Phase 1-2 Trial of AAVS3 Gene Therapy in Patients with Hemophilia B.

BACKGROUND: FLT180a (verbrinacogene setparvovec) is a liver-directed adeno-associated virus (AAV) gene therapy that uses a synthetic capsid and a gain-of-function protein to normalize factor IX levels in patients with hemophilia B. METHODS: In this multicenter, open-label, phase 1-2 trial, we assessed the safety and efficacy of varying doses of FLT180a in patients with severe or moderately severe hemophilia B (factor IX level, ≤2% of normal value). All the patients received glucocorticoids with or without tacrolimus for immunosuppression to decrease the risk of vector-related immune responses. After 26 weeks, patients were enrolled in a long-term follow-up study. The primary end points were safety and efficacy, as assessed by factor IX levels at week 26. RESULTS: Ten patients received one of four FLT180a doses of vector genomes (vg) per kilogram of body weight: 3.84×1011 vg, 6.40×1011 vg, 8.32×1011 vg, or 1.28×1012 vg. After receiving the infusion, all the patients had dose-dependent increases in factor IX levels. At a median follow-up of 27.2 months (range, 19.1 to 42.4), sustained factor IX activity was observed in all the patients except one, who resumed factor IX prophylaxis. As of the data-cutoff date (September 20, 2021), five patients had normal factor IX levels (range, 51 to 78%), three patients had levels from 23 to 43%, and one had a level of 260%. Of the reported adverse events, approximately 10% were related to FLT180a and 24% to immunosuppression. Increases in liver aminotransferase levels were the most common FLT180a-related adverse events. Late increases in aminotransferase levels occurred in patients who had received prolonged tacrolimus beyond the glucocorticoid taper. A serious adverse event of arteriovenous fistula thrombosis occurred in the patient with high factor IX levels. CONCLUSIONS: Sustained factor IX levels in the normal range were observed with low doses of FLT180a but necessitated immunosuppression with glucocorticoids with or without tacrolimus. (Funded by Freeline Therapeutics; ClinicalTrials.gov numbers, NCT03369444 and NCT03641703; EudraCT numbers, 2017-000852-24 and 2017-005080-40.).

Recombinant Adeno-Associated Viral Vectors Expressing Human Coagulation FIX-E456H Variant in Hemophilia B Mice.

Gene therapy using recombinant adeno-associated virus (AAV) has induced sustained long-term coagulation human factor IX (hFIX) levels in hemophilia B (HB) patients. However, asymptomatic transient liver toxicity was observed at high vector doses, highlighting the need to improve the potency of these vectors. We report the generation of an AAV transgene cassette containing the hyperfunctional hFIX-E456H variant showing improved binding to platelets, with a comparison to wild-type hFIX (hFIX-WT) and hFIX-R384L variant (Padua) transgenes, containing F9 truncated-intron 1 (I1). In vitro specific activity was increased by 3.2- and 4.2-fold with hFIX-E456H and hFIX-R384L variants compared with hFIX-WT, using chromogenic assay, and by 7-and 8.6-fold with hFIX-E456H and hFIX-R384L variants compared with hFIX-WT, using one-stage assay. The transgenes were packaged into single-stranded AAV2/8 vectors that were tail vein injected at 5 × 109, 2 × 1010, and 5 × 1010 vg per mouse in HB mice. Plasma FIX activity level, assessed by chromogenic assay, was up to fourfold higher for hFIX-E456H compared with hFIX-WT and was not different compared with hFIX-R384L, among the three dose cohorts. Overall, the in vivo specific activity was increased by threefold for hFIX-E456H and 4.9-fold for hFIX-R384L compared with hFIX-WT. At the lower dose of 5 × 109 vg, the blood loss was significantly lower for hFIX-E456H compared with hFIX-WT, but did not differ compared with hFIX-R384L. The results found for the hFIX-E456H variant indicate that it might be a suitable alternative for gene therapy of HB.

Potential limits of AAV-based gene therapy with the use of new transgenes expressing factor IX fusion proteins.

