Delivery Of Recombinant Adeno-associated Virus Research Articles

NLRC3 expression in macrophage impairs glycolysis and host immune defense by modulating the NF-κB-NFAT5 complex during septic immunosuppression

Impairment of innate immune cell function and metabolism underlies immunosuppression in sepsis; however, a promising therapy to orchestrate this impairment is currently lacking. In this study, high levels of NOD-like receptor family CARD domain containing-3 (NLRC3) correlated with the glycolytic defects of monocytes/macrophages from septic patients and mice that developed immunosuppression. Myeloid-specific NLRC3 deletion improved macrophage glycolysis and sepsis-induced immunosuppression. Mechanistically, NLRC3 inhibits nuclear factor (NF)-κB p65 binding to nuclear factor of activated Tcells 5 (NFAT5), which further controls the expression of glycolytic genes and proinflammatory cytokines of immunosuppressive macrophages. This is achieved by decreasing NF-κB activation-co-induced by TNF-receptor-associated factor 6 (TRAF6) or mammalian target of rapamycin (mTOR)-and decreasing transcriptional co-activator p300 activity by inducing NLRC3 sequestration of mTOR and p300. Genetic inhibition of NLRC3 disrupted the NLRC3-mTOR-p300 complex and enhanced NF-κB binding to the NFAT5 promoter in concert with p300. Furthermore, intrapulmonary delivery of recombinant adeno-associated virus harboring a macrophage-specific NLRC3 deletion vector significantly improved the defense of septic mice that developed immunosuppression upon secondary intratracheal bacterial challenge. Collectively, these findings indicate that NLRC3 mediates critical aspects of innate immunity that contribute to an immunocompromised state during sepsis and identify potential therapeutic targets.

The neuronal retromer can regulate both neuronal and microglial phenotypes of Alzheimer's disease.

SUMMARYDisruption of retromer-dependent endosomal trafficking is considered pathogenic in late-onset Alzheimer’s disease (AD). Here, to investigate this disruption in the intact brain, we turn to a genetic mouse model where the retromer core protein VPS35 is depleted in hippocampal neurons, and then we replete VPS35 using an optimized viral vector protocol. The VPS35 depletion-repletion studies strengthen the causal link between the neuronal retromer and AD-associated neuronal phenotypes, including the acceleration of amyloid precursor protein cleavage and the loss of synaptic glutamate receptors. Moreover, the studies show that the neuronal retromer can regulate a distinct, dystrophic, microglia morphology, phenotypic of hippocampal microglia in AD. Finally, the neuronal and, in part, the microglia responses to VPS35 depletion were found to occur independent of tau. Showing that the neuronal retromer can regulate AD-associated pathologies in two of AD’s principal cell types strengthens the link, and clarifies the mechanism, between endosomal trafficking and late-onset sporadic AD.

Biomaterial-Guided Recombinant Adeno-associated Virus Delivery from Poly(Sodium Styrene Sulfonate)-Grafted Poly(ɛ-Caprolactone) Films to Target Human Bone Marrow Aspirates

Scaffold-guided gene transfer offers strong systems to develop noninvasive convenient therapeutic options for the treatment of articular cartilage defects, especially when targeting bone marrow aspirates from patients containing chondroregenerative mesenchymal stromal cells in a native microenvironment. In this study, we examined the feasibility of delivering reporter (red fluorescent protein [RFP], lacZ) recombinant adeno-associated virus (rAAV) vectors over time to such samples through biocompatible mechanically stable poly(ɛ-caprolactone) (PCL) films grafted with poly(sodium styrene sulfonate) (pNaSS) for improved biological responses as clinically adapted tools for cartilage repair. Effective transgene expression (RFP, lacZ) was noted over time in human bone marrow aspirates using pNaSS-grafted films (up to 90% efficiency for at least 21 days) versus control conditions (ungrafted films, absence of vector coating on the films, free or no vector treatment), without displaying cytotoxic nor detrimental effects on the osteochondrogenic or hypertrophic potential of the samples. These findings demonstrate the potential of directly modifying therapeutic bone marrow from patients by controlled delivery of rAAV using biomaterial-guided procedures as a future noninvasive strategy for clinical cartilage repair. Impact statement Injured articular cartilage does not fully regenerate on itself and none of the currently available clinical and experimental therapeutic procedures are capable of restoring an original hyaline cartilage in sites of injury. Biomaterial-guided gene delivery has a strong potential to enhance the processes of cartilage repair. The system presented here based on the FDA-approved biocompatible poly(ɛ-caprolactone) material provides a functional scaffold for the controlled delivery of clinically adapted recombinant adeno-associated virus vectors as an off-the-shelf compound that could be applicable in a minimally invasive manner in patients.

