Liver-specific microRNA-122 target sequences incorporated in AAV vectors efficiently inhibits transgene expression in the liver

Vectors based on adeno-associated virus (AAV) are effective in gene delivery in vivo. Tissue-specific gene expression is often needed to minimize ectopic expression in unintended cells and undesirable consequences. Here, we investigated whether incorporation of target sequences of tissue-specific microRNA (miRNA) into AAV vectors could inhibit ectopic expression in tissues such as the liver and hematopoietic cells. First we inserted liver-specific miR-122 target sequences (miR-122T) into the 3'-untranslated region (UTR) of a number of AAV vectors. After intravenous delivery in mice, we found that five copies of the 20mer miR-122T reduced liver expression of luciferase by 50-fold and β-galactosidase (LacZ) by 70-fold. Five copies of miR-122T also reduced mRNA levels of a secretable protein (myostatin propeptide) from the AAV vector plasmid by 23-fold in the liver. However, gene expression in other tissues, including the heart was not inhibited. Similarly, we inserted four copies of miR-142-3pT or miR-142-5pT, both hematopoietic lineage-specific, into the 3'-UTR of the AAV-luciferase vector. We wished to see whether they could prolong transgene expression by inhibiting expression in antigen-presenting cells. However, in vivo luciferase gene expression in major tissues declined with time, regardless of the miR-142 target sequences used. Quantitative analysis of the vector DNA in various tissues revealed that the decline of transgene expression in vivo was mainly because of promoter shut-off other than loss of AAV-transduced cells by immune destruction. Moreover, transgene expression was not detected in circulating mononuclear cells after delivering AAV9 vector with or without miR142T. These results demonstrate that liver-specific miR-122 target sequence in AAV vectors was highly efficient in reducing liver expression, whereas hematopoietic miR-142 target sequences were ineffective in preventing decline of AAV vector gene expression in nonhematopoietic tissues resulted from promoter shut-off.     

Application of Mutated miR-206 Target Sites Enables Skeletal Muscle-specific Silencing of Transgene Expression of Cardiotropic AAV9 Vectors

Insertion of completely complementary microRNA (miR) target sites (miRTS) into a transgene has been shown to be a valuable approach to specifically repress transgene expression in non-targeted tissues. miR-122TS have been successfully used to silence transgene expression in the liver following systemic application of cardiotropic adeno-associated virus (AAV) 9 vectors. For miR-206–mediated skeletal muscle-specific silencing of miR-206TS–bearing AAV9 vectors, however, we found this approach failed due to the expression of another member (miR-1) of the same miR family in heart tissue, the intended target. We introduced single-nucleotide substitutions into the miR-206TS and searched for those which prevented miR-1–mediated cardiac repression. Several mutated miR-206TS (m206TS), in particular m206TS-3G, were resistant to miR-1, but remained fully sensitive to miR-206. All these variants had mismatches in the seed region of the miR/m206TS duplex in common. Furthermore, we found that some m206TS, containing mismatches within the seed region or within the 3′ portion of the miR-206, even enhanced the miR-206– mediated transgene repression. In vivo expression of m206TS-3G– and miR-122TS–containing transgene of systemically applied AAV9 vectors was strongly repressed in both skeletal muscle and the liver but remained high in the heart. Thus, site-directed mutagenesis of miRTS provides a new strategy to differentiate transgene de-targeting of related miRs.     

Augmentation of AAV-mediated cardiac gene transfer after systemic administration in adult rats

Adeno-associated virus (AAV)-6 or -9-pseudotyped vectors are suitable for efficient cardiac gene transfer after intravenous injection in mice. However, a systemic application in larger animals or humans would require very high doses of viral particles. Therefore, the aim of our study was to test if ultrasound-targeted microbubble destruction could augment cardiac transduction of AAV vectors after intravenous administration in rats. To analyze efficiency and specificity of gene transfer, microbubbles loaded with AAV-6 or -9 harboring a luciferase or enhanced green fluorescent protein (EGFP) reporter gene were infused into the jugular vein of adult Sprague-Dawley rats. During the infusion, high mechanical index ultrasound was administered to the heart. Control rats received the same amount of virus without microbubbles, but with ultrasound. After 4 weeks, organs were harvested and analyzed for reporter gene expression. In contrast to low cardiac expression after systemic transfer of the vector solution without microbubbles, ultrasound-targeted destruction of microbubbles significantly increased cardiac reporter activities between 6- and 20-fold. Analysis of spatial distribution of transgene expression using an AAV-9 vector encoding for EGFP revealed transmural expression predominantly in the left ventricular anterior wall. In conclusion, ultrasound targeted microbubble destruction augments cardiac transduction of AAV vectors in rats. This approach may be suitable for efficient, specific and noninvasive AAV-mediated gene transfer in larger animals or humans.     

