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Hemophilia Gene Therapy Trials


247-(1998-04)
Phase: I
Title: A Phase I Safety Study of Autologous Transfected Human Fibroblasts Producing Human Factor VIII in Patients with Severe Hemophilia A.
Sponsor: Transkaryotic Therapies, Inc.
Principal Investigator: David A. Roth, M.D., Beth Israel Deaconess Medical Center, 617-667-2239 or
droth@caregroup.harvard.edu
Disease: Inherited X-Linked Recessive// Hemophilia A
Vector: Plasmid DNA
Route of Administration: Intraperitoneal Implantation
Gene: Factor VIII cDNA
Status: Completed

Scientific Abstract
16 April 1998

The objective of this study is to investigate the safety of non-virally transfected autologous human fibroblasts producing human factor VIII (hFVIII) when implanted within the peritoneum of patients with severe hemophilia A. Nine (9) patients with severe hemophilia A who have a history of effective treatment with factor VIII replacement therapy will be included in the study.

Following informed consent and study enrollment, a sample of skin will be obtained by punch biopsy and transferred promptly to the pilot manufacturing facility of Transkaryotic Therapies Inc. (TKT). Dermal fibroblasts will be isolated from the skin biopsy and expanded in culture. The fibroblasts will be transfected by electroporation with a plasmid encoding hFVIII. Stably transfected fibroblasts expressing hFVIII will be selected and cloned, and one fibroblast clone will be expanded for implantation into the patient. The production of an autologous clone of fibroblasts expressing hFVIII will require approximately seven weeks.

Each patient will be implanted with a specified number of hFVIII-expressing fibroblasts derived from a single autologous clone. The autologous fibroblasts will be implanted in the peritoneum using a laparoscopic procedure. The patients will be hospitalized overnight following the implantation and will receive factor VIII replacement therapy prior to and for six days following the implantation procedure.

This is a dose escalation study, beginning with 100 x 106 transfected autologous fibroblasts and sequentially escalating to 200 x 106 and 400 x 106 transfected fibroblasts. Three patients will be included in each dose level. The patients will be evaluated during a 12 week intensive follow up period using physical examination, adverse events, routine clinical laboratory tests, specialized hematology laboratory tests, and patient diaries (evaluating bleeding episodes and factor VIII usage). Subsequent to the intensive 12 week follow up period, patients will enter into the long-term phase of the study, where they will be followed for an additional 21 months.

Non-Technical Abstract
16 April 1998

Hemophilia A is a severe inherited bleeding disorder resulting from a deficiency of factor VIII, a protein required for normal blood clotting. Current treatment is limited to the administration of the factor VIII protein at the time of a bleeding episode. Presently it is not possible to prevent the bleeding episodes.

Transkaryotic Therapy for hemophilia A represents a fundamental change in the treatment of this disorder. Transkaryotic Therapy is a gene therapy system in which a small number of skin cells are removed from a patient and stably modified with the gene for factor VIII so that the cells produce the factor VIII protein. The introduction of the factor VIII gene is done by non-viral means. The administration of cells with the correctly functioning factor VIII gene to hemophilia A patients may allow them to make sufficient factor VIII in their own bodies to prevent bleeding episodes. Thus a single administration of Transkaryotic Therapy could free the hemophilic patient from future bleeding episodes.

In the clinical study described in this application, a small skin biopsy will be performed on patients with severe hemophilia A, and fibroblasts will be isolated from the skin biopsy at TKT's manufacturing facility. A modified human factor VIII gene will then be inserted into the patient's fibroblasts. The fibroblasts will be grown and prepared for patient administration using manufacturing practices recommended by the US Food and Drug Administration. Each patient will receive his own genetically-modified fibroblasts by implantation in the abdomen using a laparoscopic technique. The laparoscopic procedure will be performed by making a small incision the patient's abdominal wall, inserting a tube into the abdomen through the incision, and pushing the patient's cells through the tube into the implantation site. The laparoscopic procedure will be performed using standard procedures:after giving the patients sufficient factor VIII to provide normal blood clotting levels.

