Eric B Kmiec
Delaware State University
Project start date: 2000-12-01
Project end date: 2013-04-30
Sponsored Links Excellgen http://Excellgen.com
REGULATION OF TARGETED GENE CORRECTION
Eric B Kmiec, Director/lead Scientist
Marshall University, 401 11th Street, Huntington, Wv 25701
Grant 7R01CA089325-08 from National Cancer Institute
Abstract: The ultimate form of gene therapy for inherited diseases is to reverse the phenotype by correcting the genetic mutation at its endogenous location in the chromosome. We have been developing a gene repair strategy that relies on DNA oligonucleotides to enter the cell, hybridize to the mutant sequence and direct single base exchanges in the target gene. During the initial grant period, we created several model systems in yeast and mammalian cells that enabled the elucidation of pathways that control the frequency of gene correction. The data indicate that the gene repair process is controlled by the activation of homologous recombination and rate at which DNA replication takes place. We now propose to transition from model systems to a clinically relevant cell type that is likely to serve as a target in the initial clinical application. Results from several laboratories indicate that liver cells, particularly hepatocytes, are highly responsive to this technique and enable gene correction to take place at robust levels. We shall target a integrated, mutant eGFP gene and the endogenous HPRT gene in clonal isolates of HepG2 and THLE cells, two established hepatocytic cell lines that have been used in the development phase of therapies aimed at liver diseases. Guided by the results of our first grant term, we will focus on the activation of homologous recombination as a means to support enhanced levels of correction in a reproducible and sustainable fashion. The experiments outlined in this grant will address the following questions; 1) are random ds breaks required for attaining high levels of gene correction?; 2) do lesions at replication forks or stalled forks themselves provide enough stimulus for elevating the levels of gene correction in the absence of DNA damage; 3) is the process of gene repair itself mutagenic at non-targeted sites and are cells undergoing gene repair more prone to genome rearrangement?; 4) how does the cell respond to the intemalization of the ssODN in terms of DNA damage response pathways. The key to developing this technique in the long term, even for liver disease and cancer, lies in the ability to regulate, predict and reliably attain correction efficiencies that have therapeutic effects. These goals support the choice of liver as a target for clinical applications of gene repair but there are other important reasons for focusing on hepatocvtes they are the target cell for gene therapy for Alpha-1 Antitrypsin Deficiency. Crigler-Naiiar. OTC. MPSVII. Hemophilia A and B and many lysosomal storage disorders among others. Our work will uncover restrictions or limitations for gene repair in hepatocvtes with the goal of treating hepatic cancer and genetic diseases of the liver
Keywords: 2-Amino 6MP; 2-Amino-1, 7-dihydro-6H-purine-6-thione; 2-Amino-6-purinethiol; 2-Aminopurin-6-thiol; 2-Aminopurine-6(1H)-thione; 2-Aminopurine-6-thiol; 6-Amino-2-mercaptopurine; 6-Mercapto-2-aminopurine; 6-Mercaptoguanine; 6-TG; 6-Thioguanine; 6H-Purine-6-thione, 2-amino-1, 7-dihydro- (9CI); Acids; Activity Cycles; Address; Adopted; Affect; Amino Acids; Animal Model; Animal Models and Related Studies; Annexins; Anti-Cancer Agents; Anti-Tumor Agents; Anti-Tumor Drugs; Antibodies; Antineoplastic Agents; Antineoplastic Drugs; Antineoplastics; Antiproliferative Agents; Antiproliferative Drugs; Apoptosis; Apoptosis Pathway; Apoptotic; Artificial Genes; Assay; Au element; Base Pairing; Behavior; Binding; Binding (Molecular Function); Bioassay; Biologic Assays; Biological Assay; Biological Models; Calcimedins; Cancer Drug; Cancers; Cell Cycle; Cell Cycle Arrest; Cell Death, Programmed; Cell Division Cycle; Cell Line; Cell Line, Transformed; Cell Lines, Strains; Cell