Gene Transfer in Rodent Nervous Tissue Following Hindlimb Intramuscular Delivery of Recombinant Adeno-Associated Virus Serotypes AAV2/6, AAV2/8, and AAV2/9

Recombinant adeno-associated virus (rAAV) vectors have emerged as the safe vehicles of choice for long-term gene transfer in mammalian nervous system. Recombinant adeno-associated virus–mediated localized gene transfer in adult nervous system following direct inoculation, that is, intracerebral or intrathecal, is well documented. However, recombinant adeno-associated virus delivery in defined neuronal populations in adult animals using less-invasive methods as well as avoiding ectopic gene expression following systemic inoculation remain challenging. Harnessing the capability of some recombinant adeno-associated virus serotypes for retrograde transduction may potentially address such limitations (Note: The term retrograde transduction in this manuscript refers to the uptake of injected recombinant adeno-associated virus particles at nerve terminals, retrograde transport, and subsequent transduction of nerve cell soma). In some studies, recombinant adeno-associated virus serotypes 2/6, 2/8, and 2/9 have been shown to exhibit transduction of connected neuroanatomical tracts in adult animals following lower limb intramuscular recombinant adeno-associated virus delivery in a pattern suggestive of retrograde transduction. However, an extensive side-by-side comparison of these serotypes following intramuscular delivery regarding tissue viral load, and the effect of promoter on transgene expression, has not been performed. Hence, we delivered recombinant adeno-associated virus serotypes 2/6, 2/8, or 2/9 encoding enhanced green fluorescent protein (eGFP), under the control of either cytomegalovirus (CMV) or human synapsin (hSyn) promoter, via a single unilateral hindlimb intramuscular injection in the bicep femoris of adult C57BL/6J mice. Four weeks post injection, we quantified viral load and transgene (enhanced green fluorescent protein) expression in muscle and related nervous tissues. Our data show that the select recombinant adeno-associated virus serotypes transduce sciatic nerve and groups of neurons in the dorsal root ganglia on the injected side, indicating that the intramuscular recombinant adeno-associated virus delivery is useful for achieving gene transfer in local neuroanatomical tracts. We also observed sparse recombinant adeno-associated virus viral delivery or eGFP transduction in lumbar spinal cord and a noticeable lack thereof in brain. Therefore, further improvements in recombinant adeno-associated virus design are warranted to achieve efficient widespread retrograde transduction following intramuscular and possibly other peripheral routes of delivery.

Five Years of Successful Inducible Transgene Expression Following Locoregional Adeno-Associated Virus Delivery in Nonhuman Primates with No Detectable Immunity.

Anti-transgene immune responses elicited after intramuscular (i.m.) delivery of recombinant adeno-associated virus (rAAV) have been shown to hamper long-term transgene expression in large-animal models of rAAV-mediated gene transfer. To overcome this hurdle, an alternative mode of delivery of rAAV vectors in nonhuman primate muscles has been described: the locoregional (LR) intravenous route of administration. Using this injection mode, persistent inducible transgene expression for at least 1 year under the control of the tetracycline-inducible Tet-On system was previously reported in cynomolgus monkeys, with no immunity against the rtTA transgene product. The present study shows the long-term follow-up of these animals. It is reported that LR delivery of a rAAV2/1 vector allows long-term inducible expression up to at least 5 years post gene transfer, with no any detectable host immune response against the transactivator rtTA, despite its immunogenicity following i.m. gene transfer. This study shows for the first time a long-term regulation of muscle gene expression using a Tet-On-inducible system in a large-animal model. Moreover, these findings further confirm that the rAAV LR delivery route is efficient and immunologically safe, allowing long-term skeletal muscle gene transfer.

