possibly other severe, IgE-mediated allergies.

Intracerebral adeno-associated virus gene delivery of apolipoprotein E2 markedly reduces brain amyloid pathology in Alzheimer's disease mouse models.

The common apolipoprotein E alleles (ε4, ε3, and ε2) are important genetic risk factors for late-onset Alzheimer's disease, with the ε4 allele increasing risk and reducing the age of onset and the ε2 allele decreasing risk and markedly delaying the age of onset. Preclinical and clinical studies have shown that apolipoprotein E (APOE) genotype also predicts the timing and amount of brain amyloid-ß (Aß) peptide deposition and amyloid burden (ε4 >ε3 >ε2). Using several administration protocols, we now report that direct intracerebral adeno-associated virus (AAV)-mediated delivery of APOE2 markedly reduces brain soluble (including oligomeric) and insoluble Aß levels as well as amyloid burden in 2 mouse models of brain amyloidosis whose pathology is dependent on either the expression of murine Apoe or more importantly on human APOE4. The efficacy of APOE2 to reduce brain Aß burden in either model, however, was highly dependent on brain APOE2 levels and the amount of pre-existing Aß and amyloid deposition. We further demonstrate that a widespread reduction of brain Aß burden can be achieved through a single injection of vector via intrathalamic delivery of AAV expressing APOE2 gene. Our results demonstrate that AAV gene delivery of APOE2 using an AAV vector rescues the detrimental effects of APOE4 on brain amyloid pathology and may represent a viable therapeutic approach for treating or preventing Alzheimer's disease especially if sufficient brain APOE2 levels can be achieved early in the course of the disease.

Genetic modification of neurons to express bevacizumab for local anti-angiogenesis treatment of glioblastoma.

The median survival of glioblastoma multiforme (GBM) is approximately 1 year. Following surgical removal, systemic therapies are limited by the blood-brain barrier. To circumvent this, we developed a method to modify neurons with the genetic sequence for therapeutic monoclonal antibodies using adeno-associated virus (AAV) gene transfer vectors, directing persistent, local expression in the tumor milieu. The human U87MG GBM cell line or patient-derived early passage GBM cells were administered to the striatum of NOD/SCID immunodeficient mice. AAVrh.10BevMab, an AAVrh.10-based vector coding for bevacizumab (Avastin), an anti-human vascular endothelial growth factor (VEGF) monoclonal antibody, was delivered to the area of the GBM xenograft. Localized expression of bevacizumab was demonstrated by quantitative PCR, ELISA and western blotting. Immunohistochemistry showed that bevacizumab was expressed in neurons. Concurrent administration of AAVrh.10BevMab with the U87MG tumor reduced tumor blood vessel density and tumor volume, and increased survival. Administration of AAVrh.10BevMab 1 week after U87MG xenograft reduced growth and increased survival. Studies with patient-derived early passage GBM primary cells showed a reduction in primary tumor burden with an increased survival. These data support the strategy of AAV-mediated central nervous system gene therapy to treat GBM, overcoming the blood-brain barrier through local, persistent delivery of an anti-angiogenesis monoclonal antibody.

A novel, live-attenuated vesicular stomatitis virus vector displaying conformationally intact, functional HIV-1 envelope trimers that elicits potent cellular and humoral responses in mice.

Though vaccination with live-attenuated SIV provides the greatest protection from progressive disease caused by SIV challenge in rhesus macaques, attenuated HIV presents safety concerns as a vaccine; therefore, live viral vectors carrying HIV immunogens must be considered. We have designed a replication-competent vesicular stomatitis virus (VSV) displaying immunogenic HIV-1 Env trimers and attenuating quantities of the native surface glycoprotein (G). The clade B Env immunogen is an Env-VSV G hybrid (EnvG) in which the transmembrane and cytoplasmic tail regions are derived from G. Relocation of the G gene to the 5'terminus of the genome and insertion of EnvG into the natural G position induced a ∼1 log reduction in surface G, significant growth attenuation compared to wild-type, and incorporation of abundant EnvG. Western blot analysis indicated that ∼75% of incorporated EnvG was a mature proteolytically processed form. Flow cytometry showed that surface EnvG bound various conformationally- and trimer-specific antibodies (Abs), and in-vitro growth assays on CD4+CCR5+ cells demonstrated EnvG functionality. Neither intranasal (IN) or intramuscular (IM) administration in mice induced any observable pathology and all regimens tested generated potent Env-specific ELISA titers of 10(4)-10(5), with an IM VSV prime/IN VSV boost regimen eliciting the highest binding and neutralizing Ab titers. Significant quantities of Env-specific CD4+ T cells were also detected, which were augmented as much as 70-fold by priming with IM electroporated plasmids encoding EnvG and IL-12. These data suggest that our novel vector can achieve balanced safety and immunogenicity and should be considered as an HIV vaccine platform.

"Triplet" polycistronic vectors encoding Gata4, Mef2c, and Tbx5 enhances postinfarct ventricular functional improvement compared with singlet vectors.