division; CellLine; Cells; Characteristics; Chemotherapeutic Agents, Neoplastic Disease; Chromosomal Breaks; Chromosome Break; Chromosomes; Cloning; Codon, Stop; Codon, Termination; Codon, Terminator; Complement; Complement Proteins; DNA; DNA Alteration; DNA Damage; DNA Damage Repair; DNA Injury; DNA Recombination; DNA Repair; DNA Replication; DNA Sequence; DNA Sequence Rearrangement; DNA Synthesis; DNA biosynthesis; DNA mutation; DNA recombination (naturally occurring); DNA, Single-Stranded; Data; Deoxyribonucleic Acid; Development; Disease; Disorder; Event; Experimental Designs; Factor VIII Deficiency; Frequencies (time pattern); Frequency; GUSB deficiency; Gene Alteration; Gene Mutation; Gene Targeting; Gene Transfer Clinical; Gene Transfer Procedure; Gene-Tx; Generalized Growth; Generations; Genes; Genes, Reporter; Genetic Alteration; Genetic Change; Genetic Condition; Genetic Diseases; Genetic Intervention; Genetic Recombination; Genetic defect; Genetic mutation; Genetics-Mutagenesis; Genome; Genomics; Goals; Gold; Grant; Growth; Hemophilia; Hemophilia A; Hemophilia As; Hepatic Cancer; Hepatic Cells; Hepatic Disorder; Hepatic Parenchymal Cell; Hepatocyte; Hereditary; Hereditary Disease; Human; Human, General; Induction of Apoptosis; Inherited; Intervention, Genetic; Laboratories; Lanvis; Lead; Lesion; Lipocortins; Liver; Liver Cells; Liver diseases; Location; MPS VII; MPSVII; Malignant Neoplasms; Malignant Tumor; Malignant neoplasm of liver; Mammalian Cell; Man (Taxonomy); Man, Modern; Measures; Methods and Techniques; Methods, Other; Model System; Modeling; Models, Biologic; Molecular Biology, Gene Therapy; Molecular Biology, Mutagenesis; Molecular Disease; Molecular Interaction; Monitor; Mucopolysaccharidosis 7; Mucopolysaccharidosis VII; Mutagenesis; Mutation; Necrosis; Necrotic; Nucleotides; Oligo; Oligonucleotides; Organ; Pathway interactions; Pattern; Pb element; Phase; Phenotype; Point Mutation; Population; Position; Positioning Attribute; Process; Processed Genes; Proteins; Protocol; Protocols documentation; RNA, Small Interfering; Reaction; Rearrangement; Recombination; Recombination, Genetic; Regulation; Relative; Relative (related person); Reporter; Reporter Genes; Resected; Resistance; Sampling; Screening procedure; Sequence Alteration; Single-Stranded DNA; Site; Sly Disease; Sly Syndrome; Small Interfering RNA; Sorting - Cell Movement; Specificity; Staining method; Stainings; Stains; Stimulus; Stop Signal, Translation; Synthetic Genes; System; System, LOINC Axis 4; TUNEL Assay; Tabloid; Target Populations; Targetings, Gene; TdT-Mediated dUTP Nick End Labeling Assay; Techniques; Terminator Codon; Testing; Therapeutic Effect; Therapy, DNA; Thioguanine; Time; Tioguanin; Tioguanine; Tissue Growth; Transformed Cell Line; Tumor-Specific Treatment Agents; Unscheduled DNA Synthesis; Work; Yeasts; alpha 1-Antitrypsin Deficiency; aminoacid; anticancer agent; anticancer drug; base; beta-glucuronidase deficiency; beta-glucuronidase deficiency mucopolysaccharidosis; body system, hepatic; cancer genetics; cell type; clinical applicability; clinical application; clinical relevance; clinically relevant; cultured cell line; disease/disorder; efficacy testing; experiment; experimental research; experimental study; gene correction; gene product; gene repair; gene therapy; gene-corrected; genetic disorder; genetic therapy; genome mutation; heavy metal Pb; heavy metal lead; hepatopathy; hereditary disorder; homologous recombination; insight; liver cancer; liver disorder; malignancy; malignant liver tumor; model organism; mucopolysaccharide storage disease VII; mucopolysaccharidosis (MPS) VII; mucopolysaccharidosis type VII; mutant; neoplasm/cancer; ontogeny; organ system, hepatic; pathway; preference; repair; repaired; research study; resistant; response; restriction enzyme; screening; screenings; siRNA; sorting; tumor
Project start date: 2000-12-01
Project end date: 2012-04-30
Budget start date: 1-MAY-2009
Budget end date: 30-APR-2010
7R01CA089325-08 (2009): $268346
Grants awarded to Eric B Kmiec
GENETIC REPAIR OF THE SICKLE CELL ANEMIA MUTATION
