SBIR-STTR Award

The current standard of care for hemophilia A is treatment with intravenous FVIII protein infusions, either prophylacticly or during bleeding episodes. Prophylactic treatment, however, is problematic due to limited availability, high costs and concerns for blood-borne diseases due to venous access. Gene transfer with viral vectors provides an attractive alternative therapeutic approach with the potential for long-term correction since only 5% of normal FVIII expression levels provide substantial clinical benefits. Success of clinical trials with adeno (Ad)- and oncoretroviral (MLV) FVIII therapy has been compromised to date by lack of transduction of non-proliferating cells (MLV) and high vector particle immunogenicity (Ad). Lentiviral vectors overcome both problems and our cumulative preclinical studies using Feline Immunodeficiency Virus (FIV)-based lentiviral vectors resulted in persistent and therapeutic FVIII expression levels in the hemophilia mouse model. The next logical steps towards the initiation of clinical trials include a detailed investigation of vector particle interactions with the host's immune system as well as the testing of FIV-based FVIII therapy in a relevant large animal model-the hemophilia dog. Our goal is to generate FIV vectors coding for canine B domain-deleted FVIII (cFVIII) optimized for high expression levels. Optimization of cFVIII expression is modeled after successful modifications of the human FVIII and thus better mimics advanced human FVIII therapy while facilitating detection of serum FVIII protein levels and therapeutic benefit in the dog model. Our second goal is aimed at gaining a better understanding of possible immune responses to the FIV vector particle itself. To date, there has been no published investigation of innate or adaptive immune responses to lentiviral vector components in any animal system. A thorough understanding of the level or extent of any immune responses to FIV particles is an important safety and efficacy concern since FVIII gene transfer therapy likely needs to be re-administered after some time. The timeframe of this proposal allows the production of functional FIV-cFVIII vectors and the testing of first innate immune responses to FIV vector particles pseudotyped with three different envelopes. Future studies beyond the scope of the current proposal will be aimed at investigating FIV-based cFVIII gene transfer in the hemophilia dog while monitoring therapeutic expression levels and adaptive anti-FIV immune responses during repeat administrations.

Project Terms:
animal genetic material tag; biotechnology; coagulation factor VIII; feline immunodeficiency virus; gene delivery system; gene therapy; green fluorescent proteins; hemophilia As; human genetic material tag; immune response; laboratory mouse; plasmids; polymerase chain reaction; technology /technique development; transfection /expression vector

Although several labs including ours have demonstrated the feasibility of gene therapy for hemophilia A in animal models, long-term expression of the transgene at therapeutic levels was not observed in the clinic. The lack of sufficient expression levels Factor VIII (FVIII) in hemophilic patients indicates that further improvements of the current gene delivery systems are needed to generate a therapeutic product. In the Phase I of this project we initiated a study to address the issues of FVIII expression level and duration based on findings showing that FVIII expression is limited by unstable mRNA, interaction with endoplasmic reticulum (ER) chaperones, and a requirement for facilitated ER to Golgi transport through interaction with the mannose- binding lectin LMAN1. Vectors for FVIII expression in gene therapy applications typically have used a B domain deleted (BDD) cDNA in which the coding sequence for this domain is removed to yield a shorter construct for protein production and gene transfer. Of note, the expression of BDD FVIII within the cell has the same limitations as the whole FVIII molecule. However, the inclusion of several asparagine-linked oligosaccharides within a short B-domain spacer increased ER to Golgi transport resulting in secretion of functional FVIII at levels 15- to 25-fold higher than full-length or B domain deleted FVIII both in vitro and in vivo. Phase I initiated the testing of the hypothesis that therapeutic FVIII production can be significantly enhanced by addition of parts of the B domain in hemophilic dogs, the most relevant animal model to the human disease and a prerequisite test for clinical studies, Two FIV vectors were constructed and tested which contain canine FVIII with different lengths of the B domain added. The constructs were prepared with the canine FVIII genetic sequence to reduce complicating cross-species factors when introduced into the hemophilic dog. Also, in Phase I the parameters for preparing high titer virus were optimized and the innate immune response to VSV- G and GP64 pseudotyped vectors were analyzed. The completion of the Phase I aims puts the project in position to conduct the FVIII expression level and duration studies in hemophilic mice and dogs and to select the most favorable FVIII cDNA for safe and long term expression. In this Phase II study the aims are to evaluate expression level, duration and clinical benefit from FIV delivery of canine FVIII with no B domain or with two different lengths of the B domain included. Also, additional elements that may further enhance FVIII production significantly from transduced cells will be incorporated into the transfer vector and tested. This Phase II also includes the development of methods to provide increased production of the virus vector for these and subsequent studies, which will require large amounts of the vector.