AAV vectors transduce hepatocytes in vivo as efficiently in cirrhotic as in healthy rat livers

In liver cirrhosis, abnormal liver architecture impairs efficient transduction of hepatocytes with large viral vectors such as adenoviruses. Here we evaluated the ability of adeno-associated virus (AAV) vectors, small viral vectors, to transduce normal and cirrhotic rat livers. Using AAV serotype-1 (AAV1) encoding luciferase (AAV1Luc) we analyzed luciferase expression with a CCD camera. AAV1Luc was injected through the hepatic artery (intra-arterial (IA)), the portal vein (intra-portal (IP)), directly into the liver (intra-hepatic (IH)) or infused into the biliary tree (intra-biliar). We found that AAV1Luc allows long-term and constant luciferase expression in rat livers. Interestingly, IP administration leads to higher expression levels in healthy than in cirrhotic livers, whereas the opposite occurs when using IA injection. IH administration leads to similar transgene expression in cirrhotic and healthy rats, whereas intra-biliar infusion is the least effective route. After 70% partial hepatectomy, luciferase expression decreased in the regenerating liver, suggesting lack of efficient integration of AAV1 DNA into the host genome. AAV1Luc transduced mainly the liver but also the testes and spleen. Within the liver, transgene expression was found mainly in hepatocytes. Using a liver-specific promoter, transgene expression was detected in hepatocytes but not in other organs. Our results indicate that AAVs are convenient vectors for the treatment of liver cirrhosis.     

Efficacy and safety of adeno-associated viral vectors based on serotype 8 and 9 vs. lentiviral vectors for hemophilia B gene therapy

BACKGROUND: Adeno-associated viral (AAV) and lentiviral vectors are promising vectors for gene therapy for hemophilia because they are devoid of viral genes and have the potential for long-term gene expression. OBJECTIVES: To compare the performance of different AAV serotypes (AAV8 and AAV9) vs. lentiviral vectors expressing factor (F) IX. METHODS AND RESULTS: AAV-based and lentiviral vectors were generated that express FIX from the same hepatocyte-specific expression cassette. AAV9 transduced the liver as efficiently as AAV8 and resulted in supra-physiological FIX levels (3000-6000% of normal) stably correcting the bleeding diathesis. Surprisingly, AAV9 resulted in unprecedented and widespread cardiac gene transfer, which was more efficient than with AAV8. AAV8 and AAV9 were not associated with any proinflammatory cytokine induction, in accordance with their minimal interactions with innate immune effectors. In contrast, lentiviral transduction resulted in modest and stable FIX levels near the therapeutic threshold (1%) and triggered a rapid self-limiting proinflammatory response (interleukin-6), which probably reflected their ability to efficiently interact with the innate immune system. CONCLUSIONS: AAV8 and 9 result in significantly higher FIX expression levels and have a reduced proinflammatory risk in comparison with lentiviral vectors. The unexpected cardiotropic properties of AAV9 have implications for gene therapy for heart disease.     

Comparative Cardiac Gene Delivery of Adeno‐Associated Virus Serotypes 1–9 reveals that AAV6 Mediates the Most Efficient Transduction in Mouse Heart