Eric B Kmiec, Professor
Biological Sciencesuniversity Of Delaware
research & Graduate Studies
newark, De 19716
Grant 5R01HL058563-04 from National Heart, Lung, And Blood Institute IRG: HEM
Abstract: This proposal seeks to investigate the feasibility of genetic repair of HbS mutations by a process of gene conversion. Dr. Kmiec and colleagues have developed an experimental strategy that centers around site-specific conversion of single base mutations using chimeric DNA-RNA oligonucleotides. This synthetic molecule folds into a double hairpin configuration that improves its stability in serum and in cells. Constructs designed to alter the HbS mutation have been found to efficiently catalyze gene conversion in lymphoblastoid cells. A similar strategy has now been shown to work in a variety of other cells, and with the alkaline phosphatase genes, the genes responsible for Crigler Najjar disease factor XIII, and factor IX deficiency. In other preliminary data, HbA to HBS conversion has been demonstrated in human CD34+ stem cells. In the proposed studies, Dr. Kmiec will explore the genotypic and phenotypic consequences of targeted gene conversion, the factors that determine conversion efficiency including the methods of delivery, and the levels of random mutagenesis produced by such constructs. Reconstitution studies using gene converted human stem cells will also be carried out in SCID/Beige mice. The long term goal of these studies is to enable gene conversion as a feasible option for gene therapy of hemoglobinopathies and other defined genetically-inherited diseases
Keywords: DNA repair, gene mutation, hemoglobin S, sickle cell anemia CD34 molecule, gene conversion, genotype, oligonucleotide, phenotype SCID mouse, hematopoietic stem cell, human tissue, polymerase chain reaction
Project start date: 1998-09-01
Project end date: 2002-06-30
5R01HL058563-04 (2000): $271482
5R01HL058563-02 (1999): $284660
REGULATION OF TARGETED GENE CORRECTION
Eric B Kmiec, Director/lead Scientist
Marshall University, 401 11th Street, Huntington, Wv 25701
Grant 5R01CA089325-09 from National Cancer Institute
Abstract: The ultimate form of gene therapy for inherited diseases is to reverse the phenotype by correcting the genetic mutation at its endogenous location in the chromosome. We have been developing a gene repair strategy that relies on DNA oligonucleotides to enter the cell, hybridize to the mutant sequence and direct single base exchanges in the target gene. During the initial grant period, we created several model systems in yeast and mammalian cells that enabled the elucidation of pathways that control the frequency of gene correction. The data indicate that the gene repair process is controlled by the activation of homologous recombination and rate at which DNA replication takes place. We now propose to transition from model systems to a clinically relevant cell type that is likely to serve as a target in the initial clinical application. Results from several laboratories indicate that liver cells, particularly hepatocytes, are highly responsive to this technique and enable gene correction to take place at robust levels. We shall target a integrated, mutant eGFP gene and the endogenous HPRT gene in clonal isolates of HepG2 and THLE cells, two established hepatocytic cell lines that have been used in the development phase of therapies aimed at liver diseases. Guided by the results of our first grant term, we will focus on the activation of homologous recombination as a means to support enhanced levels of correction in a reproducible and sustainable fashion. The experiments outlined in this grant will address the following questions; 1) are random ds breaks required for attaining high levels of gene correction?; 2) do lesions at replication forks or stalled forks themselves provide enough stimulus for elevating the levels of gene correction in the absence of DNA damage; 3) is the process of gene repair itself mutagenic at non-targeted sites and are cells undergoing gene repair more prone to genome rearrangement?; 4) how does the cell respond to the