INTRODUCTION: The variety of treatment for haemophilia B (HB) has recently improved with the emergence of both AAV-based gene therapy and bioengineered human factor IX (hFIX) molecules with prolonged half-life due to fusion to either albumin (Alb) or immunoglobulin Fc fragment (Fc). AIM: Adeno-associated viral vectors (AAV) mediating expression of hFIX-Alb and hFIX-Fc fusion proteins was investigated for gene therapy of HB to explore if their extended half-life translates to higher plasma levels of FIX. METHODS: Single-stranded cross-packaged AAV2/8 vectors expressing hFIX-Alb, hFIX-Fc and hFIX were evaluated in vitro, and in mice. RESULTS: Both hFIX-Alb and hFIX-Fc fusion proteins were synthesized and expressed as single chains of expected size following AAV-mediated gene transfer in vitro and in vivo. The procoagulant properties of these hFIX-fusion proteins were comparable to wild-type hFIX. However, their expression levels were threefold lower than wild-type hFIX in vivo most likely due to inefficient secretion. CONCLUSION: This, the first, evaluation of hFIX-fusion proteins in the context of AAV gene transfer suggests that the hFIX-fusion proteins are secreted inefficiently from the liver, thus preventing their optimal use in gene therapy approaches.

Partial pancreatic transdifferentiation of primary human hepatocytes in the livers of a humanised mouse model.

BACKGROUND: Gene therapy is one treatment that may ultimately cure type 1 diabetes. We have previously shown that the introduction of furin-cleavable human insulin (INS-FUR) to the livers in several animal models of diabetes resulted in the reversal of diabetes and partial pancreatic transdifferentiation of liver cells. The present study investigated whether streptozotocin-diabetes could be reversed in FRG mice in which chimeric mouse-human livers can readily be established and, in addition, whether pancreatic transdifferentiation occurred in the engrafted human hepatocytes. METHODS: Engraftment of human hepatocytes was confirmed by measuring human albumin levels. Following delivery of the empty vector or the INS-FUR vector to diabetic FRG mice, mice were monitored for weight and blood glucose levels. Intraperitoneal glucose tolerance tests (IPGTTs) were performed. Expression levels of pancreatic hormones and transcription factors were determined by a reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry. RESULTS: Diabetes was reversed for a period of 60 days (experimental endpoint) after transduction with INS-FUR. IPGTTs of the insulin-transduced animals were not significantly different from nondiabetic animals. Immunofluorescence microscopy revealed the expression of human albumin and insulin in transduced liver samples. Quantitative RT-PCR showed expression of human and mouse endocrine hormones and ß-cell transcription factors, indicating partial pancreatic transdifferentiation of mouse and human hepatocytes. Nonfasting human C-peptide levels were significantly higher than mouse levels, suggesting that transdifferentiated human hepatocytes made a significant contribution to the reversal of diabetes. CONCLUSIONS: These data show that human hepatocytes can be induced to undergo partial pancreatic transdifferentiation in vivo, indicating that the technology holds promise for the treatment of type 1 diabetes.

Bioengineered AAV Capsids with Combined High Human Liver Transduction In Vivo and Unique Humoral Seroreactivity.

Existing recombinant adeno-associated virus (rAAV) serotypes for delivering in vivo gene therapy treatments for human liver diseases have not yielded combined high-level human hepatocyte transduction and favorable humoral neutralization properties in diverse patient groups. Yet, these combined properties are important for therapeutic efficacy. To bioengineer capsids that exhibit both unique seroreactivity profiles and functionally transduce human hepatocytes at therapeutically relevant levels, we performed multiplexed sequential directed evolution screens using diverse capsid libraries in both primary human hepatocytes in vivo and with pooled human sera from thousands of patients. AAV libraries were subjected to five rounds of in vivo selection in xenografted mice with human livers to isolate an enriched human-hepatotropic library that was then used as input for a sequential on-bead screen against pooled human immunoglobulins. Evolved variants were vectorized and validated against existing hepatotropic serotypes. Two of the evolved AAV serotypes, NP40 and NP59, exhibited dramatically improved functional human hepatocyte transduction in vivo in xenografted mice with human livers, along with favorable human seroreactivity profiles, compared with existing serotypes. These novel capsids represent enhanced vector delivery systems for future human liver gene therapy applications.

