Long-term effect of gene therapy on Leber's congenital amaurosis.

BACKGROUND: Mutations in RPE65 cause Leber's congenital amaurosis, a progressive retinal degenerative disease that severely impairs sight in children. Gene therapy can result in modest improvements in night vision, but knowledge of its efficacy in humans is limited. METHODS: We performed a phase 1-2 open-label trial involving 12 participants to evaluate the safety and efficacy of gene therapy with a recombinant adeno-associated virus 2/2 (rAAV2/2) vector carrying the RPE65 complementary DNA, and measured visual function over the course of 3 years. Four participants were administered a lower dose of the vector, and 8 were administered a higher dose. In a parallel study in dogs, we investigated the relationship among vector dose, visual function, and electroretinography (ERG) findings. RESULTS: Improvements in retinal sensitivity were evident, to varying extents, in six participants for up to 3 years, peaking at 6 to 12 months after treatment and then declining. No associated improvement in retinal function was detected by means of ERG. Three participants had intraocular inflammation, and two had clinically significant deterioration of visual acuity. The reduction in central retinal thickness varied among participants. In dogs, RPE65 gene therapy with the same vector at lower doses improved vision-guided behavior, but only higher doses resulted in improvements in retinal function that were detectable with the use of ERG. CONCLUSIONS: Gene therapy with rAAV2/2 RPE65 vector improved retinal sensitivity, albeit modestly and temporarily. Comparison with the results obtained in the dog model indicates that there is a species difference in the amount of RPE65 required to drive the visual cycle and that the demand for RPE65 in affected persons was not met to the extent required for a durable, robust effect. (Funded by the National Institute for Health Research and others; ClinicalTrials.gov number, NCT00643747.).

Safety and liver transduction efficacy of rAAV5-cohPBGD in nonhuman primates: a potential therapy for acute intermittent porphyria.

Acute intermittent porphyria (AIP) results from haplo-insufficient activity of porphobilinogen deaminase (PBGD) and is characterized clinically by life-threatening, acute neurovisceral attacks. To date, liver transplantation is the only curative option for AIP. The aim of the present preclinical nonhuman primate study was to determine the safety and transduction efficacy of an adeno-associated viral vector encoding PBGD (recombinant AAV serotype 5-codon-optimized human porphobilinogen deaminase, rAAV5-cohPBGD) administered intravenously as part of a safety program to start a clinical study in patients with AIP. Macaques injected with either 1 × 10(13) or 5 × 10(13) vector genomes/kg of clinical-grade rAAV5-cohPBGD were monitored by standardized clinical parameters, and vector shedding was analyzed. Liver transduction efficacy, biodistribution, vector integration, and histopathology at day 30 postvector administration were determined. There was no evidence of acute toxicity, and no adverse effects were observed. The vector achieved efficient and homogenous hepatocellular transduction, reaching transgenic PBGD expression levels equivalent to 50% of the naturally expressed PBGD mRNA. No cellular immune response was detected against the human PBGD or AAV capsid proteins. Integration site analysis in transduced liver cells revealed an almost random integration pattern supporting the good safety profile of rAAV5-cohPBGD. Together, data obtained in nonhuman primates indicate that rAAV5-cohPBGD represents a safe therapy to correct the metabolic defect present in AIP patients.

A largely random AAV integration profile after LPLD gene therapy.

The clinical application of adeno-associated virus vectors (AAVs) is limited because of concerns about AAV integration-mediated tumorigenicity. We performed integration-site analysis after AAV1-LPL(S447X) intramuscular injection in five lipoprotein lipase-deficient subjects, revealing random nuclear integration and hotspots in mitochondria. We conclude that AAV integration is potentially safe and that vector breakage and integration may occur from each position of the vector genome. Future viral integration-site analyses should include the mitochondrial genome.

Transient and intensive pharmacological immunosuppression fails to improve AAV-based liver gene transfer in non-human primates.