In Vivo Monitoring of Circadian Clock Gene Expression in the Mouse Suprachiasmatic Nucleus Using Fluorescence Reporters.

This technique combines optical fiber mediated fluorescence recordings with the precise delivery of recombinant adeno-associated virus based gene reporters. This new and easy to use in vivo fluorescence monitoring system was developed to record the transcriptional rhythm of the clock gene, Cry1, in the suprachiasmatic nucleus (SCN) of freely moving mice. To do so, a Cry1 transcription fluorescence reporter was designed and packaged into Adeno-associated virus. Purified, concentrated virus was injected into the mouse SCN followed by the insertion of an optic fiber, which was then fixed onto the surface of the brain. The animals were returned to their home cages and allowed a 1-month post-operative recovery period to ensure sufficient reporter expression. Fluorescence was then recorded in freely moving mice via an in vivo monitoring system that was constructed at our institution. For the in vivo recording system, a 488 nm laser was coupled with a 1 × 4 beam-splitter that divided the light into four laser excitation outputs of equal power. This setup enabled us to record from four animals simultaneously. Each of the emitted fluorescence signals was collected via a photomultiplier tube and a data acquisition card. In contrast to the previous bioluminescence in vivo circadian clock recording technique, this fluorescence in vivo recording system allowed the recording of circadian clock gene expression during the light cycle.

Recombinant Adeno-Associated Virus-Mediated Delivery of MicroRNA-21-3p Lowers Hypertension

Hypertension is the most important risk factor for cardiovascular diseases worldwide. However, the underlying molecular mechanisms of hypertension are complex and remain largely elusive. Here, we described a novel, microRNA-dependent therapeutic strategy for hypertension. First, we found that plasma microRNA-21-3p (miR-21-3p) levels were significantly reduced both in hypertensive patients and spontaneously hypertensive rats (SHRs) when compared with normal controls. In a series of experiments to dissect the role of miR-21-3p in hypertension, we showed that intravenous delivery of recombinant adeno-associated virus (rAAV)-mediated miR-21-3p expression induced a persistent attenuation of hypertension, with marked amelioration of target organ damages, including cardiac hypertrophy and fibrosis and artery and kidney fibrosis in SHRs, whereas miR-21-3p tough decoys (TuDs) counteracted the above effects. Computational prediction coupled with biochemical experiments revealed that the miR-21-3p-mediated hypotensive reduction effect was accomplished by regulating phenotypic switch of vascular smooth muscle cells (VSMCs) via suppression of the adrenal α2B-adrenergic receptor (ADRA2B) in arteries. Furthermore, we observed that activation of transcription factor NF-κB and SRF significantly increased the expression of miR-21-3p in VSMCs. In summary, our study is the first to identify a novel role and mechanism of miR-21-3p in blood pressure control and provides a possible strategy for hypertension therapy using rAAV-miR-21-3p.

In Vivo Genome Editing Restores Dystrophin Expression and Cardiac Function in Dystrophic Mice.