While participating in the study patients will continue to receive their regular factor VIII replacement therapy as needed. The patients will be monitored intensively for twelve weeks following the cell implantation. After the three month intensive evaluation period, the patients will be evaluated periodically during the next 21 months. The total observational period will be of two years duration.

279-(1999-01)
Phase: I
Title: A Phase I Safety Study in Patients with Severe Hemophilia B (Factor IX Deficiency) Using Adeno-Associated Viral Vector to Deliver the Gene for Human Factor IX to Skeletal Muscle.
Principal Investigator: Catherine S. Manno, M.D., The Children's Hospital of Philadelphia, 215-590-2263 or manno@email.chop.edu
Disease: Inherited X-Linked Recessive// Hemophilia B
Vector: Adeno-Associated Virus
Route of Administration: Intramuscular Injection
Gene: Factor IX
Gene Status: Completed

Scientific Abstract
January 4, 1999

This clinical protocol is a phase I trial to determine the safety of an adenoviral associated viral (AAV) vector in delivery of the gene for human coagulation factor IX to muscle. Hemophilia B is an X-linked bleeding disorder resulting from the deficiency of coagulation factor IX. Current therapy using factor IX protein concentrates is suboptimal because of the inevitable delay between the onset of bleeding and hemostasis following infusion of factor IX. Such delays result in tissue damage. A newer strategy of prophylactic infusion of factor IX is effective in decreasing central nervous bleeding and the development of chronic arthropathy. This approach suffers from the high cost of recombinant or plasma-derived factor IX concentrates and in some patients with limited venous access, the requirement for placement of a central venous catheter with the attendant risks of infection. Although plasma-derived factor IX concentrates undergo a series of viral inactivation steps and are safe with respect to hepatitis B, C and HIV, there are still real and theoretical risks of contamination of these products with blood-borne agents such as hepatitis A, parvovirus B-19 and new agents that may escape inactivation by heat or solvent/detergent treatment. By resulting in a steady plasma l evel of factor IX, a gene therapy approach to the treatment of hemophilia may provide the benefits observed with prophylactic infusion therapy without the expense of factor IX concentrate or the inconvenience of obtaining intravenous access. Hemophilia B is a good model for gene therapy because tissue specific (i.e. hepatic) expression of the transgene is not required and precise regulation of expression is not necessary. Even low levels of plasma factor IX (2-3%) resulting from transgene expression are adequate to change the phenotype of an individual with severe hemophilia B, such that spontaneous hemarthroses and soft tissue hemorrhages (including central nervous system bleeds) will be prevented. The vector contains the human factor IX minigene consisting of all sequences coding for the mature protein, the signal sequence which directs the protein for secretion, the pro-peptide which is required for proper g-carboxylation, a portion of intron 1, and the first 227 nucleotides of the 3' untranslated region. The transgene is under the control of a cytomegalovirus immediate early promoter and the cassette contains an SV40 polyadenylation signal. Pre-clinical toxicity studies in rodents and dogs with hemophilia B have shown no local or systemic toxicity related to vector administration. Efficacy studies Rag-1 mice treated at a dose of I x 1013 vector genomes/kg demonstrated expression of factor IX at levels of 250-350 ng/ml (5-7% of normal human plasma levels), and studies in hemophilia B dogs treated at a dose of 8.5 x 1012 vector genomes/ kg have demonstrated levels of canine factor IX between 12% of normal human factor IX. A low-titer transient inhibitory antibody to canine factor IX developed in one of five hemophilic dogs. The inhibitory antibody lasted for a period of --8 weeks and disappeared without any specific therapy. Given our data on safety and promising data on efficacy of AAV-mediated muscle directed gene transfer of the factor IX gene, a phase I/II trial is proposed. Three patients will be enrolled in three dose escalation groups beginning with a low dose of 2.0 x 1011 vector genomes/kg and a high dose of 1.0 x 1013 vector genomes/kg (a dose expected to result in factor IX levels of >1 % based on dose response observed in mice and dogs). All patients will be observed closely for 12 months for signs of local toxicity (including biopsy of the injection site) and systemic toxicity (including evidence for the development of inhibitory antibodies to factor IX). Factor IX antigen and activity levels will be determined, and transgene presence and expression will be assessed on muscle biopsy.

