Margaret V. Ragni

Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response

We have previously shown that a single portal vein infusion of a recombinant adeno-associated viral vector (rAAV) expressing canine Factor IX (F.IX) resulted in long-term expression of therapeutic levels of F.IX in dogs with severe hemophilia B. We carried out a phase 1/2 dose-escalation clinical study to extend this approach to humans with severe hemophilia B. rAAV-2 vector expressing human F.IX was infused through the hepatic artery into seven subjects. The data show that: (i) vector infusion at doses up to 2 × 1012 vg/kg was not associated with acute or long-lasting toxicity; (ii) therapeutic levels of F.IX were achieved at the highest dose tested; (iii) duration of expression at therapeutic levels was limited to a period of ∼8 weeks; (iv) a gradual decline in F.IX was accompanied by a transient asymptomatic elevation of liver transaminases that resolved without treatment. Further studies suggested that destruction of transduced hepatocytes by cell-mediated immunity targeting antigens of the AAV capsid caused both the decline in F.IX and the transient transaminitis. We conclude that rAAV-2 vectors can transduce human hepatocytes in vivo to result in therapeutically relevant levels of F.IX, but that future studies in humans may require immunomodulation to achieve long-term expression*.

Evidence for gene transfer and expression of factor IX in haemophilia B patients treated with an AAV vector.

Pre-clinical studies in mice and haemophilic dogs have shown that introduction of an adeno-associated viral (AAV) vector encoding blood coagulation factor IX (F.IX) into skeletal muscle results in sustained expression of F.IX at levels sufficient to correct the haemophilic phenotype. On the basis of these data and additional pre-clinical studies demonstrating an absence of vector-related toxicity, we initiated a clinical study of intramuscular injection of an AAV vector expressing human F.IX in adults with severe haemophilia B. The study has a dose-escalation design, and all patients have now been enrolled in the initial dose cohort (2×1011 vg/kg). Assessment in the first three patients of safety and gene transfer and expression show no evidence of germline transmission of vector sequences or formation of inhibitory antibodies against F.IX. We found that the vector sequences are present in muscle by PCR and Southern-blot analyses of muscle biopsies and we demonstrated expression of F.IX by immunohistochemistry. We observed modest changes in clinical endpoints including circulating levels of F.IX and frequency of F.IX protein infusion. The evidence of gene expression at low doses of vector suggests that dose calculations based on animal data may have overestimated the amount of vector required to achieve therapeutic levels in humans, and that the approach offers the possibility of converting severe haemophilia B to a milder form of the disease.

CD8 + T-cell responses to adeno-associated virus capsid in humans

Hepatic adeno-associated virus (AAV)-serotype 2 mediatedgene transfer results in transgene product expression that is sustained in experimental animals but not in human subjects. We hypothesize that this is caused by rejection of transduced hepatocytes by AAV capsid–specific memory CD8+ T cells reactivated by AAV vectors. Here we show that healthy subjects carry AAV capsid–specific CD8+ T cells and that AAV-mediated gene transfer results in their expansion. No such expansion occurs in mice after AAV-mediated gene transfer. In addition, we show that AAV-2 induced human T cells proliferate upon exposure to alternate AAV serotypes, indicating that other serotypes are unlikely to evade capsid-specific immune responses.

Impact of Human Immunodeficiency Virus Infection on Progression to End-Stage Liver Disease in Individuals with Hemophilia and Hepatitis C Virus Infection

Hepatitis C virus (HCV) is the major cause of chronic liver disease in hemophiliacs. To determine the effect of human immunodeficiency virus (HIV) on the natural history of HCV infection, we evaluated end-stage liver disease (ESLD) in 157 hemophiliacs (85 HIV positive and 72 HIV negative) with HCV infection for an average of 24 years. After adjusting for age at HCV infection, past or current hepatitis B surface antigen positivity, and history of alcohol abuse, we determined that the rate of ESLD was significantly greater among HIV-positive than among HIV-negative hemophiliacs (relative risk [RR], 3.72; 95% confidence interval [CI], 1.25-11.09), as was the adjusted RR for death due to ESLD (RR, 3.81; 95% CI, 1.19-12.16). Among HIV-positive hemophiliacs, crude RR for ESLD was lower, but not significantly so, with antiretroviral treatment (RR, 0.19; 95% CI, 0.03-1.14; P=.069) and increased with each decade of HCV infection (RR, 2.26; 95% CI, 1.42-3.59; P=.0006) and HIV infection (RR, 2.18; 95% CI, 1.36-3.49; P=.0013). These findings suggest that HIV accelerates HCV disease progression.