Duchenne muscular dystrophy is a severe inherited form of muscular dystrophy caused by mutations in the reading frame of the dystrophin gene disrupting its protein expression. Dystrophic cardiomyopathy is a leading cause of death in Duchenne muscular dystrophy patients, and currently no effective treatment exists to halt its progression. Recent advancement in genome editing technologies offers a promising therapeutic approach in restoring dystrophin protein expression. However, the impact of this approach on Duchenne muscular dystrophy cardiac function has yet to be evaluated. Therefore, we assessed the therapeutic efficacy of CRISPR (clustered regularly interspaced short palindromic repeats)-mediated genome editing on dystrophin expression and cardiac function in mdx/Utr +/- mice after a single systemic delivery of recombinant adeno-associated virus. To examine the efficiency and physiological impact of CRISPR-mediated genome editing on cardiac dystrophin expression and function in dystrophic mice. Here, we packaged SaCas9 (clustered regularly interspaced short palindromic repeat-associated 9 from Staphylococcus aureus) and guide RNA constructs into an adeno-associated virus vector and systemically delivered them to mdx/Utr +/- neonates. We showed that CRIPSR-mediated genome editing efficiently excised the mutant exon 23 in dystrophic mice, and immunofluorescence data supported the restoration of dystrophin protein expression in dystrophic cardiac muscles to a level approaching 40%. Moreover, there was a noted restoration in the architecture of cardiac muscle fibers and a reduction in the extent of fibrosis in dystrophin-deficient hearts. The contractility of cardiac papillary muscles was also restored in CRISPR-edited cardiac muscles compared with untreated controls. Furthermore, our targeted deep sequencing results confirmed that our adeno-associated virus-CRISPR/Cas9 strategy was very efficient in deleting the ≈23 kb of intervening genomic sequences. This study provides evidence for using CRISPR-based genome editing as a potential therapeutic approach for restoring dystrophic cardiomyopathy structurally and functionally.

Transfer of Therapeutic Genes into Fetal Rhesus Monkeys Using Recombinant Adeno-Associated Type I Viral Vectors.

Neuromuscular disorders such as Pompe disease (glycogen storage disease, type II), result in early and potentially irreversible cellular damage with a very limited opportunity for intervention in the newborn period. Pompe disease is due to deficiency in acid α-glucosidase (GAA) leading to lysosomal accumulation of glycogen in all cell types, abnormal myofibrillogenesis, respiratory insufficiency, neurological deficits, and reduced contractile function in striated muscle. Previous studies have shown that fetal delivery of recombinant adeno-associated virus (rAAV) encoding GAA to the peritoneal cavity of Gaa-/- mice resulted in high-level transduction of the diaphragm. While progression of other genetic disorders may occur later in life, the potential of fetal gene delivery to avoid the onset of irreversible damage suggests it is an attractive option for many inherited diseases. In this study, rhesus monkey fetuses were administered 4.5 × 1012 particles of rAAV type 1 expressing human GAA (rAAV1-CMV-hGAA), human α-1-antitrypsin (rAAV1-CBA-hAAT), or human mini-dystrophin (rAAV1-CMV-miniDMD) in the late first trimester using an established intraperitoneal ultrasound-guided approach. Fetuses were monitored sonographically and newborns delivered at term for postnatal studies. All animals remained healthy during the study period (growth, hematology, and clinical chemistry), with no evidence of adverse effects. Tissues were collected at a postnatal age of 3 months (∼7 months post-fetal gene transfer) for immunohistochemistry (IHC) and quantitative PCR. Both the diaphragm and peritoneum from vector-treated animals were strongly positive for expression of human GAA, AAT, or dystrophin by IHC, similar to findings when reporter genes were used. Protein expression in the diaphragm and peritoneum correlated with high vector copy numbers detected by real-time PCR. Other anatomical areas were negative, although the liver showed minimal evidence of human GAA, AAT, and DMD, vector genomes. In summary, delivery of rAAV vectors provided stable transduction of the muscular component of the diaphragm without any evidence of adverse effects.