intemalization of the ssODN in terms of DNA damage response pathways. The key to developing this technique in the long term, even for liver disease and cancer, lies in the ability to regulate, predict and reliably attain correction efficiencies that have therapeutic effects. These goals support the choice of liver as a target for clinical applications of gene repair but there are other important reasons for focusing on hepatocvtes they are the target cell for gene therapy for Alpha-1 Antitrypsin Deficiency. Crigler-Naiiar. OTC. MPSVII. Hemophilia A and B and many lysosomal storage disorders among others. Our work will uncover restrictions or limitations for gene repair in hepatocvtes with the goal of treating hepatic cancer and genetic diseases of the liver
Project start date: 2000-12-01
Project end date: 2012-04-30
Budget start date: 1-MAY-2010
Budget end date: 30-APR-2011
5R01CA089325-09 (2010): $269932
5R01CA089325-06 (2007): $210384
2R01CA089325-05A2 (2006): $210264
NEW GENE THERAPY FOR CONNECTIVE TISSUE DISEASES
Eric B Kmiec, Professor
Thomas Jefferson University 201 South 11th St Philadelphia, Pa 191075587
Grant 5R01AR044092-03 from National Institute Of Arthritis And Musculoskeletal And Skin Diseases IRG: MEDB
Abstract: Many inherited connective tissue diseases result from a point mutation or frame-shift mutation. A gene therapy capable of correcting such mutations would be an advance in treating severe and nondurable diseases such as hypophosphatasia, chondrodysplasia and perhaps even osteogenesis imperfecta. Current protocols rely on the expression of a normal gene under the control of a heterologous promoter in cells that harbor a defective copy of the gene. One obstacle in such treatments is the failure of the transferred gene to recapitulate faithfully the expression pattern of the normal endogenous gene. Thus, gene therapy, capable of correcting such the mutation at the genetic level is preferable. The success rate of this approach however in mammalian cells has been hampered by an extremely low frequency of targeting and interference from the illegitimate recombination pathway that takes place independent of DNA sequence homology. We developed an experimental strategy that centers around site-specific correction of mutations using a unique chimeric oligonucleotide containing both RNA and DNA and the enzymatic activity of a eukaryotic recombinase. These oligonucleotides increase the specificity of homologous alignment by the recombinase and facilitate the recognition and correction of the mismatched base on the genomic strand by the endogenous DNA repair system. We have also cloned and over-expressed a eukaryotic analog of the RecA protein from Ustilago maydis, designated as Rec2. This protein catalyzes homologous pairing of a wide variety of DNA substrates and promotes homologous alignment of the chimeric molecule with its genomic target. Thus, used in conjunction with chimeric oligonucleotides, the Rec2 protein can provide a highly specific and regulatable gene targeting system. In our preliminary studies, we demonstrated that a chimeric oligonucleotide designed to correct a point mutation (G to A) at position 711 of the human bone/liver/kidney alkaline phosphatase cDNA converted an enzymatically inactive alkaline phosphatase cDNA to wild type form. A high frequency of a correction event was observed at low concentration of oligonucleotide of specific sequence. Furthermore, we demonstrated that the chimeric oligonucleotides were extremely stable in both serum and inside cells. The high efficacy, specificity and stability of chimeric oligonucleotides make them an attractive candidate for gene therapy utilizing nucleic-acid-based therapeutics. This particular mutation at position 711 has been identified in patients with the prenatal form of hypophosphatasia, which results in defective skeletal mineralization. In this proposal, we will extend our preliminary results to more clinically relevant protocols by attempting to correct mutations of genomic sequences involved in several inherited connective tissue diseases.