Phase 3 study of recombinant factor VIII Fc fusion protein in severe hemophilia A

This phase 3 pivotal study evaluated the safety, efficacy, and pharmacokinetics of a recombinant FVIII Fc fusion protein (rFVIIIFc) for prophylaxis, treatment of acute bleeding, and perioperative hemostatic control in 165 previously treated males aged ≥12 years with severe hemophilia A. The study had 3 treatment arms: arm 1, individualized prophylaxis (25-65 IU/kg every 3-5 days, n = 118); arm 2, weekly prophylaxis (65 IU/kg, n = 24); and arm 3, episodic treatment (10-50 IU/kg, n = 23). A subgroup compared recombinant FVIII (rFVIII) and rFVIIIFc pharmacokinetics. End points included annualized bleeding rate (ABR), inhibitor development, and adverse events. The terminal half-life of rFVIIIFc (19.0 hours) was extended 1.5-fold vs rFVIII (12.4 hours; P < .001). Median ABRs observed in arms 1, 2, and 3 were 1.6, 3.6, and 33.6, respectively. In arm 1, the median weekly dose was 77.9 IU/kg; approximately 30% of subjects achieved a 5-day dosing interval (last 3 months on study). Across arms, 87.3% of bleeding episodes resolved with 1 injection. Adverse events were consistent with those expected in this population; no subjects developed inhibitors. rFVIIIFc was well-tolerated, had a prolonged half-life compared with rFVIII, and resulted in low ABRs when dosed prophylactically 1 to 2 times per week.

Phase 3 Study of Recombinant Factor IX Fc Fusion Protein in Hemophilia B

BACKGROUND Prophylactic factor replacement in patients with hemophilia B improves outcomes but requires frequent injections. A recombinant factor IX Fc fusion protein (rFIXFc) with a prolonged half-life was developed to reduce the frequency of injections required. METHODS We conducted a phase 3, nonrandomized, open-label study of the safety, efficacy, and pharmacokinetics of rFIXFc for prophylaxis, treatment of bleeding, and perioperative hemostasis in 123 previously treated male patients. All participants were 12 years of age or older and had severe hemophilia B (endogenous factor IX level of ≤2 IU per deciliter, or ≤2% of normal levels). The study included four treatment groups: group 1 received weekly dose-adjusted prophylaxis (50 IU of rFIXFc per kilogram of body weight to start), group 2 received interval-adjusted prophylaxis (100 IU per kilogram every 10 days to start), group 3 received treatment as needed for bleeding episodes (20 to 100 IU per kilogram), and group 4 received treatment in the perioperative period. A subgroup of group 1 underwent comparative sequential pharmacokinetic assessments of recombinant factor IX and rFIXFc. The primary efficacy end point was the annualized bleeding rate, and safety end points included the development of inhibitors and adverse events. RESULTS As compared with recombinant factor IX, rFIXFc exhibited a prolonged terminal half-life (82.1 hours) (P<0.001). The median annualized bleeding rates in groups 1, 2, and 3 were 3.0, 1.4, and 17.7, respectively. In group 2, 53.8% of participants had dosing intervals of 14 days or more during the last 3 months of the study. In groups 1, 2 and 3, 90.4% of bleeding episodes resolved after one injection. Hemostasis was rated as excellent or good during all major surgeries. No inhibitors were detected in any participants receiving rFIXFc; in groups 1, 2, and 3, 73.9% of participants had at least one adverse event, and serious adverse events occurred in 10.9% of participants. These events were mostly consistent with those expected in the general population of patients with hemophilia. CONCLUSIONS Prophylactic rFIXFc, administered every 1 to 2 weeks, resulted in low annualized bleeding rates in patients with hemophilia B. (Funded by Biogen Idec; ClinicalTrials.gov number, NCT01027364.).

Safety and prolonged activity of recombinant factor VIII Fc fusion protein in hemophilia A patients

Current factor VIII (FVIII) products display a half-life (t(1/2)) of ∼ 8-12 hours, requiring frequent intravenous injections for prophylaxis and treatment of patients with hemophilia A. rFVIIIFc is a recombinant fusion protein composed of a single molecule of FVIII covalently linked to the Fc domain of human IgG(1) to extend circulating rFVIII t(1/2). This first-in-human study in previously treated subjects with severe hemophilia A investigated safety and pharmacokinetics of rFVIIIFc. Sixteen subjects received a single dose of rFVIII at 25 or 65 IU/kg followed by an equal dose of rFVIIIFc. Most adverse events were unrelated to study drug. None of the study subjects developed anti-rFVIIIFc antibodies or inhibitors. Across dose levels, compared with rFVIII, rFVIIIFc showed 1.54- to 1.70-fold longer elimination t(1/2), 1.49- to 1.56-fold lower clearance, and 1.48- to 1.56-fold higher total systemic exposure. rFVIII and rFVIIIFc had comparable dose-dependent peak plasma concentrations and recoveries. Time to 1% FVIII activity above baseline was ∼ 1.53- to 1.68-fold longer than rFVIII across dose levels. Each subject showed prolonged exposure to rFVIIIFc relative to rFVIII. Thus, rFVIIIFc may offer a viable therapeutic approach to achieve prolonged hemostatic protection and less frequent dosing in patients with hemophilia A. This trial was registered at www.clinicaltrials.gov as NCT01027377.