373. Optimization of AAV-Gene Therapy for GM1-Gangliosidosis

GM1-gangliosidosis is an autosomal recessive disease caused by mutations in the GLB1 gene encoding for the lysosomal acid beta-galactosidase (βgal) enzyme. The resulting enzymatic deficiency leads to accumulation of GM1-ganglioside in neurons and eventually massive neurodegeneration. The incidence of GM1-gangliosidosis is estimated at 1:100,000-200,000 live births, and there is currently no available treatment. Intracranial delivery of recombinant adeno-associated virus (AAV) vectors has been shown to be highly effective in animal models of this disease. The choice of injection sites and promoters driving transgene expression are important parameters to translate into clinical trials a safe and effective AAV-based therapy for GM1 gangliosidosis. We performed a study using AAVrh10 vectors to determine the safest and most effective promoter and delivery route combination for AAVrh10-βgal gene therapy in a GM1-mouse model. We used a combination of two direct intracranial injection site - bilateral thalamic and deep cerebral nuclei infusions (Th+DCN), or bilateral thalamic and CSF infusion into one cerebral lateral ventricle (Th+ICV). We tested three different promoters of varying strengths. The CBA promoter, composed of the chicken beta actin promoter with an enhancer element and an artificial intron; the CBi, identical to the previous promoter but lacking the enhancer element; the CB, the basic chicken beta-actin promoter without an enhancer or intron. All vectors were delivered at a total dose of 4E9 (Th+DCN) or 1E10 (Th+ICV) vector genomes. At four weeks post injection, we analyzed the brain histologically, for beta-galactosidase expression and decrease in lysosomal storage. Additionally, we analyzed the enzyme activity of beta-galactosidase, as well as the total GM1-gangliosidosis storage by enzymatic assays and LC-MS/MS mass spectrometry, respectively. The AAVrh10-CBA-βgal vector restored GM1 ganglioside levels to normal, and generated the highest βgal activity of all three vectors. Additionally, the distribution was more extensive for the AAVrh10-CBA vector, showing distribution to the frontal cortex, while for the other vectors the enzyme was mostly restricted to the injection site and the immediate area. However, at this vector dose we did not detect any apparent toxicity or cell death. Thus, we determined that the AAVrh10-CBA-βgal vector is safe and effective, and thus appropriate to carry out long-term therapeutic studies in GM1 mice toward a clinical trial.

Convection Enhanced Delivery of Recombinant Adeno-associated Virus into the Mouse Brain.

Recombinant adeno-associated virus (rAAV) has become an extremely useful tool for the study of gene over expression or knockdown in the central nervous system of experimental animals. One disadvantage of intracranial injections of rAAV vectors into the brain parenchyma has been restricted distribution to relatively small volumes of the brain. Convection enhanced delivery (CED) is a method for delivery of clinically relevant amounts of therapeutic agents to large areas of the brain in a direct intracranial injection procedure. CED uses bulk flow to increase the hydrostatic pressure and thus improve volume distribution. The CED method has shown robust gene transfer and increased distribution within the CNS and can be successfully used for different serotypes of rAAV for increased transduction of the mouse CNS. This chapter details the surgical injection of rAAV by CED into a mouse brain.

Intraparenchymal Stereotaxic Delivery of rAAV and Special Considerations in Vector Handling.

Stereotaxic surgery enables precise and consistent microinjections to discrete neural nuclei. Using stereotaxic surgery to deliver viral vectors is a powerful tool that provides the ability to manipulate gene expression in specific regions, or even specific cell types in the brain. Here, we describe the proper handling and stereotaxic delivery of recombinant adeno-associated virus to various neuroanatomical structures of the rodent brain.

Widespread and Efficient Transduction of Spinal Cord and Brain Following Neonatal AAV Injection and Potential Disease Modifying Effect in ALS Mice

The architecture of the spinal cord makes efficient delivery of recombinant adeno-associated virus (rAAV) vectors throughout the neuraxis challenging. We describe a paradigm in which small amounts of virus delivered intraspinally to newborn mice result in robust rAAV-mediated transgene expression in the spinal cord. We compared the efficacy of rAAV2/1, 2/5, 2/8, and 2/9 encoding EGFP delivered to the hindlimb muscle (IM), cisterna magna (ICM), or lumbar spinal cord (IS) of neonatal pups. IS injection of all four capsids resulted in robust transduction of the spinal cord with rAAV2/5, 2/8, and 2/9 vectors appearing to be transported to brain. ICM injection resulted in widespread expression of EGFP in the brain, and upper spinal cord. IM injection resulted in robust muscle expression, with only rAAV2/8 and 2/9 transducing spinal motor and sensory neurons. As proof of concept, we use the IS paradigm to express murine Interleukin (IL)-10 in the spinal cord of the SOD1-G93A transgenic mouse model of amyotrophic lateral sclerosis. We show that expression of IL-10 in the spinal axis of SOD1-G93A mice altered the immune milieu and significantly prolonged survival. These data establish an efficient paradigm for somatic transgene delivery of therapeutic biologics to the spinal cord of mice.