Non-technical Abstract
January 4, 1999

Hemophilia B is the bleeding diathesis caused by a deficiency of functional blood coagulation factor IX. Currently, hemophilia B is treated by giving intravenous injections of clotting factor concentrates to hemophilia patients when they are bleeding. A difficulty with this treatment approach is that the clotting factors last in the circulation for only 12-18 hours, while the disease is lifelong. The goal of a gene therapy approach is to treat the disease by transferring to the patient the gene that makes factor IX protein, so that the protein is made constantly in the patient's own cells. The goal of this proposed phase I study is to examine the safety of one gene therapy approach, injection of an adeno-associated viral vector expressing human factor IX (AAV-hFIX) into the muscles of the leg. In the proposed study, men with severe hemophilia B will be admitted to the Clinical Research Center of The Children's Hospital of Philadelphia, where they will undergo a series of intramuscular injections of the AAV vector that results in the production of human factor IX. The design of the trial is a dose escalation study, beginning with doses that are not expected to result in therapeutic levels of factor IX. This means that the first group will receive the lowest dose, and each subsequent group will receive a higher dose until a therapeutic dose is achieved or until unacceptable toxicity occurs. The injections will be given into the muscles of the thigh, at anywhere from 6 to 20 injection sites, depending on the dose of vector to be administered. Patients will be monitored in the hospital for at least 24 hours after injection, then discharged and followed closely as outpatients. Parameters to be monitored include plasma factor IX levels, presence of inhibitory antibodies or factor IX "inhibitors", and blood tests to look for evidence of toxicity in organs such as the liver, kidneys and muscle, and blood cell counts. In addition, patients will undergo muscle biopsy (a small piece of the injected muscle tissue will be removed) at 2, 6 and possibly 12 months after injection, to analyze the injected muscle for the presence of the vector DNA sequences, and production of the factor IX protein. Patients will be seen in follow-up in outpatient clinics at frequent intervals for the first 12 months, then every 6 months for the rest of their lives. Ongoing evaluation at a hemophilia center is a part of routine management of hemophilia.

284-(1999-02)
Phase: I
Title: Phase I Multi-Center, Single Treatment Dose Escalation Study of Factor VIII Vector [HFVIII(V)] for Treatment of Severe Hemophilia A.
Sponsor: Chiron Corporation
Principal Investigators: Bruce M. Ewenstein, MD, PhD, Brigham & Women's Hospital, 617-732-5844 or bewenstein@partners.org; Jeanne M. Lusher, MD, Children's Hospital of Michigan, 313-745-5515 or jlusher@med.wayne.edu; Jerry S. Powell, MD, University of California Davis Medical Center, 916-734-8616 or jspowell@ucdavis.edu; Margaret V Ragni, MD, University of Pittsburgh Hemophilia Center, 412-209-7288 or ragni@msx.dept-med.pitt.edu; Gilbert White, MD, University of North Carolina - Chapel Hill, 919-966-3311 or gcwhite@med.unc.edu
Disease: Inherited X-Linked Recessive// Hemophilia A
Vector: Retrovirus
Route of Administration: Intravenous Injection
Gene: Factor VIII cDNA
Status: Completed

Technical Abstract
1. Agent
The agent, hFVIII(V), is a purified formulated retroviral vector preparation encoding only a gene for human factor FVIII (hFVIII) intended for the treatment of severe hemophilia A (congenital FVIII deficiency). The hFVIII gene is a truncated gene and codes for a functional protein. The vector is a Murine Leukemia Virus (MLV) derived system which is produced by an amphotropic packaging line (HAII) derived from a human cell line (HT1080). Expression is driven by the vector Long Terminal Repeat (LTR). The packaging cell line and vector are constructed to minimize regions of homology between vector and packaging components in order to reduce the chance of a recombination event. The producer cell line has been rigorously tested for the accidental production of replication competent retrovirus by standard cocultivation assays at multiple steps, and has been uniformly negative. The producer cell line has been banked and tested according to conventional FDA guidelines for these agents. Cells from the working cell bank were grown at large scale in medium containing fetal calf serum, the supernatant harvested, processed, formulated and stored at -70�C, pending QC testing and release.