Cardiac gene transfer is an attractive tool for developing novel heart disease treatments. Adeno‐associated viral (AAV) vectors are widely used to mediate transgene expression in animal models and are being evaluated for human gene therapy. However, it is not clear which serotype displays the best cardiac tropism. Therefore, we curried out this study to directly compare AAV serotypes 1–9 heart transduction efficiency after indirect intracoronary injection. AAV‐cytomegalovirus immediate early enhancer promoter (CMV)‐luciferase serotypes 1–9 were injected in the left ventricular cavity of adult mice, after cross‐clamping the ascending aorta and pulmonary artery. An imaging system was used to visualize luciferase expression at 3, 7, 21, 70, and 140 days postinjection. Echocardiography was performed to evaluate cardiac function on day 140. At the end of the study, luciferase enzyme activity and genome copies of the different AAV serotypes were assessed in several tissues and potential AAV immunogenicity was evaluated on heart sections by staining for macrophage and lymphocyte antigens. Among AAV serotypes 1–9, AAV6 showed the best capability of achieving high transduction levels in the myocardium in a tissue‐specific manner, whereas the other serotypes had less cardiac transduction and more extracardiac expression, especially in the liver. Importantly, none of the serotypes tested with this marker gene affected cardiac function nor was associated with inflammation.     

Cardio-specific long-term gene expression in a porcine model after selective pressure-regulated retroinfusion of adeno-associated viral (AAV) vectors

Cornerstone for an efficient cardiac gene therapy is the need for a vector system, which enables selective and long-term expression of the gene of interest. In rodent animal models adeno-associated viral (AAV) vectors like AAV-6 have been shown to efficiently transduce cardiomyocytes. However, since significant species-dependent differences in transduction characteristics exist, large animal models are of imminent need for preclinical evaluations. We compared gene transfer efficiencies of AAV-6 and heparin binding site-deleted AAV-2 vectors in a porcine model. Application of the AAVs was performed by pressure-regulated retroinfusion of the anterior interventricular cardiac vein, which has been previously shown to efficiently deliver genes to the myocardium (3.5 x 10(10) viral genomes per animal; n=5 animals per group). All vectors harbored a luciferase reporter gene under control of a cytomegalovirus (CMV)-enhanced 1.5 kb rat myosin light chain promoter (CMV-MLC2v). Expression levels were evaluated 4 weeks after gene transfer by determining luciferase activities. To rule out a systemic spillover peripheral tissue was analyzed by PCR for the presence of vector genomes. Selective retroinfusion of AAV serotype 6 vectors into the anterior cardiac vein substantially increased reporter gene expression in the targeted distal left anterior descending (LAD) territory (65 943+/-31 122 vs control territory 294+/-69, P<0.05). Retroinfusion of AAV-2 vectors showed lower transgene expression, which could be increased with coadministration of recombinant human vascular endothelial growth factor (1365+/-707 no vascular endothelial growth factor (VEGF) vs 38 760+/-2448 with VEGF, P<0.05). Significant transgene expression was not detected in other organs than the heart, although vector genomes were detected also in the lung and liver. Thus, selective retroinfusion of AAV-6 into the coronary vein led to efficient long-term myocardial reporter gene expression in the targeted LAD area of the porcine heart. Coapplication of VEGF significantly increased transduction efficiency of AAV-2.     

Characterization of the relationship of AAV capsid domain swapping to liver transduction efficiency.

Recombinant adeno-associated virus (AAV) vectors show promise for use in gene therapy. For liver-targeted gene transfer in animals, AAV vectors pseudotyped with the AAV serotype 8 (AAV8) capsid have definite advantages over the widely used but less efficient serotype AAV2, even though the capsid amino acid sequences are 82% conserved. To demonstrate the mechanism behind the higher liver transduction efficiency associated with AAV8 capsids, we adopted a domain-swapping strategy that would generate 27 chimeric capsid genes containing exchanged domains between AAV2 and AAV8. The resulting chimeric capsids were then used to package AAV genomes with a liver-specific human coagulation factor IX (hFIX) expression cassette. By comparing the transduction efficiencies between vectors pseudotyped with chimeric, AAV2 and AAV8 capsids, we found that the more efficient liver transduction achieved by AAV8 was closely related to the components of its interstrand Loop IV domain, particularly the subloops 1 and 4. These subloops are exposed on opposite sides of a threefold proximal peak on the virion surface, which may function as a critical structural determinant for AAV transduction. Because a single specific peptide component could not explain all the observed differences in the transduction parameters, we suggest that important subloop regions require interaction with other portions of the capsid for their functioning.     