Expanded Central Nervous System Gene Transfer in Rats by Intravenous Delivery of Recombinant Adeno-Associated Virus

Viral vector gene transfer is an important tool for studying protein function in vivo. Recombinant adeno-associated virus (AAV) is advantageous for gene transfer to the central nervous system. Over the past few years it has been shown that an intravenous vector delivery can transduce neurons throughout the central nervous system [1-3]. The images are of rats administered AAV serotype 9 vector encoding green fluorescent protein (GFP). The rats were injected intravenously on post-natal day one and imaged for GFP expression 4-12 weeks later. There is robust expression in the spinal cord and dorsal root ganglia and the cerebellum (Figures 1-3). We have been exploiting the efficient spinal cord transduction to study a spinal cord disease, amyotrophic lateral sclerosis [2-4].

Generation and in vivo evaluation of IL10-treated dendritic cells in a nonhuman primate model of AAV-based gene transfer

Preventing untoward immune responses against a specific antigen is a major challenge in different clinical settings such as gene therapy, transplantation, or autoimmunity. Following intramuscular delivery of recombinant adeno-associated virus (rAAV)-derived vectors, transgene rejection can be a roadblock to successful clinical translation. Specific immunomodulation strategies potentially leading to sustained transgene expression while minimizing pharmacological immunosuppression are desirable. Tolerogenic dendritic cells (TolDC) are potential candidates but have not yet been evaluated in the context of gene therapy, to our knowledge. Following intramuscular delivery of rAAV-derived vectors expressing an immunogenic protein in the nonhuman primate model, we assessed the immunomodulating potential of autologous bone marrow-derived TolDC generated in the presence of IL10 and pulsed with the transgene product. TolDC administered either intradermally or intravenously were safe and well tolerated. While the intravenous route showed a modest ability to modulate host immunity against the transgene product, intradermally delivery resulted in a robust vaccination of the macaques when associated to intramuscular rAAV-derived vectors-based gene transfer. These findings demonstrate the critical role of TolDC mode of injection in modulating host immunity. This study also provides the first evidence of the potential of TolDC-based immunomodulation in gene therapy.

The dual effect of ultrasound-targeted microbubble destruction in mediating recombinant adeno-associated virus delivery in renal cell carcinoma: transfection enhancement and tumor inhibition

Recombinant adeno-associated virus (rAAV) is recognized as a promising vector for cancer gene therapy, although its low transfer efficiency in less permissive cells limits extensive application. Our previous studies reported that ultrasound-targeted microbubble (MB) destruction (UTMD) enhanced rAAV transfer in its permissive retinal cells. In the present study, we investigated whether UTMD increased rAAV transfer in less permissive human renal cell carcinoma (hRCC) cells and tumors. hRCC cells were treated with rAAV2 under different conditions of UTMD, and the viral transfer efficiency and cell viability were analyzed. Fifty-two male nude mice (BALB/c) implanted with hRCC cells were randomly assigned to four groups consisting of rAAV, rAAV + ultrasound and rAAV + UTMD (20 µl and 40 µl of MBs). UTMD was initiated immediately after intratumoral viral injection, and viral transfer efficiency and tumor volumes were analyzed at 12 weeks after infection. The efficiency of non-augmented transfer of rAAV2 into hRCC cells was low (17.28 ± 2.44%). The use of UTMD enhanced viral transfer efficiency by two- to three-fold, and enhanced viral genomic DNA by more than nine-fold, without decreasing cell viability. In vivo studies also showed that UTMD increased rAAV2 transfer in tumor. The enhancements were maintained for a period of 12 weeks. Tumor growth in mice was inhibited by UTMD treatment, and UTMD treatment augmented by MBs (40 µl) produced an even stronger effect. UTMD enhanced rAAV2 transfer into less permissive RCC cells and tumors, resulting in inhibition of tumor growth, which suggests that UTMD may be a useful delivery tool for cancer gene therapy.