BACKGROUND: Adeno-associated vectors (rAAV) have been used to attain long-term liver gene expression. In humans, the cellular immune response poses a serious obstacle for transgene persistence while neutralizing humoral immunity curtails re-administration. Porphobilinogen deaminase (PBGD) haploinsufficiency (acute intermittent porphyria) benefits from liver gene transfer in mouse models and clinical trials are about to begin. In this work, we sought to study in non-human primates the feasibility of repeated gene-transfer with intravenous administration of rAAV5 vectors under the effects of an intensive immunosuppressive regimen and to analyze its ability to circumvent T-cell immunity and thereby prolong transgene expression. METHODS: Three female Macaca fascicularis were intravenously injected with 1 x 10(13) genome copies/kg of rAAV5 encoding the human PBGD. Mycophenolate mofetil (MMF), anti-thymocyte immunoglobulin, methylprednisolone, tacrolimus and rituximab were given in combination during 12 weeks to block T- and B-cell mediated adaptive immune responses in two macaques. Immunodeficient and immunocompetent mice were intravenously injected with 5 x 10(12) genome copies/kg of rAAV5-encoding luciferase protein. Forty days later MMF, tacrolimus and rituximab were daily administrated to ascertain whether the immunosuppressants or their metabolites could interfere with transgene expression. RESULTS: Macaques given a rAAV5 vector encoding human PBGD developed cellular and humoral immunity against viral capsids but not towards the transgene. Anti-AAV humoral responses were attenuated during 12 weeks but intensely rebounded following cessation of the immunosuppressants. Accordingly, subsequent gene transfer with a rAAV5 vector encoding green fluorescent protein was impossible. One macaque showed enhanced PBGD expression 25 weeks after rAAV5-pbgd administration but overexpression had not been detected while the animal was under immunosuppression. As a potential explanation, MMF decreases transgene expression in mouse livers that had been successfully transduced by a rAAV5 several weeks before MMF onset. Such a silencing effect was independent of AAV complementary strand synthesis and requires an adaptive immune system. CONCLUSIONS: These results indicate that our transient and intensive pharmacological immunosuppression fails to improve AAV5-based liver gene transfer in non-human primates. The reasons include an incomplete restraint of humoral immune responses to viral capsids that interfere with repeated gene transfer in addition to an intriguing MMF-dependent drug-mediated interference with liver transgene expression.

Development of a liver-specific Tet-on inducible system for AAV vectors and its application in the treatment of liver cancer.

Recombinant adeno-associated virus (rAAV) are effective gene delivery vehicles that can mediate long-lasting transgene expression. However, tight regulation and tissue-specific transgene expression is required for certain therapeutic applications. For regulatable expression from the liver we designed a hepatospecific bidirectional and autoregulatory tetracycline (Tet)-On system (Tet(bidir)Alb) flanked by AAV inverted terminal repeats (ITRs). We characterized the inducible hepatospecific system in comparison with an inducible ubiquitous expression system (Tet(bidir)CMV) using luciferase (luc). Although the ubiquitous system led to luc expression throughout the mouse, luc expression derived from the hepatospecific system was restricted to the liver. Interestingly, the induction rate of the Tet(bidir)Alb was significantly higher than that of Tet(bidir)CMV, whereas leakage of Tet(bidir)Alb was significantly lower. To evaluate the therapeutic potential of this vector, an AAV-Tet(bidir)-Alb-expressing interleukin-12 (IL-12) was tested in a murine model for hepatic colorectal metastasis. The vector induced dose-dependent levels of IL-12 and interferon-γ (IFN-γ), showing no significant toxicity. AAV-Tet(bidir)-Alb-IL-12 was highly efficient in preventing establishment of metastasis in the liver and induced an efficient T-cell memory response to tumor cells. Thus, we have demonstrated persistent, and inducible in vivo expression of a gene from a liver-specific Tet-On inducible construct delivered via an AAV vector and proved to be an efficient tool for treating liver cancer.

Adeno-associated virus liver transduction efficiency measured by in vivo [18F]FHBG positron emission tomography imaging in rodents and nonhuman primates.

Recombinant adeno-associated virus 5 (rAAV5) represents a candidate vector with unique advantages for the treatment of hepatic disorders because of its narrow hepatic tropism. Noninvasive in vivo imaging of transgene expression provides an important tool with which to quantify the transduction efficiency, and duration and location, of transgene expression. In this study, we used positron emission tomography (PET) and positron emission tomography-computed tomography (PET-CT) imaging to monitor liver transduction efficacy in rodents and nonhuman primates that received rAAV5 vector encoding herpes simplex virus thymidine kinase (HSV-TK). HSV-TK expression in liver was also measured by immunohistochemistry. Notable differences in liver transduction efficiency were found, dependent on the animal species and sex. Male rodents were better transduced than females, as previously described. Moreover, male nonhuman primates also displayed increased hepatic expression of the rAAV5-delivered transgene, indicating that differences in rAAV-mediated liver transduction can be anticipated in humans. Our results demonstrate the high sensitivity and reproducibility of PET, using HSV-TK and [(18)F]FHBG, to detect gene expression after rAAV vector administration into living animals, confirming the utility of this technology in the quantification of transgene expression, even at low expression levels. However, we also describe how an immune response against HSV-TK hampered analysis of long-term expression in nonhuman primates.