Identification of liver-specific enhancer-promoter activity in the 3' untranslated region of the wild-type AAV2 genome.

Vectors based on adeno-associated virus type 2 (AAV2) are powerful tools for gene transfer and genome editing applications. The level of interest in this system has recently surged in response to reports of therapeutic efficacy in human clinical trials, most notably for those in patients with hemophilia B (ref. 3). Understandably, a recent report drawing an association between AAV2 integration events and human hepatocellular carcinoma (HCC) has generated controversy about the causal or incidental nature of this association and the implications for AAV vector safety. Here we describe and functionally characterize a previously unknown liver-specific enhancer-promoter element in the wild-type AAV2 genome that is found between the stop codon of the cap gene, which encodes proteins that form the capsid, and the right-hand inverted terminal repeat. This 124-nt sequence is within the 163-nt common insertion region of the AAV genome, which has been implicated in the dysregulation of known HCC driver genes and thus offers added insight into the possible link between AAV integration events and the multifactorial pathogenesis of HCC.

Selection and evaluation of clinically relevant AAV variants in a xenograft liver model.

Recombinant adeno-associated viral (rAAV) vectors have shown early promise in clinical trials. The therapeutic transgene cassette can be packaged in different AAV capsid pseudotypes, each having a unique transduction profile. At present, rAAV capsid serotype selection for a specific clinical trial is based on effectiveness in animal models. However, preclinical animal studies are not always predictive of human outcome. Here, in an attempt to further our understanding of these discrepancies, we used a chimaeric human-murine liver model to compare directly the relative efficiency of rAAV transduction in human versus mouse hepatocytes in vivo. As predicted from preclinical and clinical studies, rAAV2 vectors functionally transduced mouse and human hepatocytes at equivalent but relatively low levels. However, rAAV8 vectors, which are very effective in many animal models, transduced human hepatocytes rather poorly-approximately 20 times less efficiently than mouse hepatocytes. In light of the limitations of the rAAV vectors currently used in clinical studies, we used the same murine chimaeric liver model to perform serial selection using a human-specific replication-competent viral library composed of DNA-shuffled AAV capsids. One chimaeric capsid composed of five different parental AAV capsids was found to transduce human primary hepatocytes at high efficiency in vitro and in vivo, and provided species-selected transduction in primary liver, cultured cells and a hepatocellular carcinoma xenograft model. This vector is an ideal clinical candidate and a reagent for gene modification of human xenotransplants in mouse models of human diseases. More importantly, our results suggest that humanized murine models may represent a more precise approach for both selecting and evaluating clinically relevant rAAV serotypes for gene therapeutic applications.

Adeno-associated virus-mediated rescue of neonatal lethality in argininosuccinate synthetase-deficient mice.

Viral vectors based on adeno-associated virus (AAV) are showing exciting promise in gene therapy trials targeting the adult liver. A major challenge in extending this promise to the pediatric liver is the loss of episomal vector genomes that accompanies hepatocellular proliferation during liver growth. Hence maintenance of sufficient transgene expression will be critical for success in infants and children. We therefore set out to explore the therapeutic efficacy and durability of liver-targeted gene transfer in the challenging context of a neonatal lethal urea cycle defect, using the argininosuccinate synthetase deficient mouse. Lethal neonatal hyperammonemia was prevented by prenatal and early postnatal vector delivery; however, hyperammonemia subsequently recurred limiting survival to no more than 33 days despite vector readministration. Antivector antibodies acquired in milk from vector-exposed dams were subsequently shown to be blocking vector readministration, and were avoided by crossfostering vector-treated pups to vector-naive dams. In the absence of passively acquired antivector antibodies, vector redelivery proved efficacious with mice surviving to adulthood without recurrence of significant hyperammonemia. These data demonstrate the potential of AAV vectors in the developing liver, showing that vector readministration can be used to counter growth-associated loss of transgene expression provided the challenge of antivector humoral immunity is addressed.

Gene therapy for metabolic disorders: an overview with a focus on urea cycle disorders.