Abstract 18798: Gata4 and Gata6 Are Necessary for Neonatal but Not Adult Cardiac Function Under Normal Physiological Conditions

Objective: Fetal inactivation of the cardiac transcription factors Gata4 and Gata6 has been shown to cause postnatal heart dysfunction. However, the postnatal requirement for these transcription factors has not been defined. Methods: Using systemic delivery of recombinant adeno-associated virus 9 expressing Cre from the cardiac specific TNNT2 promoter (1.2E x 12VG/gram body weight; n=6), we inactivated floxed Gata4 and Gata6 alleles in neonatal or adult hearts. Left ventricular performance was followed serially using M-mode echocardiography and two-dimensional speckle tracking-derived myocardial tissue deformation (Visualsonics Vevo 2100) under light isofluorane anesthesia. The intervention group was compared to a control AAV-TNNT2 encoding luciferase. Findings: In the neonatal heart (P1-P5), effective knock-down of Gata4 and Gata6 (n=6 mice) was confirmed by Western blot and quantitative reverse-transcription PCR of myocardial tissue: We observed a 4.9-fold reduction in Gata4 expression; (95% C.I. +/-1.2) and a 6-fold reduction in Gata6 expression (95% C.I. +/- 1.2). Over the course of 5 days, left ventricular ejection fraction decreased significantly from a mean of 81% to 39% +/- 3% SEM (p<0.0001). Strain analysis confirmed a global reduction in myocardial performance after knock-down. The expression of heart failure markers, m-ANF and MYH7, were markedly up-regulated at 17-fold and 24-fold, respectively. In the adult heart (postnatal weeks 6-9, n=6), despite a technically successful systemic delivery of rAAV9, no noted left ventricular dysfunction was apparent on subsequent serial follow-up by m-mode echocardiography. Similarly, myocardial performance, as measured by strain & strain rate analysis in the longitudinal, circumferential and radial planes, remained equal in the intervention group to that of the control group. Conclusions: AAV9-TNNT2-Cre is an effective means of achieving temporally controlled inactivation of floxed alleles in the postnatal heart. Gata4 and Gata6 are required to maintain normal left ventricular systolic function during neonatal life, but are no longer required in adulthood under normal physiological conditions.

No tumour-initiating risk associated with scAAV transduction in newborn rat liver

Delivery of recombinant adeno-associated virus (rAAV) vectors to the newborn liver is followed by a rapid loss of episomal vector copies because of hepatocyte proliferation. In selected hepatocytes, integration of rAAV genomes can lead to a sustained expression of the transgene. The safety of in vivo gene therapy with single-stranded AAV vectors has been questioned in a study reporting a high incidence of hepatocellular carcinoma, associated with provirus integration events in mice that receive an single-stranded AAV injection at birth. To investigate the tumour-initiating potential of the newly established self-complementary AAV (scAAV) vectors in the liver, groups of newborn rats received intravenous injection of a scAAV vector encoding the green fluorescent protein (GFP), or were injected with phosphate-buffered saline (PBS) or diethylnitrosamine (DEN), a well-known liver tumour initiator. The rats were fed on a diet containing 2-acetylaminofluorene, a potent liver tumour-promoting agent to accelerate the carcinogenic process. After 2 months, the animals were killed and their livers analysed. Preneoplastic nodules were identified by glutathion S-transferase-p (GSTp) staining, and GFP expression was detected by immunohistochemistry. Vector genome integration events were analysed. The numbers of GSTp-positive foci were comparable in the PBS and the scAAV-GFP groups and significantly higher in the DEN group. The proportion of GSTp-positive foci that also expressed GFP was low and in the range expected for random occurrence. No specific integration hot spots were detected by linear amplification-mediated-PCR in transduced liver. In conclusion, scAAV transduction of newborn rat liver does not trigger preneoplastic lesions suggesting an absence of liver tumourigenesis.