2. Preclinical Efficacy and Toxicology
Treatment of normal rabbits and dogs with hFVIII(V) by peripheral vein administration, resulted in therapeutic levels of hFVIII. These levels persisted for as long as two years. Both adult and juvenile animals produced hFVIII following hFVIII(V) treatment. There is evidence of a dose response. and re-administration of hFVIII(V), after hFVIII levels had dropped, increased the level of circulating hFVIII. Hemophilic dogs showed shortening of the whole blood clotting time for variable periods after administration of hFVIII(V). The expected species-specific immune response to hFVIII was demonstrated in these dogs and complicated any observation of clinical efficacy. There were no effects on general health, hematology or clinical chemistry parameters up to 21 months after hFVIII(V) treatment. hFVIII(V) was generally well tolerated in mice up to 26.7 x 108 TU/kg. Overt toxicity was seen in 2/10 rabbits treated with a dose of 16.7 x 108 TU/kg which is seven times higher than planned for human administration. Doses planned for human administration were not associated with acute or chronic toxicity in mice, dogs or rabbits.

3. Biolocalization
PCR analysis to detect vector-specific sequences in the tissues of rabbits treated with hFVIII(V) showed that the spleen and liver were the most highly positive. Vectorspecific sequences were seen less frequently in other tissues, including kidney, lung, bone marrow, lymph node and testis. Localization of vector-specific sequences to tissues was not associated with any changes in histopathology. In testis, the low frequency with which vector-specific sequences were detected suggests that they are present near the limit of assay sensitivity (1 in 150,000 cells).

4. Clinical Study
Phase I is an uncontrolled, open label dose escalation study to establish the safety of intravenous infusions of hFVIII(V) at escalating doses in the range of 2.8 x 107 TU/kg to 2.2 x 108 TU/kg. A second study objective will be to determine whether one or more doses results in the therapeutic target response of at least 7% FVIII sustained over a 12 week period. Each subject will receive a single course of treatment by intravenous administration of equally divided doses on three successive days. The same total dose will be administered to three subjects. These three will be monitored for inhibitor formation and other adverse effects for a period of 4-7 weeks before three additional subjects are treated at the next higher dose. Subjects eligible for Phase I must have a diagnosis of severe hemophilia A (<1% FVIII); be at least 25 years old; be previously treated on at least 100 occasions with FVIII concentrates; have no present or past FVIII inhibitor; and be sterile (due to vasectomy or some other medically documented condition). Subjects who are human immunodeficiency virus (HIV) positive must have CD4 cell counts >300 cells/mm3 and not be treated with reverse transcriptase inhibitor medication. Subjects who are hepatitis C (HCV) positive must not have clinical or laboratory signs of liver failure. Phase I safety endpoints include adverse clinical events and laboratory tests for: FVIII inhibitor activity; hepatic, renal, hematologic and other organ function; replication competent retrovirus (RCR); CD4 cell count and viral load in HIV-positive subjects; and viral load in HCV-positive subjects. Abnormal laboratory values will be interpreted with reference to underlying chronic conditions. The study will be stopped if any subject develops a positive RCR test, or if more than one subject develops a clinically significant FV111 inhibitor. Efficacy will be assessed by measurement of FVIII activity three times weekly using a one-stage coagulation assay. In addition, FVIII response will be measured by chromogenic assay for FVIII activity, by activated partial thromboplastin time (aPTT), and by ELISA for FVIII antigen. Subjects will record all bleeding episodes, FVIII concentrate treatments, and adverse events on a home diary record. The trial will have two stages: Phase I (approximately 13 weeks); and Phase I-Extension (approximately 40 weeks). Subjects completing the Phase I-Extension will be enrolled in a lifelong surveillance registry for evaluation of long-term safety.