Keywords: connective tissue disorder, fusion gene, gene expression, gene mutation, gene targeting, gene therapy, oligonucleotide, DNA repair, alkaline phosphatase, collagen, disease model, frameshift mutation, hypophosphatasia, osteogenesis imperfecta, point mutation, recombinase, laboratory mouse, nucleic acid sequence, tissue /cell culture
Project start date: 1996-04-01
Project end date: 1999-08-31
5R01AR044092-03 (1998): $149911
5R01AR044092-02 (1997): $144144
Selectable Gene Editing For Muscular Dystrophy
Eric B Kmiec, Professor
University Of Delaware Research And Graduate Studies Newark, De 19716
Grant 5R21NS053608-02 from National Institute Of Neurological Disorders And Stroke IRG: NSD
Abstract: Inherited diseases, occurring in solid tissues, are often caused by simple recessive mutations derived from two carrier parents with a normal phenotype. In X-linked diseases, hemizygous males with a mutant allele will be affected while the female carrier is not. Gene therapy for recessive diseases has typically centered on a DNA-based gene system to deliver the normal allele in a mammalian expression vector or via an allogenic cell transplant. Numerous problems have been identified with these gene complementing systems arising from the lack of proper gene regulation, poor distribution of the transfecting vector, antigenic response from host immune system and the oncogenic potential of viral integration. These adverse events coupled with our long-standing interest in using the efficient endogenous DMA repair systems to correct mutations in mammalian cells led us to develop a new approach to gene therapy. This alternative strategy employs a synthetic oligonucleotide to direct the exchange of the mutant base in the affected gene or allele. The process, termed, gene repair or gene editing, has proven to be efficacious in a number of model systems and disease targets, including muscular dystrophy. The work in DMD, however, has not achieved levels of correction that enable therapeutic benefit. Thus, a primary challenge for this technique is to increase the frequency of repair or to isolate cells that have been corrected and expand them in culture. In this proposal, we aim to carry out preliminary investigations into the feasibility of meeting both challenges using primary muscle cells obtained from mdxScv and normal mice. We will examine the most efficient way of transfecting both a reporter plasmid and an oligonucleotide and achieving high levels of correction in either dividing or differentiating cells. We aim to develop a dual correction protocol in which a mutant reporter gene eYFP or DsRFP and the dystrophin mutation are simultaneously repaired. We shall select cells displaying functional gene editing activity by the emergence of fluorescence and assess the coordinate repair of the dystrophin mutation at the phenotypic and genotypic levels. Our long term goal is to develop a protocol in which primary muscle cells can be isolated and propagated ex vivo, then eventually transplanted back into the patient.