Systemic elimination of de novo capsid protein synthesis from replication-competent AAV contamination in the liver

The capsid protein synthesis in targeted tissues resulting from residual contaminating replication-competent adeno-associated virus particles (rcAAV) remains a concern for hazardous immune responses that shut down the factor IX expression in the hemophilia B clinical trial. To systematically reduce/eliminate the effects of potential contaminating rcAAV particles, we designed a novel adeno-associated virus (AAV) helper (pH22mir) with a microRNA binding cassette containing multiple copies of liver-specific (hsa-mir-122) and hematopoietic-specific (has-mir-142-3p) sequences to specifically control cap gene expression. In 293 cells, the rep and cap gene from pH22mir functioned similarly to that of conventional helper pH22. The vector yields and compositions from pH22mir and pH22 were indistinguishable. The performance of vector produced in this new system was comparable to that of similar vectors produced by conventional methods. In the human hepatic cell line, the capsid expression was reduced significantly from cap-mir cassette driven by a cytomegalovirus promoter. In the liver, 99.9% of capsid expression could be suppressed and no cap expression could be detected by western blot. In summary, we demonstrated a new concept in reducing de novo capsid synthesis in the targeted tissue. This strategy may not only help AAV vectors in controlling undesirable capsid gene expression, but can also be adopted for lentiviral or adenoviral vector production.     

Transendocardial delivery of AAV6 results in highly efficient and global cardiac gene transfer in rhesus macaques

Heart disease is the leading cause of morbidity and mortality, and cardiac gene transfer has potential as a novel therapeutic approach. We previously demonstrated safe and efficient gene transfer to the canine heart using a percutaneous transendocardial injection procedure to deliver self-complementary (sc) adeno-associated virus 6 (AAV6) vector. In the present study, we proceed with our vertical translation study to evaluate cardiac gene transfer in nonhuman primates (NHPs). We screened approximately 30 adult male rhesus macaques for the presence of neutralizing antibodies against AAV6, AAV8, and AAV9, and then selected seven monkeys whose antibody titers against these three serotypes were lower than 1/5. The animals were then randomized to receive either scAAV6 (n=3), scAAV8 (n=1), or scAAV9 (n=3) vector expressing the enhanced green fluorescent protein (EGFP) reporter gene at a dose of 5.4×10(12) genome copies/kg, which was administered according to a modified version of our previously developed transendocardial injection procedure. One animal treated with scAAV6 died secondary to esophageal intubation. The remaining animals were euthanized 7 days after gene transfer, at which time tissue was collected for analysis of EGFP expression, histopathology, and biodistribution of the vector genome. We found that (i) transendocardial delivery of AAV is safe in the NHP, (ii) AAV6 and AAV8 provide efficient cardiac gene transfer at similar levels and are superior to AAV9, and (iii) AAV6 is more cardiac-specific than AAV8 and AAV9. The results of this NHP study may help guide the development AAV vectors for the treatment of cardiovascular disease in humans.     

Topoisomerase inhibition accelerates gene expression after adeno-associated virus-mediated gene transfer to the mammalian heart.

Utility of adeno-associated virus 2 (AAV2) vectors for cardiac gene therapy is limited by the prolonged lag phase before maximal gene expression. Topoisomerase inhibition can induce AAV2-mediated gene expression in vivo, but with variable success in different tissues. In this study, we demonstrate that topoisomerase inhibition can accelerate AAV2-mediated gene expression in the mouse heart. We used an AAV2 vector expressing firefly luciferase and monitored expression kinetics using non-invasive bioluminescence imaging. In the group receiving vector alone, cardiac luciferase activity was evident from week 2 onward and increased progressively to reach a steady plateau by 9 weeks postinjection. In the group receiving vector and camptothecine (CPT), luciferase expression was evident from days 2 to 4 onward and increased rapidly to reach a steady plateau by 3-4 weeks postinjection, nearly three times faster than in the absence of CPT (P<0.05). Southern blot analysis of AAV2 genomes in cardiac tissue showed rapid conversion of the AAV2 genome from its single-stranded to double-stranded form in CPT-treated mice. Non-invasive determinations of luciferase expression correlated well with in vitro luciferase assays. Direct injection of the AAV2 vector and long-term luciferase gene expression had no detectable effects on normal cardiac function as assessed by magnetic resonance imaging.     

Differential myocardial gene delivery by recombinant serotype-specific adeno-associated viral vectors.