Non-techincal Abstract
Severe hemophilia A is a congenital bleeding disorder that results from the lack of a protein in the blood (known as Factor VIII [FVIII]) that is necessary for clotting. Affected individuals are born with a defective gene controlling FVIII production. Because the FVIII gene is located on the X chromosome, virtually all affected individuals are men (X-linked inheritance). Conventional treatment of hemophilia A requires intravenous injection of FVIII to stop bleeding whenever it starts. An alternative treatment approach would be to provide a working copy of the FVIII gene to the individual so that his body could produce FVIII continuously at a level sufficient to prevent bleeding. This would protect him from developing progressive joint disease, pain, and disability-usual complications of hemophilia treated in the conventional manner.

We have produced a retroviral vector that carries a gene for functional human FVIII protein. It is known as the human FVIII retroviral vector. The retroviral vector is a gene delivery vehicle that can enter human cells one time but cannot cause infection or disease, because key components of the virus have been removed or altered. The retroviral vector can carry a FVIII gene into a cell without damaging the cell and make the FVIII gene integrate permanently into the DNA of the cell. Then the gene provides permanent instructions for production of FVIII protein inside the cell and its subsequent export to the bloodstream. This process resembles that by which normal FVIII arrives in the bloodstream of unaffected individuals. The FVIII gene provided in our vector is truncated. This smaller hFVIII gene allows more potent vector preparations to be made, and higher levels of FVIII protein to be produced. Preparations of a similar protein have undergone clinical testing in hundreds of hemophilia patients and these preparations have normal safety and effectiveness.

To evaluate whether a therapeutic amount of FVIII could be produced by treatment with our vector, normal dogs and rabbits received various doses by intravenous injection, and then the amount of human FVIII protein was measured in the blood. Levels of FVIII that lessen disease severity in man were measured in some animals, and persisted for as long as two years. Levels of human FVIII protein varied between animals, but appeared to be higher if more of the vector was injected. In dogs that suffer from severe hemophilia A, treatment appeared to increase the ability of their blood to clot; however, this did not always happen. The variability may well be due to dog antibodies to the foreign human FVIII protein that interfere with the clotting action of FVIII. To test the safety of the human FVIII retroviral vector, we injected rabbits and mice with various doses and monitored them for signs of illness. Treatment with the vector at doses planned for the Phase I clinical trial did not cause changes suggestive of toxicity in body weight, the results of blood tests, or the microscopic or macroscopic appearance of body tissues. At doses about seven times higher than the highest dose planned for human use, two out of ten rabbits died. All mice given doses twelve times higher survived. In animals, very sensitive methods were used to determine which organs contained traces of the vector. Genes from the vector were found primarily in the liver and spleen, (organs which are known to normally produce active FVIII), with very small amounts occasionally detected in other organs, including the testis. The type of cells containing the vector-derived genes in each of these organs is not known.

The next step in testing the human FVIII retroviral vector will be a Phase I clinical study, primarily designed to evaluate whether administration is safe in humans at doses within the range shown to be safe in animals. Also, blood levels of FVIII will be measured to evaluate whether FVIII is produced at levels potentially protective against bleeding. The goal would be to reach at least 7% of the normal blood level, sustained for at least 12 weeks. This level would be expected to prevent day-to-day bleeding that would otherwise commonly occur. Any bleeding episodes during the study period will be treated as usual with FV111 infusions and reported on home diary forms. Participants in the initial Phase I study are required to have a diagnosis of severe hemophilia A (defined as < 1% FVIII), to be adults over 25 years of age, to have no inhibitor antibody to FVIII, and to be sterile. If they are HIV positive, they must not be severely immunosuppressed and, if they are positive for hepatitis C virus, they must have no signs of liver failure. The first subjects receiving the vector would receive the lowest dose and then be monitored for harmful side effects for at least 4 weeks before additional subjects are treated. Each participant would receive a single dose, administered intravenously into an arm or hand vein, in three equal parts on each of three successive days. After the highest dose is reached, all subjects will continue to be monitored closely for approximately one year, and then less intensively in a lifelong surveillance registry to evaluate long-term safety. Patients will be monitored for any allergic reactions or other change in physical condition during or after administration, changes in blood tests reflecting liver, kidney, or blood function abnormalities, appearance of an antibody in the blood which would block FVIII function (known as a FVIII inhibitor), and appearance in the blood of replication competent retrovirus (RCR). If any participant developed a positive RCR test or if more than one developed an inhibitor that interfered with treatment for bleeding, then the study would be stopped immediately and no additional subjects would receive the vector.