Many metabolic diseases are compelling candidates for gene therapy, and are the subject of vigorous pre-clinical research. Successful phenotype correction in mouse models is now commonplace and research effort is increasingly being directed towards addressing the translational challenges inherent in human clinical trials. This paper places current efforts to develop gene therapy approaches to metabolic disease in historical context and describes contemporary research in the authors' laboratory on urea cycle defects, particularly ornithine transcarbamylase deficiency, in a manner that is illustrative of the general state of the field.

Induction and prevention of severe hyperammonemia in the spfash mouse model of ornithine transcarbamylase deficiency using shRNA and rAAV-mediated gene delivery.

Urea cycle defects presenting early in life with hyperammonemia remain difficult to treat and commonly necessitate liver transplantation. Gene therapy has the potential to prevent hyperammonemic episodes while awaiting liver transplantation, and possibly also to avert the need for transplantation altogether. Ornithine transcarbamylase (OTC) deficiency, the most prevalent urea cycle disorder, provides an ideal model for the development of liver-targeted gene therapy. While we and others have successfully cured the spf(ash) mouse model of OTC deficiency using adeno-associated virus (AAV) vectors, a major limitation of this model is the presence of residual OTC enzymatic activity which confers a mild phenotype without clinically significant hyperammonemia. To better model severe disease we devised a strategy involving AAV2/8-mediated delivery of a short hairpin RNA (shRNA) to specifically knockdown residual endogenous OTC messenger RNA (mRNA). This strategy proved highly successful with vector-treated mice developing severe hyperammonemia and associated neurological impairment. Using this system, we showed that the dose of an AAV rescue construct encoding the murine OTC (mOTC) cDNA required to prevent hyperammonemia is fivefold lower than that required to control orotic aciduria. This result is favorable for clinical translation as it indicates that the threshold for therapeutic benefit is likely to be lower than indicated by earlier studies.

Lymphomagenesis in SCID-X1 mice following lentivirus-mediated phenotype correction independent of insertional mutagenesis and gammac overexpression.

The development of leukemia as a consequence of vector-mediated genotoxicity in gene therapy trials for X-linked severe combined immunodeficiency (SCID-X1) has prompted substantial research effort into the design and safety testing of integrating vectors. An important element of vector design is the selection and evaluation of promoter-enhancer elements with sufficient strength to drive reliable immune reconstitution, but minimal propensity for enhancer-mediated insertional mutagenesis. In this study, we set out to explore the effect of promoter-enhancer selection on the efficacy and safety of human immunodeficiency virus-1-derived lentiviral vectors in gammac-deficient mice. We observed incomplete or absent T- and B-cell development in mice transplanted with progenitors expressing gammac from the phosphoglycerate kinase (PGK) and Wiscott-Aldrich syndrome (WAS) promoters, respectively. In contrast, functional T- and B-cell compartments were restored in mice receiving an equivalent vector containing the elongation factor-1-alpha (EF1alpha) promoter; however, 4 of 14 mice reconstituted with this vector subsequently developed lymphoma. Extensive analyses failed to implicate insertional mutagenesis or gammac overexpression as the underlying mechanism. These findings highlight the need for detailed mechanistic analysis of tumor readouts in preclinical animal models assessing vector safety, and suggest the existence of other ill-defined risk factors for oncogenesis, including replicative stress, in gene therapy protocols targeting the hematopoietic compartment.

Sexually dimorphic patterns of episomal rAAV genome persistence in the adult mouse liver and correlation with hepatocellular proliferation.

Recombinant adeno-associated virus vectors (rAAVs) show exceptional promise for liver-targeted gene therapy, with phenotype correction in small and large animal disease models being reported with increasing frequency. Success in humans, however, remains a considerable challenge that demands greater understanding of host-vector interactions, notably those governing the efficiency of initial gene transfer and subsequent long-term persistence of gene expression. In this study, we examined long-term enhanced green fluorescent protein (eGFP) expression and vector genome persistence in the mouse liver after rAAV2/8-mediated gene transfer in early adulthood. Two intriguing findings emerged of considerable scientific and clinical interest. First, adult female and male mice showed distinctly different patterns of persistence of eGFP expression across the hepatic lobule after exhibiting similar patterns initially. Female mice retained a predominantly perivenous pattern of expression, whereas male mice underwent inversion of this pattern with preferential loss of perivenous expression and relative retention of periportal expression. Second, these changing patterns of expression correlated with sexually dimorphic patterns of genome persistence that appear linked both spatially and temporally to underlying hepatocellular proliferation. Observation of the equivalent phenomenon in man could have significant implications for the long-term therapeutic efficacy of rAAV-mediated gene transfer, particularly in the context of correction of liver functions showing metabolic zonation.