OBJECTIVE: The in situ reprogramming of cardiac fibroblasts into induced cardiomyocytes by the administration of gene transfer vectors encoding Gata4 (G), Mef2c (M), and Tbx5 (T) has been shown to improve ventricular function in myocardial infarction models. The efficacy of this strategy could, however, be limited by the need for fibroblast targets to be infected 3 times--once by each of the 3 transgene vectors. We hypothesized that a polycistronic "triplet" vector encoding all 3 transgenes would enhance postinfarct ventricular function compared with use of "singlet" vectors. METHODS: After validation of the polycistronic vector expression in vitro, adult male Fischer 344 rats (n=6) underwent coronary ligation with or without intramyocardial administration of an adenovirus encoding all 3 major vascular endothelial growth factor (VEGF) isoforms (AdVEGF-All6A positive), followed 3 weeks later by the administration to AdVEGF-All6A-positive treated rats of singlet lentivirus encoding G, M, or T (1×10(5) transducing units each) or the same total dose of a GMT "triplet" lentivirus vector. RESULTS: Western blots demonstrated that triplet and singlet vectors yielded equivalent GMT transgene expression, and fluorescence activated cell sorting demonstrated that triplet vectors were nearly twice as potent as singlet vectors in generating induced cardiomyocytes from cardiac fibroblasts. Echocardiography demonstrated that GMT triplet vectors were more effective than the 3 combined singlet vectors in enhancing ventricular function from postinfarct baselines (triplet, 37%±10%; singlet, 13%±7%; negative control, 9%±5%; P<.05). CONCLUSIONS: These data have confirmed that the in situ administration of G, M, and T induces postinfarct ventricular functional improvement and that GMT polycistronic vectors enhance the efficacy of this strategy.

Vaccination with Gag, Vif, and Nef gene fragments affords partial control of viral replication after mucosal challenge with SIVmac239.

UNLABELLED: Broadly targeted cellular immune responses are thought to be important for controlling replication of human and simian immunodeficiency viruses (HIV and SIV). However, eliciting such responses by vaccination is complicated by immunodominance, the preferential targeting of only a few of the many possible epitopes of a given antigen. This phenomenon may be due to the coexpression of dominant and subdominant epitopes by the same antigen-presenting cell and may be overcome by distributing these sequences among several different vaccine constructs. Accordingly, we tested whether vaccinating rhesus macaques with "minigenes" encoding fragments of Gag, Vif, and Nef resulted in broadened cellular responses capable of controlling SIV replication. We delivered these minigenes through combinations of recombinant Mycobacterium bovis BCG (rBCG), electroporated recombinant DNA (rDNA) along with an interleukin-12 (IL-12)-expressing plasmid (EP rDNA plus pIL-12), yellow fever vaccine virus 17D (rYF17D), and recombinant adenovirus serotype 5 (rAd5). Although priming with EP rDNA plus pIL-12 increased the breadth of vaccine-induced T-cell responses, this effect was likely due to the improved antigen delivery afforded by electroporation rather than modulation of immunodominance. Indeed, Mamu-A*01(+) vaccinees mounted CD8(+) T cells directed against only one subdominant epitope, regardless of the vaccination regimen. After challenge with SIVmac239, vaccine efficacy was limited to a modest reduction in set point in some of the groups and did not correlate with standard T-cell measurements. These findings suggest that broad T-cell responses elicited by conventional vectors may not be sufficient to substantially contain AIDS virus replication. IMPORTANCE: Immunodominance poses a major obstacle to the generation of broadly targeted, HIV-specific cellular responses by vaccination. Here we attempted to circumvent this phenomenon and thereby broaden the repertoire of SIV-specific cellular responses by vaccinating rhesus macaques with minigenes encoding fragments of Gag, Vif, and Nef. In contrast to previous mouse studies, this strategy appeared to minimally affect monkey CD8(+) T-cell immundominance hierarchies, as seen by the detection of only one subdominant epitope in Mamu-A*01(+) vaccinees. This finding underscores the difficulty of inducing subdominant CD8(+) T cells by vaccination and demonstrates that strategies other than gene fragmentation may be required to significantly alter immunodominance in primates. Although some of the regimens tested here were extremely immunogenic, vaccine efficacy was limited to a modest reduction in set point viremia after challenge with SIVmac239. No correlates of protection were identified. These results reinforce the notion that vaccine immunogenicity does not predict control of AIDS virus replication.

Intrapleural administration of an AAVrh.10 vector coding for human α1-antitrypsin for the treatment of α1-antitrypsin deficiency.

