Final Report Summary - GENOPHILIA (AAV-mediated Gene Therapy for Haemophilia A)
Genophilia Project
Haemophilia is a group of hereditary disorders that leads to deficiency of a plasma protein needed for normal blood clotting and coagulation. The two most common forms of haemophilia are haemophilia A and B. Both are classically transmitted in an X-linked recessive pattern. Haemophilia A (clotting factor VIII deficiency) is the most common form of the disorder, occurring in ~1 in 5,000 males in Europe. Haemophilia B (clotting factor IX deficiency) occurs in ~1 in 25,000 males. Prophylaxis treatment including frequent infusions of plasma-derived or recombinant factors has been shown to extend the lifespan and quality of life of patients. Several strategies are being pursued to bioengineer clotting factors to enhance the half-life. Despite these improvements, development of antibodies inhibiting coagulation factor activity is frequently observed. Patients with haemophilia are ideal candidates for gene therapy, because a small increase in protein production can lead to significantly decreased bleeding diathesis. Although different gene transfer strategies for factor replacement have been evaluated, several properties make adeno-associated virus (AAV) vectors the most promising. Most importantly, AAV directs long-term transgene expression from non-dividing cells in animal models after a single administration of vector. In addition, AAV has an excellent safety profile. Unlike other vectors of viral origin, AAV has never been associated with any human disease and is naturally replication deficient.
The overriding goal of the proposal is to improve the probability that AAV-mediated FVIII gene transfer can be used to successfully treat patients with haemophilia A. To achieve this goal, we established a simple and efficient GMP compliant manufacturing platform for rAAV-HLP-codop-hFVIII pseudotyped with serotype 8 capsid (aim 1) and we assessed the safety and efficacy of an AAV vector encoding a novel, potent FVIII-variant in mice (aim 2).
Aim 1 : To optimize the rAAV production yield, we evaluated several parameters such as the transduction DNA ratio, the presence of FBS in the culture media, and the harvesting time. Then, we adapted the process to the cell factory systems that provide a large growth surface in a small space for large-scale cell culture. In this way we generated sufficient rAAV-HLP-codop-hFVIII to perform the GLP toxicity study in C56Bl/6 mice and FVIII-/- mice.
Aim 2 : Toxicology study in C57BL/6 + FVIII-KO
To carry out a GLP study in C57Bl/6 mice and a non-GLP study in FVIII deficient mice. The report of the GLP study is not yet finalised as results are outstanding for FVIII antigen and FVIII antibodies. The reporting for macropathology showed no test substance related lesions, there was however a small increase in liver and spleen weight in the treated animals. There were significant changes in the haematology parameters with increase in neutrophil, basophil, monocyte and large unstained cell counts, although only a small number of treated mice were outside the normal range. There was also a significantly lower platelet count in the treated mice compared to controls. Blood chemistry analysis also showed significant changes, which were again associated with 3 of the treated mice, increases were detected in liver enzymes, bilirubin and phosphorus. A decrease in creatinine, glucose, albumin, urea, cholesterol and triglyceride levels were also observed.
The non-GLP study in FVIII KO mice showed an increase in blood cell counts at 2 weeks in some of the treated mice, but had returned to normal levels at 4 weeks. At termination of the study a significant reduction in the number of platelets was observed in some of the treated mice. A supraphysiological levels of FVIII antigen (20-254x normal levels) were detected at the end of the study with full activity in a FVIII chromogenic assay. Despite the supraphysiological levels of FVIII there was not correction of the bleeding phenotype in 6 out of the 10 treated mice. This is an unexpected finding which we hypothesis is due to the supraphysiological levels of FVIII and a decrease in the von Willebrand factor:FVIII ratio. Histology of the organs showed evidence of thrombus formation in the heart in 6 animals and in liver and lung in 2 treated animals. Again the this is probably due to the supraphysiological FVIII levels. Previous experience with the Haemophilia B trial has shown considerably higher levels of expression in mice and non-human primates than is achieved in man.
The preclinical data generated in mice are sufficient to support a clinical trial application of AAV-mediated FVIII gene transfer for haemophilia A.