371-(2000-01)
Phase: I
Title: A Phase I Safety Study in Patients with Severe Hemophilia B (Factor IX Deficiency) Using Adeno-Associated Viral Vector to Deliver the Gene for Human Factor IX into the Liver.
Principal Investigator: Bertil Glader, MD, PhD, Stanford University, 650-723-5535 or bglader@leland.stanford.edu
Disease: Inherited X-Linked Recessive// Hemophilia B
Vector: Adeno-Associated Virus
Route of Administration: Intrahepatic Artery Injection
Gene: Factor IX Gene Elibility/Exclusion
Criteria: Open to individuals 18 years of age or older with severe hemophilia B. Subjects with a known history of allergic reaction to X-ray dye may not participate.
Status: Ongoing

Scientific Abstract
Hemophilia B is the bleeding diathesis that results from a deficiency of blood coagulation factor IX. The disease is X-linked and affects approximately 1 in 30,000 males. Most individuals with hemophilia B have severe disease, with factor IX levels of <1% of normal. The major morbidity is arthropathy from recurrent spontaneous joint bleeds; the major morbidity (and most common cause of premature death before the AIDS era) is central nervous system hemorrhage. The prevalence of CNS bleeding ranges from 2.6 and 13.8% with mortality rates between 20 and 50% and morbidity rates (seizures, motor impairment or mental retardation) of 40-50% in survivors. These bleeds occur predominantly in patients with severe disease (<1% factor level), thus, supporting the concept that raising the levels of factor even slightly would improve the chances to avoid this life-threatening complication of the disease. The incidence of arthropathy and of CNS hemorrhage can be reduced by the use of prophylactic regimens, the goal of which are to maintain trough factor levels >1% of normal. Since there is direct correlation of the severity of the disease with the level of factor IX, analyses of hemostatic parameters (particularly, whole blood clotting time and activated partial thromboplastin time) and of human factor IX (by ELISA) provide readily quantifiable measurements of treatment efficacy.

Recombinant AAV vectors show great promise for therapeutic success in the treatment of hemophilia and certain genetic diseases when delivered to the liver. The exact mechanism(s) involved in transduction is not known. There is strong evidence that at least a portion of the proviral genomes are integrated in head to tail concatemers. Episomal non-integrated, transciptionally active concatemers also are likely to exist. In mice, the vector genomes appear to enter almost every hepatocyte after intraportal administration but only about 5% of the hepatocytes become stably transduced over a period of about 5 to 6 weeks. In mice, curative levels of factor IX have been achieved while in dogs, therapeutic levels of about 1-2% of the normal level of factor IX have been accomplished with relatively small doses compared to the rodent studies.

The overall purpose of this research is to determine the safety of hepatic artery injection of an AAV vector expressing human factor IX into patients with severe hemophilia B. 1) Evaluate the safety of inter-patient dose escalations of an adenoassociated virus (AAV) vector containing the gene for human factor IX (AAV-hFIX) administered into the hepatic artery. Toxicity related to the delivery of AAV-hFIX will be evaluated locally and systemically. 2) Determine whether inhibitory antibodies against factor IX develop in patients receiving AAV-hFIX by hepatic artery administration. 3) Determine whether gene transfer is affected by the presence of preexisting antibodies against AAV. 4) Determine duration of expression of an AAV vector delivered to the liver in humans. 5) Determine whether therapy with AAV vector results in transfer to human germline cells. 6) Evaluate potential efficacy by measuring presence and activity of the transgene product. Analyses will be done to detect the presence of protein expression in blood by measurement of hemostatic parameters and factor IX antigen by ELISA.