Local gene delivery of heme oxygenase-1 by adeno-associated virus into osteoarthritic mouse joints exhibiting synovial oxidative stress

To evaluate the role of synovial oxidative stress on joint pathology in a spontaneous mouse model of osteoarthritis (OA) by intra-articular (IA) delivery of recombinant adeno-associated virus (rAAV) expressing anti-oxidant protein heme oxygenase-1 (HO-1). Joint transduction by rAAV vectors was evaluated with serotype 1, 2, 5 and 8 capsids carrying LacZ gene administered by IA injections into STR/ort mice. Transduced cell types were identified by β-galactosidase staining in sectioned joints. Effect of oxidative stress on AAV transduction of primary synoviocytes in vitro was quantitated by fluorescence-activated cell sorting (FACS) analysis. In vivo, the efficacy of rAAV1/HO-1 was tested by IA administration into STR/ort mice followed by histopathological scoring of cartilage. Levels of 3-nitrotyrosine (3-NT) and HO-1 were assessed by immunohistochemistry (IHC) of joint sections. Administration of a rAAV1 based vector into OA mouse joints resulted in transduction of the synovium, joint capsule, adipocytes and skeletal muscle while none of the serotypes showed significant cartilage transduction. All OA joints exhibited significantly elevated levels of oxidative stress marker, 3-NT, in the synovium compared to OA-resistant CBA-strain of mice. In vitro studies demonstrated that AAV transgene expression in primary synoviocytes was augmented by oxidative stress induced by H(2)O(2) and that a rAAV expressing HO-1 reduced the levels of oxidative stress. In vivo, HO-1 was increased in the synovium of STR/ort mice. However, delivery of rAAV1/HO-1 into OA joints did not reduce cartilage degradation. AAV-mediated HO-1 delivery into OA joints during active disease was not sufficient to improve cartilage pathology in this model.

Broad Transgenic, and Cardiac-Specific Viral Mediated, Over-Expression of Ribonucleotide Reductase Increases In Vivo Cardiac Contractility

We have previously demonstrated that substitution of ATP with 2 deoxy-ATP (dATP) increased the magnitude and rate of force production at all activating [Ca2+] in demembranated cardiac muscle via increased crossbridge binding and cycling rates. Furthermore, we demonstrated that cellular [dATP] is increased by adenoviral-driven overexpression of the ribonucleotide reductase (Rrm) complex, which increased contractility with no decrement of relaxation in adult rat cardiomyocytes from normal or infarcted hearts. In this study we hypothesized that in vivo overexpression of Rrm would increase cardiac contractility. Elevated cardiac Rrm was accomplished by 1) transgenic (Tg) overexpression in mice via the chicken beta-actin promoter and 2) intravenous delivery of recombinant adeno-associated virus (rAAV6) to drive Rrm production using a cardiac specific promoter (cTnT455). Echocardiographic fractional shortening was significantly increased in 3-month old Tg animals (43.5 ± 1.4%) as compared to wild-type (WT) littermates (34.5 ± 2.2%), and this increase persisted at 6 months of age (42.8 ± 2.5% vs 28.8 ± 2.7%). Similar increases in FS were seen in rAAV6-Rrm transduced animals (43.0 ± 2.1%) as compared to sham-injected control animals (32.1 ± 2.9%). LV pressure development (balloon catheterization) was elevated in Tg-Rrm vs. WT Langendorff perfused hearts, and concurrent NMR spectra indicated no difference in PCr:ATP ratio during high workload challenge. Upregulation of Rrm was confirmed via RT-PCR, western blot analysis, and immunohistochemistry. Preliminary western blot results also suggest no difference in SERCA expression, but increased Ser-16 phosphorylation of phospholamban, in Tg-Rrm vs WT hearts. This may, in part, explain our previously reported acceleration of Ca2+ transient decay seen in Rrm-overexpressing cardiomyocytes in vitro. Overall, these experiments demonstrate the feasibility of directly targeting the actin-myosin crossbridge to enhance cardiac contractility without impairing relaxation. HL65497, HL091368, AHA2310117.