Gene delivery to the juvenile mouse liver using AAV2/8 vectors.

Recombinant adeno-associated viral (rAAV) vectors have shown promise for use in liver-targeted gene delivery, but their effects have not been extensively investigated in the immature liver. Understanding the impact of liver growth on the efficacy of transduction is essential, because many monogenic liver diseases that are amenable to gene therapy will require treatment early in life. Here we show that rAAV2/8 transduces the neonatal mouse liver with high efficiency. With just one doubling in liver weight, however, there is a rapid reduction in vector genome numbers, irrespective of form, and the loss of episomal vector is almost complete by 2 weeks. Stable transgene expression is observed in a small percentage of hepatocytes, often in two- to eight-cell clusters, suggestive of genomic integration. Delivery at serially older ages was associated with progressively improved episome persistence and transgene expression. Vector re-administration was possible following initial neonatal administration, albeit at reduced efficacy because of an anticapsid humoral immune response. We also found that intraperitoneal (i.p.) delivery of rAAV2/8 was highly effective at all ages, and that promoter selection is the critical determinant of the intensity and pattern of transgene expression across the hepatic lobule. We conclude that successful use of rAAV to treat liver disease in early childhood will require optimally efficient vector constructs and probable re-administration.

Gene Delivery to the Juvenile Mouse Liver Using AAV2/8 Vectors.

Recombinant adeno-associated viral (rAAV) vectors have shown promise for use in liver-targeted gene delivery, but their effects have not been extensively investigated in the immature liver. Understanding the impact of liver growth on the efficacy of transduction is essential, because many monogenic liver diseases that are amenable to gene therapy will require treatment early in life. Here we show that rAAV2/8 transduces the neonatal mouse liver with high efficiency. With just one doubling in liver weight, however, there is a rapid reduction in vector genome numbers, irrespective of form, and the loss of episomal vector is almost complete by 2 weeks. Stable transgene expression is observed in a small percentage of hepatocytes, often in two- to eight-cell clusters, suggestive of genomic integration. Delivery at serially older ages was associated with progressively improved episome persistence and transgene expression. Vector re-administration was possible following initial neonatal administration, albeit at reduced efficacy because of an anticapsid humoral immune response. We also found that intraperitoneal (IP) delivery of rAAV2/8 was highly effective at all ages, and that promoter selection is the critical determinant of the intensity and pattern of transgene expression across the hepatic lobule. We conclude that successful use of rAAV to treat liver disease in early childhood will require optimally efficient vector constructs and probable re-administration.

Purine nucleoside phosphorylase and fludarabine phosphate gene-directed enzyme prodrug therapy suppresses primary tumour growth and pseudo-metastases in a mouse model of prostate cancer.

Gene-directed enzyme prodrug therapy based on the E. coli purine nucleoside phosphorylase (PNP) gene produces efficient tumour cell killing. PNP converts adenosine analogs into toxic metabolites that diffuse across cell membranes to kill neighbouring untransduced cells (PNP-GDEPT). Interference with DNA, RNA and protein synthesis kills dividing and non-dividing cells, an important consideration for slow-growing prostate tumours. This study examined the impact of administering PNP-GDEPT into orthotopically grown RM1 prostate cancers (PCas) on the growth of lung pseudo-metastases of immunocompetent mice. C57BL/6 mice bearing orthotopic RM1 PCas received a single intraprostatic injection of OAdV220 (10(10) particles), a recombinant ovine atadenovirus containing the PNP gene controlled by the Rous Sarcoma virus promoter, followed by fludarabine phosphate (approximately 600 mg/m(2)/day) administered intraperitoneally (ip) once daily for 5 days. Pseudo-metastases were induced 2 days after intraprostatic vector administration by tail-vein injection of untransduced RM1 cells. Mice given PNP-GDEPT showed a significant reduction both in prostate volume (approximately 50%) and in lung colony counts (approximately 60%). Apoptosis was increased two-fold in GDEPT-treated prostates compared with controls (P < 0.01), but was absent in the lungs. Staining for proliferating cell nuclear antigen (PCNA) indicated that proliferation of both RM1 prostate tumours (P < 0.01) and lung colonies (P < 0.01) was significantly suppressed after GDEPT. Although prostate tumour immune cell infiltration did not differ significantly between treatments, immunostaining for Thy-1.2 (CD90) showed that GDEPT promoted Thy-1.2(+) cell infiltration into the prostate tumour site. This study showed that a single course of PNP-GDEPT significantly suppressed local PCa growth and reduced lung colony formation in the aggressive RM1 tumour model.