Recombinant factor IX-Fc fusion protein (rFIXFc) demonstrates safety and prolonged activity in a phase 1/2a study in hemophilia B patients

Current factor IX (FIX) products display a half-life (t(1/2)) of ∼ 18 hours, requiring frequent intravenous infusions for prophylaxis and treatment in patients with hemophilia B. This open-label, dose-escalation trial in previously treated adult subjects with hemophilia B examined the safety and pharmacokinetics of rFIXFc. rFIXFc is a recombinant fusion protein composed of FIX and the Fc domain of human IgG(1), to extend circulating time. Fourteen subjects received a single dose of rFIXFc; 1 subject each received 1, 5, 12.5, or 25 IU/kg, and 5 subjects each received 50 or 100 IU/kg. rFIXFc was well tolerated, and most adverse events were mild or moderate in intensity. No inhibitors were detected in any subject. Dose-proportional increases in rFIXFc activity and Ag exposure were observed. With baseline subtraction, mean activity terminal t(1/2) and mean residence time for rFIXFc were 56.7 and 71.8 hours, respectively. This is ∼ 3-fold longer than that reported for current rFIX products. The incremental recovery of rFIXFc was 0.93 IU/dL per IU/kg, similar to plasma-derived FIX. These results show that rFIXFc may offer a viable therapeutic approach to achieve prolonged hemostatic protection and less frequent dosing in patients with hemophilia B. The trial was registered at www.clinicaltrials.gov as NCT00716716.

Transplantation In Hiv+ Patients

Twenty-five whole-organ recipients treated from 1981 through September 1988 were HIV carriers. Eleven were infected before transplantation, although this was not known until later in 8 recipients. The other 14 were infected perioperatively. Ten of the 25 recipients were infants or children. The organs transplanted were the liver (n = 15), and the heart or kidney (n = 5, each). After a mean follow-up of 2.75 years (range, 0.7-6.6 years), 13 recipients are alive. Survival is 7/15, 2/5, and 4/5 of the liver, heart, and kidney recipients, respectively. The best results were in the pediatric group (70% survival) in which only 1 of 10 patients died of AIDS. In contrast, AIDS caused the death of 5 of 15 adult recipients and was the leading cause of death. Transplantation plus immunosuppression appeared to shorten the AIDS-free time in HIV+ patients as compared to nontransplant hemophiliac and transfusion control groups. Accrual of HIV+ transplant recipients has slowed markedly since the systematic screening of donors, recipients, and blood products was begun in 1985.

Outcomes of liver transplant recipients with hepatitis C and human immunodeficiency virus coinfection

Hepatitis C virus (HCV) is a controversial indication for liver transplantation (LT) in human immunodeficiency virus (HIV)–infected patients because of reportedly poor outcomes. This prospective, multicenter US cohort study compared patient and graft survival for 89 HCV/HIV‐coinfected patients and 2 control groups: 235 HCV‐monoinfected LT controls and all US transplant recipients who were 65 years old or older. The 3‐year patient and graft survival rates were 60% [95% confidence interval (CI) = 47%‐71%] and 53% (95% CI = 40%‐64%) for the HCV/HIV patients and 79% (95% CI = 72%‐84%) and 74% (95% CI = 66%‐79%) for the HCV‐infected recipients (P < 0.001 for both), and HIV infection was the only factor significantly associated with reduced patient and graft survival. Among the HCV/HIV patients, older donor age [hazard ratio (HR) = 1.3 per decade], combined kidney‐liver transplantation (HR = 3.8), an anti‐HCV–positive donor (HR = 2.5), and a body mass index < 21 kg/m2 (HR = 3.2) were independent predictors of graft loss. For the patients without the last 3 factors, the patient and graft survival rates were similar to those for US LT recipients. The 3‐year incidence of treated acute rejection was 1.6‐fold higher for the HCV/HIV patients versus the HCV patients (39% versus 24%, log rank P = 0.02), but the cumulative rates of severe HCV disease at 3 years were not significantly different (29% versus 23%, P = 0.21). In conclusion, patient and graft survival rates are lower for HCV/HIV‐coinfected LT patients versus HCV‐monoinfected LT patients. Importantly, the rates of treated acute rejection (but not the rates of HCV disease severity) are significantly higher for HCV/HIV‐coinfected recipients versus HCV‐infected recipients. Our results indicate that HCV per se is not a contraindication to LT in HIV patients, but recipient and donor selection and the management of acute rejection strongly influence outcomes. Liver Transpl 18:716–726, 2012.