Phenotypic correction of a mouse model for primary hyperoxaluria with adeno-associated virus gene transfer.

Primary hyperoxaluria type I (PH1) is an inborn error of metabolism caused by deficiency of the hepatic enzyme alanine-glyoxylate aminotransferase (AGXT or AGT) which leads to overproduction of oxalate by the liver and subsequent urolithiasis and renal failure. The current therapy largely depends on liver transplantation, which is associated with significant morbidity and mortality. To explore an alternative treatment, we used somatic gene transfer in a mouse genetic model for PH1 (Agxt1KO). Recombinant adeno-associated virus (AAV) vectors containing the human AGXT complementary DNA (cDNA) were pseudotyped with capsids from either serotype 8 or 5, and delivered to the livers of Agxt1KO mice via the tail vein. Both AAV8-AGXT and AAV5-AGXT vectors were able to reduce oxaluria to normal levels. In addition, treated mice showed blunted increase of oxaluria after challenge with ethylene glycol (EG), a glyoxylate precursor. In mice, AGT enzyme activity in whole liver extracts were restored to normal without hepatic toxicity nor immunogenicity for the 50 day follow-up. In summary, this study demonstrates the correction of primary hyperoxaluria in mice treated with either AAV5 or AAV8 vectors.

Sustained enzymatic correction by rAAV-mediated liver gene therapy protects against induced motor neuropathy in acute porphyria mice.

Acute intermittent porphyria (AIP) is characterized by a hereditary deficiency of hepatic porphobilinogen deaminase (PBGD) activity. Clinical features are acute neurovisceral attacks accompanied by overproduction of porphyrin precursors in the liver. Recurrent life-threatening attacks can be cured only by liver transplantation. We developed recombinant adeno-associated virus (rAAV) vectors expressing human PBGD protein driven by a liver-specific promoter to provide sustained protection against induced attacks in a predictive model for AIP. Phenobarbital injections in AIP mice induced porphyrin precursor accumulation, functional block of nerve conduction, and progressive loss of large-caliber axons in the sciatic nerve. Hepatocyte transduction showed no gender variation after rAAV2/8 injection, while rAAV2/5 showed lower transduction efficiency in females than males. Full protection against induced phenobarbital-attacks was achieved in animals showing over 10% of hepatocytes expressing high amounts of PBGD. More importantly, sustained hepatic expression of hPBGD protected against loss of large-caliber axons in the sciatic nerve and disturbances in nerve conduction velocity as induced by recurrent phenobarbital administrations. These data show for the first time that porphyrin precursors generated in the liver interfere with motor function. rAAV2/5-hPBGD vector can be produced in sufficient quantity for an intended gene therapy trial in patients with recurrent life-threatening porphyria attacks.

AAV gene therapy as a means to increase apolipoprotein (Apo) A-I and high-density lipoprotein-cholesterol levels: correction of murine ApoA-I deficiency.

BACKGROUND: Inherited apolipoprotein (Apo) A-I deficiency is an orphan disorder characterized by high-density lipoprotein (HDL)-cholesterol deficiency and premature atherosclerosis. Constitutive over-expression of ApoA-I might provide a means to treat this disease. The present study provides a comprehensive evaluation of adeno-associated virus (AAV)-mediated ApoA-I gene delivery to express human (h)ApoA-I and correct the low HDL-cholesterol phenotype associated with ApoA-I deficiency. METHODS: In an effort to maximize AAV-mediated gene expression, we performed head-to-head comparisons of recombinant AAVs with pseudotype capsids 1, 2, 6 and 8 administered by different routes with the use of five different liver-specific promoters in addition to cytomegalovirus as single-stranded or as self-complementary (sc) AAV vectors. RESULTS: Intravenous administration of 1 x 10(13) gc/kg scAAV8, in combination with the liver-specific promoter LP1, in female ApoA-I(-/-) mice resulted in hApoA-I expression levels of 634 +/- 69 mg/l, which persisted for the duration of the study (15 weeks). This treatment resulted in full recovery of HDL-cholesterol levels with correction of HDL particle size and apolipoprotein composition. In addition, we observed increased adrenal cholesterol content and a significant increase in bodyweight in treated mice. CONCLUSIONS: The present study demonstrates that systemic delivery of a scAAV8 vector provides a means for efficient liver expression of hApoA-I, thereby correcting the lipid abnormalities associated with murine ApoA-I deficiency. Importantly, the study demonstrates that AAV-based gene therapy can be used to express therapeutic proteins at a high level for a prolonged period of time and, as such, provides a basis for further development of this strategy to treat hApoA-I deficiency.