Haemophilia is a group of hereditary disorders that leads to deficiency of a plasma protein needed for normal blood clotting and coagulation. The two most common forms of haemophilia are haemophilia A and B. Both are classically transmitted in an X-linked recessive pattern. Haemophilia A (clotting factor VIII deficiency) is the most common form of the disorder, occurring in ~1 in 5,000 males in Europe. Haemophilia B (clotting factor IX deficiency) occurs in ~1 in 25,000 males. Prophylaxis treatment including frequent infusions of plasma-derived or recombinant factors has been shown to extend the lifespan and quality of life of patients. Several strategies are being pursued to bioengineer clotting factors to enhance the half-life. Despite these improvements, development of antibodies inhibiting coagulation factor activity is frequently observed. Patients with haemophilia are ideal candidates for gene therapy, because a small increase in protein production can lead to significantly decreased bleeding diathesis. Although different gene transfer strategies for factor replacement have been evaluated, several properties make adeno-associated virus (AAV) vectors the most promising. Most importantly, AAV directs long-term transgene expression from non-dividing cells in animal models after a single administration of vector. In addition, AAV has an excellent safety profile. Unlike other vectors of viral origin, AAV has never been associated with any human disease and is naturally replication deficient.
The overriding goal of the proposal is to improve the probability that AAV-mediated FVIII gene transfer can be used to successfully treat patients with haemophilia A. To achieve this goal, we established a simple and efficient GMP compliant manufacturing platform for rAAV-HLP-codop-hFVIII pseudotyped with serotype 8 capsid (aim 1) and we assessed the safety and efficacy of an AAV vector encoding a novel, potent FVIII-variant in mice (aim 2).
Aim 1 : To optimize the rAAV production yield, we evaluated several parameters such as the transduction DNA ratio, the presence of FBS in the culture media, and the harvesting time. Then, we adapted the process to the cell factory systems that provide a large growth surface in a small space for large-scale cell culture. In this way we generated sufficient rAAV-HLP-codop-hFVIII to perform the GLP toxicity study in C56Bl/6 mice and FVIII-/- mice.
Aim 2 : Toxicology study in C57BL/6 + FVIII-KO
To carry out a GLP study in C57Bl/6 mice and a non-GLP study in FVIII deficient mice. The report of the GLP study is not yet finalised as results are outstanding for FVIII antigen and FVIII antibodies. The reporting for macropathology showed no test substance related lesions, there was however a small increase in liver and spleen weight in the treated animals. There were significant changes in the haematology parameters with increase in neutrophil, basophil, monocyte and large unstained cell counts, although only a small number of treated mice were outside the normal range. There was also a significantly lower platelet count in the treated mice compared to controls. Blood chemistry analysis also showed significant changes, which were again associated with 3 of the treated mice, increases were detected in liver enzymes, bilirubin and phosphorus. A decrease in creatinine, glucose, albumin, urea, cholesterol and triglyceride levels were also observed.
The non-GLP study in FVIII KO mice showed an increase in blood cell counts at 2 weeks in some of the treated mice, but had returned to normal levels at 4 weeks. At termination of the study a significant reduction in the number of platelets was observed in some of the treated mice. A supraphysiological levels of FVIII antigen (20-254x normal levels) were detected at the end of the study with full activity in a FVIII chromogenic assay. Despite the supraphysiological levels of FVIII there was not correction of the bleeding phenotype in 6 out of the 10 treated mice. This is an unexpected finding which we hypothesis is due to the supraphysiological levels of FVIII and a decrease in the von Willebrand factor:FVIII ratio. Histology of the organs showed evidence of thrombus formation in the heart in 6 animals and in liver and lung in 2 treated animals. Again the this is probably due to the supraphysiological FVIII levels. Previous experience with the Haemophilia B trial has shown considerably higher levels of expression in mice and non-human primates than is achieved in man.
The preclinical data generated in mice are sufficient to support a clinical trial application of AAV-mediated FVIII gene transfer for haemophilia A.