Recombinant cross-packaging of adeno-associated virus (AAV) genome of one serotype into other AAV serotypes has the potential to optimize tissue-specific gene transduction and expression in the heart. To evaluate the role of AAV1 to 5 virion shells on AAV2 transgene transduction, we constructed hybrid vectors in which each serotype capsid coding domain was cloned into a common vector backbone containing AAV2 replication genes. Constructs were tested for expression in: (1) adult murine heart in vivo using direct injection of virus, (2) neonatal and adult murine ventricular cardiomyocytes in vitro, and (3) adult human ventricular cardiomyocytes in vitro, using green fluorescent protein (GFP) as the measurable transgene. Serotype 1 virus demonstrated the highest transduction efficiency in adult murine cardiomyocytes both in vitro and in vivo, while serotype 2 virus had the greater transduction efficiency in neonatal cardiomyocytes in vitro. Prolonged in vivo myocardial GFP expression was observed for up to 12 months using serotype 1 and 2 vectors only. In human cardiomyocytes, serotype 1 vector was superior in transduction efficiency, followed by types 2, 5, 4, and 3. These data establish a hierarchy for efficient serotype-specific vector transduction in myocardial tissue. AAV1 serotype packaging results in more efficient transduction of genes in the murine and human adult heart, compared to other AAV serotypes. Our results suggest that adult human cardiac gene therapy may be enhanced by the use of serotype 1-specific AAV vectors.     

Engineering Liver-detargeted AAV9 Vectors for Cardiac and Musculoskeletal Gene Transfer

We report the generation of a new class of adeno-associated virus serotype 9 (AAV9)-derived vectors displaying selective loss of liver tropism and demonstrating potential for cardiac and musculoskeletal gene transfer applications. Random mutagenesis of residues within a surface-exposed region of the major AAV9 capsid protein yielded a capsid library with mutations clustered at the icosahedral threefold symmetry axis. Using a combination of sequence analysis, structural models, and in vivo screening, we identified several functionally diverse AAV9 variants. The latter were classified into three functional subgroups, with respect to parental AAV9 displaying: (i) decreased transduction efficiency across multiple tissues; (ii) a selective decrease in liver transduction, or (iii) a similar transduction profile. Notably, variants 9.45 and 9.61 (subgroup II) displayed 10- to 25-fold lower gene transfer efficiency in liver, while transducing cardiac and skeletal muscle as efficiently as AAV9. These results were further corroborated by quantitation of vector genome copies and histological analysis of reporter (tdTomato) gene expression. The study highlights the feasibility of generating AAV vectors with selectively ablated tissue tropism, which when combined with other targeting strategies could allow sharply segregated gene expression. Liver-detargeted AAV9 variants described herein are excellent candidates for preclinical evaluation in animal models of cardiac and musculoskeletal disease.     

Long-term preservation of cardiac structure and function after adeno-associated virus serotype 9-mediated microdystrophin gene transfer in mdx mice

Dystrophin plays an important role in muscle contraction, linking the intracellular cytoskeleton to the extracellular matrix. Mutations of the dystrophin gene leading to a complete loss of the protein cause Duchenne muscular dystrophy (DMD), frequently associated with severe cardiomyopathy. Early clinical trials in DMD using gene transfer to skeletal muscle are underway, but gene transfer to dystrophic cardiac muscle has not yet been tested in humans. The aim of this study was to develop an optimized protocol for cardiac gene therapy in the mouse model of dystrophin deficiency (mdx), using a cardiac promoter for expression of a microdystrophin (μDys) transgene packaged into an adeno-associated virus serotype 9 vector (AAV9). In this study adult mdx mice were intravenously injected with 1×10(12) genomic particles of AAV9 vectors carrying a cDNA encoding μDys under the control of either a ubiquitously active cytomegalovirus (CMV) promoter or a cardiac-specific CMV-enhanced myosin light chain (MLC0.26) promoter. After 10 months, both AAV9 vectors led to sustained μDys expression in cardiac muscle, but the MLC promoter conferred about 4-fold higher protein levels. AAV9-CMV-MLC0.26-μDys resulted in significant protection of cardiac morphology and function as assessed by histopathology, echocardiography, and left ventricular catheterization. In conclusion, we established an AAV9-mediated gene transfer approach for efficient and specific long-term μDys expression in the hearts of mdx mice, resulting in a sustained therapeutic effect. Thus, this approach might be a basis for further translation into a treatment strategy for DMD-associated cardiomyopathy.     