Effect of adeno-associated virus serotype and genomic structure on liver transduction and biodistribution in mice of both genders.

Recombinant adeno-associated viral (AAV) vectors have unique properties, which make them suitable vectors for gene transfer. Here we assess the liver transduction efficiency and biodistribution of AAV-pseudotyped capsids (serotypes) 1, 5, 6, and 8, combined with single-stranded and double-stranded genomic AAV2 structures carrying the luciferase reporter gene after systemic administration. The analysis was performed in vivo and ex vivo, in male and female mice. Gender-related differences in AAV-mediated transduction and biodistribution were shown for the four serotypes. Our data confirm the superiority of AAV8 over the rest of the serotypes, as well as a significant advantage of double-stranded genomes in terms of liver transduction efficiency, particularly in females. Regarding biodistribution, AAV5 displayed a narrower tropism than the other serotypes tested, transducing, almost exclusively, the liver. Interestingly, AAV1 and AAV8, in particular those having single-stranded genomes, showed high transduction efficiency of female gonads. However, no inadvertent germ line transmission of AAV genomes was observed after breeding single-stranded AAV8-injected female mice with untreated males. In conclusion, double-stranded AAV8 vectors led to the highest levels of liver transduction in mice, as demonstrated by luciferase expression. Nevertheless, the transduction of other organs with AAV8 vectors could favor the use of less efficient serotypes, such as AAV5, which display a narrow tropism.

Recombinant adeno-associated virus-mediated gene delivery of long chain acyl coenzyme A dehydrogenase (LCAD) into LCAD-deficient mice.

BACKGROUND: Very long chain acyl coenzyme A (CoA) dehydrogenase (VLCAD) deficiency is a relatively common mitochondrial beta-oxidation disorder. The most severe form of VLCAD deficiency presents with neonatal cardiomyopathy and hepatic failure and is generally fatal within the first year of life. Mice deficient for long chain acyl CoA dehydrogenase (LCAD) closely resemble the clinical syndrome observed in VLCAD-deficient humans. Recombinant adeno-associated viral (rAAV) vectors with pseudotype capsids were investigated for their potential towards correcting the phenotype observed in mice heterozygous (+/-) for LCAD (i.e. liver and muscle steatosis). METHODS: rAAV containing the mouse LCAD cDNA (mLCAD) under the transcriptional control of the CMV/chicken beta-actin hybrid promoter were injected intramuscularly into the tibialis anterior (TA) muscle of LCAD(+/-) mice or injected into the portal vein to transduce hepatocytes. RESULTS: Ten weeks post-injection of rAAV1-mLCAD into the TA muscle, significantly increased levels of mLCAD within mitochondria were demonstrated by immunostaining of TA sections, immunoblotting of mitochondrial isolates and by the electron transfer flavoprotein (ETF) fluorescence reduction enzyme activity assay. Magnetic resonance spectroscopy of vector-injected TA muscle demonstrated a reduction in the lipid content compared to phosphate-buffered saline-injected mice, whereas a systemic effect was observed as a reduction in liver macrosteatosis. Eight weeks after portal vein injection of rAAV8-mLCAD into LCAD(+/-) mice, increased levels of mLCAD within hepatocyte mitochondria were demonstrated by immunostaining and also by the ETF assay. Scoring of the hepatosteatosis observed in partially deficient LCAD mice indicated a reduction in the lipid content within livers of vector-treated mice. CONCLUSIONS: These studies show that rAAV-mediated delivery of mLCAD was efficient and led to an amelioration of local and systemic pathologies observed in partially deficient LCAD mice.

Biochemical correction of short-chain acyl-coenzyme A dehydrogenase deficiency after portal vein injection of rAAV8-SCAD.