Non-technical Abstract
Hemophilia B is the bleeding diathesis that results from a deficiency of blood coagulation factor IX. The disease is X-linked and affects approximately 1 in 30,000 males. Most individuals with hemophilia B have severe disease, with factor IX levels of <1% of normal. The major morbidity is arthropathy from recurrent spontaneous joint bleeds; the major morbidity (and most common cause of premature death before the AIDS era) is central nervous system hemorrhage. The prevalence of CNS bleeding ranges from 2.6 and 13.8% with mortality rates between 20 and 50% and morbidity rates (seizures, motor impairment or mental retardation) of 40-50% in survivors. These bleeds occur predominantly in patients with severe disease (<1% factor level), thus, supporting the concept that raising the levels of factor even slightly would improve the chances to avoid this life-threatening complication of the disease. The incidence of arthropathy and of CNS hemorrhage can be reduced by the use of prophylactic regimens, the goal of which are to maintain trough factor levels >1% of normal. Since there is direct correlation of the severity of the disease with the level of factor IX, analyses of hemostatic parameters (particularly, whole blood clotting time and activated partial thromboplastin time) and of human factor IX (by ELISA) provide readily quantifiable measurements of treatment efficacy.

Recombinant AAV vectors have been shown to result in safe and efficacious gene transfer when administered into the liver of animals that suffer from hemophilia B. The overall purpose of this research is to determine the safety of hepatic artery injection of an AAV vector expressing human factor IX into patients with severe hemophilia B. 1) Evaluate the safety of inter-patient dose escalations of an adeno-associated virus (AAV) vector containing the gene for human factor IX (AAV-hFIX) administered into the hepatic artery. Toxicity related to the delivery of AAV-hFIX will be evaluated locally and systemically. 2) Determine whether inhibitory antibodies against factor IX develop in patients receiving AAV-hFIX by hepatic artery administration. 3) Determine whether gene transfer. is affected by the presence of pre-existing antibodies against AAV. 4) Determine duration of expression of an AAV vector delivered to the liver in humans. 5) Determine whether therapy with AAV vector results in transfer to human germline cells. 6) Evaluate potential efficacy by measuring presence and activity of the transgene product. Analyses will be done to detect the presence of protein expression in blood by measurement of hemostatic parameters and factor IX antigen by ELISA.

372-(2000-01)
Phase: I
Title: A Phase 1, Single-Dose, Dose-Escalation Study of MiniAdFVIII Vector in Patients with Severe Hemophilia A.
Sponsor: GenStar Therapeutics Corporation
Principal Investigator: Gilbert White, MD, University of North Carolina - Chapel Hill, 919-966-3311 or gcwhite@med.unc.edu
Disease: Inherited X-Linked Recessive// Hemophilia A
Vector: Helper-Dependent (Gutted) Adenovirus
Route of Administration: Intravenous Injection
Gene: Factor VIII cDNA Eligibility/Exclusion
Criteria: Open to those 18 years of age or older with severe hemophilia A. Subjects are excluded if they 1) have a history of an inhibitor to factor VIII, 2) have received gene transfer in the past, 3) have a current infection with a temperature of 100.5 degrees or more, 4) are hepatitis C positive, or 5) are being treated with antibiotics, antifungal, or antiviral medication (except HIV medication).
Status: Terminated February 2003 because of lack of success in reruiting subjects

Scientific Abstract
The inherited blood coagulation disorder hemophilia A results from deficiency in the expression or function of MIL Treatment of moderate and severe hemophilia A involves intravenous infusion of plasma-derived or recombinant FVIII concentrates. A major limitation of current therapy is the short half-life of infused FVIII.

The MiniAdFVIII vector is a minimal (gutless) adenovirus vector designed to restore production of human FVIII by delivering the entire FVDIII complementary deoxyribonucleic acid to somatic cells. Pre-clinical data indicates that the vector will not express adenoviral antigens in vivo, thus minimizing potential immune responses and resulting in long-term persistence of the vector and expression of the transgene. Nonclinical pharmacology studies have indicated that physiological levels of hFVIII were produced in vivo, and these levels persisted for an extended period of time (approximately 1 year), resulting in phenotypic correction in hemophilic mice. Importantly, pre-clinical studies performed at several independent laboratories (including our own) have indicated that gutless adenoviral vectors have improved safety and efficacy profiles compared to earlier generation adenoviral vectors currently in clinical trails.