Gene-directed enzyme prodrug therapy for prostate cancer in a mouse model that imitates the development of human disease.

BACKGROUND: Gene-directed enzyme prodrug therapy (GDEPT) based on the E. coli enzyme purine nucleoside phosphorylase (PNP) represents a new approach for treating slow growing tumours like prostate cancer (PCa). Expressed enzyme converts a systemically administered prodrug, fludarabine phosphate, to a toxic metabolite, 2-fluoroadenine. Infected and neighbouring cells are killed by a bystander effect that results from the inhibition of DNA and RNA synthesis. METHODS: These studies were carried out using the transgenic adenocarcinoma of the prostate (TRAMP) model that mimics human PCa development and progression. Control TRAMP mice were injected intraprostatically with vector vehicle and thereafter intraperitoneally with saline or fludarabine phosphate ( approximately 600 mg/m(2)/day) once daily for 5 consecutive days. Treated mice received a single intraprostatic injection containing 10(10) particles of OAdV220, an ovine atadenovirus which expresses the E. coli PNP gene under the control of the Rous sarcoma virus promoter, followed by systemic fludarabine treatment. The weight of the genitourinary tract, seminal vesicles and the prostate as well as animal survival were monitored. Tumours were also analysed histologically. RESULTS: Preliminary studies showed that fludarabine alone caused no significant change in genitourinary (GU) tract weight in TRAMP mice. Animals injected with vector and prodrug showed a significant reduction (36-47%) in GU tract weight (ANOVA p = 0.0002) and a 35-50% reduction in seminal vesicle weight (ANOVA p = 0.0007). In particular, the target organ showed a significant 57% reduction in prostate weight (ANOVA p = 0.0007). PNP-GDEPT mice also showed a survival advantage over control mice. Histological analysis suggested that the cancer progression was slowed in GDEPT-treated animals. CONCLUSION: A single course of GDEPT based on OAdV-delivered PNP and fludarabine produced highly significant suppression of PCa progression in immune-competent TRAMP mice.

Application of the transgenic adenocarcinoma mouse prostate (TRAMP) model for pre-clinical therapeutic studies.

BACKGROUND: The suitability of (C57BL/6 TRAMP x C57BL/6)F1 transgenic (TRAMP+) mice with well- to moderately-differentiated prostate cancer (PCa) was assessed for pre-clinical therapeutic studies. MATERIALS AND METHODS: TRAMP+ and TRAMP- mice were assessed for variability in genitourinary tract weight, seminal vesicle weight, prostate weight/volume and histopathology. Time-points included the reported ages of average tumour onset (approximately 25 weeks) and PCa-induced death (approximately 33 weeks). RESULTS: Seventy % of TRAMP+ mice aged 25-33 weeks had well- to moderately-differentiated PCa. At 25-28 weeks, the mean genitourinary tract weight was 2X greater and the mean prostate weight/volume was 1.5X more in TRAMP+ than in TRAMP- mice, respectively. Prostate weight/volume showed significant increases (p < 0.0001) by 2X and 3X, respectively by 31-33 weeks of age. CONCLUSION: The window for using the TRAMP model successfully for pre-clinical experimentation is 25-33 weeks provided that mice with poorly-differentiated PCa showing a large tumour burden are excluded.