Adeno-associated virus vector serotypes mediate sustained correction of bilirubin UDP glucuronosyltransferase deficiency in rats.

Crigler-Najjar (CN) patients have no bilirubin UDP glucuronosyltransferase (UGT1A1) activity and suffer brain damage because of bilirubin toxicity. Vectors based on adeno-associated virus (AAV) serotype 2 transduce liver cells with relatively low efficiency. Recently, AAV serotypes 1, 6, and 8 have been shown to be more efficient for liver cell transduction. We compared AAV serotypes 1, 2, 6, and 8 for correction of UGT1A1 deficiency in the Gunn rat model of CN disease. Adult Gunn rats were injected with CMV-UGT1A1 AAV vectors. Serum bilirubin was decreased over the first year by 64% for AAV1, 16% for AAV2, 25% for AAV6, and 35% for AAV8. Antibodies to UGT1A1 were detected after injection of all AAV serotypes. An AAV1 UGT1A1 vector with the liver-specific albumin promoter corrected serum bilirubin levels but did not induce UGT1A1 antibodies. Two years after injection of AAV vectors all animals had large lipid deposits in the liver. These lipid deposits were not seen in age-matched control animals. AAV1 vectors are promising candidates for CN gene therapy because they can mediate a reduction in serum bilirubin levels in Gunn rats that would be therapeutic in humans.     

In vivo high-efficiency transcoronary gene delivery and Cre-LoxP gene switching in the adult mouse heart

High-efficiency somatic gene transfer in adult mouse heart has not yet been achieved in vivo. Here, we demonstrate high-efficiency in vivo transcoronary gene delivery to the adult murine myocardium using a catheter-based technique with recombinant adenovirus (AdV) and adeno-associated virus (AAV) vectors in normal and genetically engineered mice. The method involves immersion hypothermia followed by transient aortic and pulmonary artery occlusion with proximal intra-aortic segmental injection of cardioplegic solution containing substance P and viral vectors. Gene expression measured using a LacZ marker gene was observed throughout both ventricles. The expression efficiency of a cytoplasmic LacZ marker gene in the left ventricular myocardium was 56.4+/-14.5% (mean+/-s.d.) at 4 days with an AdV vector, and with an AAV vector it was 81.0+/-5.9% at 4 weeks. Following AAV gene transfer, no gene expression was found in kidney, brain, lung, and spleen, but there was slight expression in liver. In addition, we demonstrate temporally controlled genetic manipulation in the heart with an efficiency of 54.6+/-5.2%, by transferring an AdV vector carrying Cre recombinase in ROSA26 flox-LacZ reporter mice. Procedure-related mortality was 16% for AdV and zero for AAV transfer. Thus, this method provides efficient, relatively homogeneous gene expression in both ventricles of the adult mouse heart, and offers a novel approach for conditional gene rescue or ablation in genetically engineered mouse models.     

Adeno-associated virus (AAV) serotype 9 provides global cardiac gene transfer superior to AAV1, AAV6, AAV7, and AAV8 in the mouse and rat

Heart disease is the leading cause of morbidity and mortality. Cardiac gene transfer may serve as a novel therapeutic approach. This investigation was undertaken to compare cardiac tropisms of adeno-associated virus (AAV) serotypes 1, 6, 7, 8, and 9. Neonatal mice were injected with 2.5 x 10(11) genome copies (GC) of AAV serotype 1, 6, 7, 8, or 9 expressing LacZ under the control of the constitutive chicken beta-actin promoter with cytomegalovirus enhancer promoter via intrapericardial injection and monitored for up to 1 year. Adult rats were injected with 5 x 10(11) GC of the AAV vectors via direct cardiac injection and monitored for 1 month. Cardiac distribution of LacZ expression was assessed by X-Gal histochemistry, and beta-galactosidase activity was quantified in a chemiluminescence assay. Cardiac functional data and biodistribution data were also collected in the rat. AAV9 provided global cardiac gene transfer stable for up to 1 year that was superior to other serotypes. LacZ expression was relatively cardiac specific, and cardiac function was unaffected by gene transfer. AAV9 provides high-level, stable expression in the mouse and rat heart and may provide a simple alternative to the creation of cardiac-specific transgenic mice. AAV9 should be used in rodent cardiac studies and may be the vector of choice for clinical trials of cardiac gene transfer.