Public Health Relevance:
Clinical application of Factor VIII gene therapies to hemophilia A patients have suffered from insufficient production of Factor VIII. Using an FIV lentiviral vector capable of long-term expression in host cells, Factor VIII gene sequences with improvements designed to overcome the problem of poor expression will be tested in hemophilic dogs, the animal model which closely resembles the human disease. The goal is to advance the best product as determined from this study toward application in the clinic to human patients.

Thesaurus Terms:
A Mouse; Acute; Address; Advanced Development; Affect; Animal Model; Animal Models And Related Studies; Antibodies; Antihemophilic Factor; Apoptosis; Apoptosis Pathway; Asparagine; B-Domain-Deleted Factor Viii; Baculoviruses; Biomedical Engineering; Bleeding; Blood Clot; Blood Clotting; Blood Coagulation Disorders; Blood Coagulation Factor Viii; Blood Plasma; Blood Coagulation; Blood-Coagulation Factor Viii, Complex; Body Tissues; Canine Species; Canis Familiaris; Cell Death, Programmed; Cells; Chaperone; Clinic; Clinical; Clinical Research; Clinical Study; Clinical Trials; Clinical Trials, Unspecified; Coagulation Disorder; Coagulation Factor Viii; Coagulation Factor Viiic; Coagulopathy; Code; Coding System; Complementary Dna; Dna, Complementary; Development; Dogs; Elements; Endoplasmic Reticulum; Ergastoplasm; Fiv; Ftlv; Factor Viii; Factor Viii Deficiency; Factor Viii F8b; Feline Immunodeficiency Virus; Feline T-Lymphotropic Lentivirus; Feline T-Lymphotropic Virus; Frequencies (Time Pattern); Frequency; Funding; Future; Gc-Rahf; Gene Transfer; Gene Transfer Clinical; Gene Transfer Procedure; Gene-Tx; Genes; Genetic; Genetic Intervention; Goals; Golgi; Golgi Apparatus; Golgi Complex; Hemophilia; Hemophilia A; Hemophilia As; Hemorrhage; Hepatic Cells; Hepatic Parenchymal Cell; Hepatocyte; Human; Human, General; Immune Response; Immunodeficiency Virus, Feline; Immunologic, Immunochemical; Immunologics; In Vitro; Incidence; Intervention, Genetic; L-Asparagine; Length; Lentiviral Vector; Lentivirus Vector; Life; Link; Liver; Liver Cells; Lytotoxicity; Mbl; Mammals, Dogs; Mammals, Mice; Man (Taxonomy); Man, Modern; Mannan-Binding Lectin; Mannan-Binding Protein; Mannose Binding Lectin; Mannose-Binding Lectins; Mannose-Binding Protein; Mannose-Specific Lectin; Maps; Measures; Messenger Rna; Metabolic Glycosylation; Methods; Mice; Modeling; Modification; Molecular Biology, Gene Therapy; Molecular Chaperones; Morbidity; Morbidity - Disease Rate; Murine; Mus; Oligosaccharides; Operation; Operative Procedures; Operative Surgical Procedures; Outcome; Patients; Phase; Phenotype; Plasma; Position; Positioning Attribute; Procoagulant Component; Production; Promoter; Promoters (Genetics); Promotor; Promotor (Genetics); Protein Secretion; Proteins; Rna, Messenger; Reticuloendothelial System, Serum, Plasma; Series; Serum, Plasma; Shapes; Site; Staging; Surgical; Surgical Interventions; Surgical Procedure; System; System, Loinc Axis 4; Testing; Therapeutic; Therapy, Dna; Thromboplastinogen; Time; Tissues; Toxic Effect; Toxicities; Vsv; Vesicular Stomatitis Virus; Vesicular Stomatitis Indiana Virus; Virus; Viruses, General; Whblood; Whole Blood; Antihemophilic Factor A; Base; Bioengineering; Bioengineering/Biomedical Engineering; Biopsy Of Liver; Blood Loss; Body System, Hepatic; Cdna; Cdna Expression; Canine; Cell Transduction; Cellular Transduction; Clinical Applicability; Clinical Application; Clinical Investigation; Clotting Disorder; Cytokine; Cytotoxicity; Design; Designing; Domestic Dog; Endoplasmic Reticulum Stress; Enhancing Factor; Experiment; Experimental Research; Experimental Study; Expression Vector; Gene Delivery System; Gene Product; Gene Replacement; Gene Therapy; Genetic Therapy; Glycosylation; Host Response; Human Disease; Immunoresponse; Improved; In Vivo; Liver Biopsy; Mrna; Male; Method Development; Model Organism; Organ System, Hepatic; Phase 2 Study; Platelet Cofactor I; Protein Expression; Public Health Relevance; Rfviii (B-Domain-Deleted); Research Study; Response; Surgery; Therapeutic Protein; Thromboplastinogen A; Transduced Cells; Transfer Of A Gene; Transgene Expression; Vector