Alpha-1 antitrypsin (α1AT) deficiency is a common autosomal recessive disorder characterized by a marked reduction in serum α1AT levels, lung and liver disease. α1AT is mainly produced and secreted by hepatocytes, with its primary function to protect the lung against the proteolytic activity of neutrophil elastase. Serum α1AT levels <11 µM are associated with progressive destruction of lung parenchyma and early-onset of panacinar emphysema in the age range 35-45. The current approved treatment for α1AT deficiency is a costly protein augmentation therapy requiring weekly intravenous infusion of purified α1AT from pooled human plasma. Gene therapy offers the advantage of a single vector administration, eliminating the burden of the repeated purified protein infusions, with the consequent reduced overall drug cost and improved compliance. We have developed a novel, highly efficient gene therapy approach for α1AT deficiency based on the administration of AAVrh.10hα1AT, an adeno-associated viral vector serotype rh.10 coding for normal M-type human α1AT via the intrapleural route. On the basis of prior murine studies, this approach provides sustained α1AT proximal to the lung with a highly efficient vector. In support of a clinical trial for this approach, we carried out a study to assess the safety of intrapleural administration of AAVrh.10hα1AT to 280 mice and 36 nonhuman primates. The data demonstrate that this approach is safe, with no toxicity issues. Importantly, there was persistent expression of the human α1AT mRNA in chest cavity cells for the duration of the study (6 months in mice and 1 year in nonhuman primates). Together, these data support the initiation of a clinical trial of intrapleural human AAVrh.10hα1AT for the treatment of α1AT deficiency.

Gene therapy to stimulate angiogenesis to treat diffuse coronary artery disease.

Cardiac gene therapy offers a strategy to treat diffuse coronary artery disease (CAD), a disorder with no therapeutic options. The use of genes to revascularize the ischemic myocardium has been the focus of two decades of preclinical research with a variety of angiogenic mediators, including vascular endothelial growth factor, fibroblast growth factor, hepatocyte growth factor, and others encoded by DNA plasmids or adenovirus vectors. The multifaceted challenge for developing efficient induction of collateral vessels in the ischemic heart requires a choice for route of delivery, dosing level, a relevant animal model, duration of treatment, and assessment of phenotype for efficacy. Overall, studies of gene therapy for ischemia in experimental models are very encouraging, with clear evidence of safety and efficacy, strongly supporting the concept that gene therapy to induce angiogenesis is a viable therapeutic approach for CAD. Clinical studies of cardiac gene therapy with angiogenic factors have added substantially to the evidence for efficacy, but definitive studies have not yet led to commercial approval. This review provides the general concepts for angiogenesis-based therapeutic approaches for diffuse CAD and summarizes the results from key studies in the field with recommendations for refinement to a successful product design and evaluation.

Membrane-bound SIV envelope trimers are immunogenic in ferrets after intranasal vaccination with a replication-competent canine distemper virus vector.

We are investigating canine distemper virus (CDV) as a vaccine vector for the delivery of HIV envelope (Env) that closely resembles the native trimeric spike. We selected CDV because it will promote vaccine delivery to lymphoid tissues, and because human exposure is infrequent, reducing potential effects of pre-existing immunity. Using SIV Env as a model, we tested a number of vector and gene insert designs. Vectors containing a gene inserted between the CDV H and L genes, which encoded Env lacking most of its cytoplasmic tail, propagated efficiently in Vero cells, expressed the immunogen on the cell surface, and incorporated the SIV glycoprotein into progeny virus particles. When ferrets were vaccinated intranasally, there were no signs of distress, vector replication was observed in the gut-associated lymphoid tissues, and the animals produced anti-SIV Env antibodies. These data show that live CDV-SIV Env vectors can safely induce anti-Env immune responses following intranasal vaccination.

SIV antigen-specific effects on immune responses induced by vaccination with DNA electroporation and plasmid IL-12.

Molecular adjuvants are important for augmenting or modulating immune responses induced by DNA vaccination. Promising results have been obtained using IL-12 expression plasmids in a variety of disease models including the SIV model of HIV infection. We used a mouse model to evaluate plasmid IL-12 (pIL-12) in a DNA prime, recombinant adenovirus serotype 5 (rAd5) boost regimen specifically to evaluate the effect of IL-12 expression on cellular and humoral immunity induced against both SIVmac239 Gag and Env antigens. Priming with electroporated (EP) DNA+pIL-12 resulted in a 2-4-fold enhanced frequency of Gag-specific CD4 T cells which was maintained through the end of the study irrespective of the pIL-12 dose, while memory Env-specific CD4+T cells were maintained only at the low dose of pIL-12. There was little positive effect of pIL-12 on the humoral response to Env, and in fact, high dose pIL-12 dramatically reduced SIV Env-specific IgG. Additionally, both doses of pIL-12 diminished the frequency of CD8 T-cells after DNA prime, although a rAd5 boost recovered CD8 responses regardless of the pIL-12 dose. In this prime-boost regimen, we have shown that a high dose pIL-12 can systemically reduce Env-specific humoral responses and CD4T cell frequency, but not Gag-specific CD4+ T cells. These data indicate that it is important to independently characterize individual SIV or HIV antigen immunogenicity in multi-antigenic vaccines as a function of adjuvant dose.