The objective of this Phase 1 study is to evaluate through dose escalation in defined increments the safety of intravenous infusion of MiniAdFVIII vector in severe hemophilia A patients without inhibitors. Additional objectives of this study are as follows: (1) to evaluate through dose escalation in defmed increments the ability of an intravenous infusion of MiniAdFVIII vector to produce circulating, functional levels of FVIII, (2) to evaluate the effect of MiniAdFVIII vector therapy on the frequency and severity of bleeding events following defined dose escalation, (3) to evaluate immunologic responses following the administration of MiniAdFVIII vector by monitoring anti-adenoviral and anti-FVM antibody titers in blood, and (4) to determine the functional FVIII expression profile by measuring the level, time course, and duration of functional and circulating FVIII.

The proposed study consists of a 7-day Screening Phase, a 1-day Treatment-Phase, a 12-week Post-treatment Phase and a 2-year Follow-up Phase. During the Screening Phase, the general clinical status of the patient will be determined. In addition, FVIII levels and FVIII inhibitor levels will be assessed. Two dose levels will be evaluated in this study (6 patients enrolled into two cohorts). In the Treatment Phase, MiniAdFVIII (1.4 x 1010 vp/kg or 4.3 x 1010 vp/kg) will be administered by intravenous infusion. The study will begin with the first cohort of three patients receiving a single dose of MiniAdFVIII. Dosing of each of the three patients in each cohort will be separated by at least two weeks. After 28 days of observations of each study subject at the first dose level to evaluate safety and immunologic responses, the second cohort of three patients will be administered the higher dose level. During the Post-treatment Phase of the study, patients will be closely monitored for adverse events. Weekly monitoring until Week 12 will include physical examinations, vital signs, liver function assessment, as well as clinical chemistry, and hematology, and urinalysis assessments. Serum FVIII levels and the development of FVIII inhibitors will be assessed. MiniAdFVIII levels will be monitored in the blood and urine. Anti-adenoviral serotype 5 antibodies will also be monitored in the blood. Follow-up Phase monitoring will be performed monthly for approximately 2 years or until no ongoing safety concerns exist; liver function, clinical chemistry, and hematology, and urinalysis will be assessed monthly.

Non-technical Abstract
The primary purpose of this clinical investigation is to assess the safety of a novel, adenoviral vector (so called minimal or gutless vector), termed MiniAdFVIII.

The secondary purpose of this clinical investigation is to determine if gene transfer can be used to cause the production of circulating, functional levels of Factor VIII (FVIII). FVIII is a protein that aids in the clotting of blood that is deficient in patients with severe hemophilia A. Patients who have hemophilia A are currently treated with clotting factors, but most patients receive treatment only for bleeding events and not for prevention of bleeding.

Gene transfer experiments have been carried out in people with various diseases including hemophilia. Some of these studies have used a virus to carry the gene into the patient's cells. One of the viruses used in gene transfer is the adenovirus that, in its natural form, can cause illness such as flu. Importantly, preclinical studies performed at several independent laboratories (including our own) have indicated that gutless adenoviral vectors have improved safety and efficacy profiles compared to earlier generation adenoviral vectors which are currently in clinical trails.

The investigators will determine if MiniAdFVIII can transfer the FVIII gene to produce the FVIII clotting factor in severe hemophilia A patients. MiniAdFVIII has most of the virus DNA removed and replaced with the FVIII gene, a normal gene that is designed to assist the liver cell to make FVIII through gene transfer. The MiniAdFVIII is designed not to multiply and spread in the body. This approach has been previously tested in laboratory animals. The experiments suggested that administration of the MiniAdFVIII could produce circulating, functional levels of FVIII in the blood.

16 January 2002

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