Recombinant adeno-associated viral vectors pseudotyped with serotype 5 and 8 capsids (AAV5 and AAV8) have been shown to be efficient gene transfer reagents for the liver. We have produced AAV5 and AAV8 vectors that express mouse short-chain acyl-CoA dehydrogenase (mSCAD) cDNA under the transcriptional control of the cytomegalovirus-chicken beta-actin hybrid promoter. We hypothesized that these vectors would produce sufficient hepatocyte transduction (after administration via the portal vein) and thus sufficient SCAD enzyme to correct the phenotype observed in the SCAD-deficient (BALB/cByJ) mouse, which includes elevated blood butyrylcarnitine and hepatic steatosis. Ten weeks after portal vein injection into 8-week-old mice, AAV8-treated livers contained acyl-CoA dehydrogenase activity (14.3 mU/mg) toward butyryl-CoA, compared with 7.6 mU/mg in mice that received phosphate-buffered saline. Immunohistochemistry showed expression of mSCAD within rAAV8-mSCAD-transduced hepatocytes, as seen by light microscopy. A significant reduction of circulating butyrylcarnitine was seen in AAV5-mSCAD- and AAV8-mSCAD-injected mice. Magnetic resonance spectroscopy of fasted mice demonstrated a significant reduction in relative lipid content within the livers of AAV8-mSCAD-treated mice. These results demonstrate biochemical correction of SCAD deficiency after AAV8-mediated SCAD gene delivery.

In vivo post-transcriptional gene silencing of alpha-1 antitrypsin by adeno-associated virus vectors expressing siRNA.

alpha-1 Antitrypsin (AAT) deficiency is one of the most common genetic diseases in North America, with a carrier frequency of approximately 4% in the US population. Homozygosity for the most common mutation (Glu342Lys, PI(*)Z) leads to the synthesis of a mutant protein, which accumulates and polymerizes within hepatocytes rather than being efficiently secreted. This lack of secretion causes severe serum deficiency predisposing to chronic lung disease. Twelve to fifteen percent of patients with PI(*)ZZ also develop liver disease, which can be severe, even in infancy. This is thought to be due to toxic effects of the accumulated mutant Z-AAT within the hepatocyte. Thus, an approach to reduce AAT-deficient liver disease will likely require some mechanism to decrease the amount of Z-AAT within hepatocytes. In this report, we describe studies of small-interfering RNAs (siRNAs) designed to downregulate endogenous AAT within hepatocytes. Three different siRNA sequences were identified and cloned into a recombinant adeno-associated virus (rAAV) backbone, either singly or as a trifunctional (3X) construct. Each had activity independently, but the levels of AAT expression in cell culture models showed the greatest decrease with the 3X construct, resulting in levels that were five-fold lower than controls. The rAAV-3X-siRNA was then packaged into AAV8 capsids and used in vivo to transduce the livers of human Z-AAT overexpressing transgenic mice. Those studies showed a decrease in total human AAT, a clearing of Z-AAT accumulation by immunohistochemistry, and a decrease in monomer Z-AAT within the liver within 3 weeks after vector injection. The rAAV8-3X-siRNA vector may hold promise as a potential therapy for patients with AAT liver disease.

Reversal of RNA missplicing and myotonia after muscleblind overexpression in a mouse poly(CUG) model for myotonic dystrophy.

RNA-mediated pathogenesis is a recently developed disease model that proposes that certain types of mutant genes produce toxic transcripts that inhibit the activities of specific proteins. This pathogenesis model was proposed first for the neuromuscular disease myotonic dystrophy (DM), which is associated with the expansion of structurally related (CTG)(n) and (CCTG)(n) microsatellites in two unrelated genes. At the RNA level, these expansions form stable hairpins that alter the pre-mRNA splicing activities of two antagonistic factor families, the MBNL and CELF proteins. It is unclear which altered activity is primarily responsible for disease pathogenesis and whether other factors and biochemical pathways are involved. Here, we show that overexpression of Mbnl1 in vivo mediated by transduction of skeletal muscle with a recombinant adeno-associated viral vector rescues disease-associated muscle hyperexcitability, or myotonia, in the HSA(LR) poly(CUG) mouse model for DM. Myotonia reversal occurs concurrently with restoration of the normal adult-splicing patterns of four pre-mRNAs that are misspliced during postnatal development in DM muscle. Our results support the hypothesis that the loss of MBNL1 activity is a primary pathogenic event in the development of RNA missplicing and myotonia in DM and provide a rationale for therapeutic strategies designed either to overexpress MBNL1 or inhibit MBNL1 interactions with CUG and CCUG repeat expansions.