Keywords: cell, gene, muscular dystrophy, DNA, allele, antibiotic, antibody, antigen, back, base, birth, cell differentiation, cell line, cell membrane, child rearing, choice, chromophore, chromosome, complement, conditioning, creatine kinase, culture, dystrophin, element, emotion, experience, female, fluorescence, gene frequency, gene mutation, gene targeting, gene therapy, human, immune response, immune system, lead, learning, male, measurement, model, molecular genetics, motivation, muscle, muscle cell, mutant, myoblast, myotube, natural selection, nucleic acid sequence, oligonucleotide, parent, peptide, phenotype, plasmid, protein, reporter gene, sectioning, staining, stem cell, striated muscle, success, therapy, tissue, tissue /cell culture, transfection, transfection /expression vector, transplantation, university, yeast
Project start date: 2006-01-01
Project end date: 2008-12-31
5R21NS053608-02 (2007): $131959
1R21NS053608-01 (2006): $135900
Regulation Of Targeted Gene Correction
Eric B Kmiec, Professor
Biological Sciencesuniversity Of Delaware
research & Graduate Studies
newark, De 19716
Grant 1R01CA089325-01A1 from National Cancer Institute IRG: ET
Abstract: Chimeric DNAJRNA double hairpins are synthetic oligonucleotides that direct targeted nucleotide substitutions in bacteria, yeast, plants and animals. We have postulated that cellular recombinases catalyze joint molecule formation between these oligonucleotides and homologous dsDNA. According to our model, resolution of these joints can be accompanied by point mutation of the target DNA when the oligonucleotide is mismatched within the intermediate joint. Since chimeric hairpins are effective gene repair agents in yeast, we propose to validate our model in this microorganism. Experiments will be carried out with whole cells, cell-free extracts, and purified protein with the goal of delineating the mechanism of joint molecule formation and gene substitution or frameshift mutations in yeast will be monitored by targeting episomal or chromosomal genes that express a fluorescent signal or an antibiotic resistance marker. Targeting of the episomal target in different knock out strains will drive the discovery of formation between chimeric hairpins and dsDNA will be optimized using gene products from the RAD52 epistasis group either alone or in combination with other related enzymes. Nucleoprotein intermediates formed during strand exchange will be probed to elucidate how a chimeric DNAIRNA backbone enhances recombination. Performed double D-loop joints that contain a specific mismatch or unpaired base will be used as substrates to study targeted nucleotides exchange. The mechanism which emerges from this study should provide guidance in the use of chimeric hairpin oligonucleotides in other systems including plant, animal and human cells
Keywords: DNA repair, frameshift mutation, fungal genetics, point mutation, transposon /insertion element DNA binding protein, bacterial protein, crosslink, drug resistance, fungal protein, fusion gene, oligonucleotide, psoralen, synapse cell free system, yeast
Project start date: 2001-07-01
Project end date: 2005-06-30
1R01CA089325-01A1 (2001): $271410
Sponsored Links Excellgen http://Excellgen.com
5R01CA089325-04 (2004): $256885
5R01CA089325-03 (2003): $256885
MECHANISM OF GENE CORRECTION BY RNA/DNA OLIGONUCLEOTIDES
Eric B Kmiec, Professor
Biological Sciencesuniversity Of Delaware
research & Graduate Studies
newark, De 19716
Grant 5R01DK056134-02 from National Institute Of Diabetes And Digestive And Kidney Diseases IRG: MEDB
Abstract: Adapted from applicant´s ) A novel strategy for gene therapy has been developed and shown promise as a potential therapeutic for inherited disorders. Unique synthetic molecules, known as chimeric oligonucleotides, are now in the pipeline toward clinical trials. Recent reports from the investigator´s and other laboratories have described the use of chimeric RNA/DNA oligonucleotides to introduce targeted base pair substitutions into episomal or genomic DNA of mammalian cells in culture or in animals. With a frequency of targeted mutagenesis greater than 1 percent, chimeraplasty represents a promising new approach to potentially correct genetic defects attributable to point or frameshift mutations. In this grant, the investigators propose to study the mechanism of gene correction relying upon studies with purified enzymes and with human cell-free extracts. Preliminary work indicates that correction proceeds by a two step pathway consisting of homologous recombination of the chimeric oligonucleotide with dsDNA followed by resolution and repair of the mismatched joint molecule. In the repair step the target DNA acquires the sequence of the chimera thus leading to correction. The investigators will use standard in vitro assays to investigate how RecA and hsRad51/52 proteins catalyze homologous pairing and strand exchange between chimeras and homologous double-stranded DNA targets. This reaction is believed to generate a novel joint molecule with a complement-stabilized displacement loop. The properties of this joint molecule would be studied at both the biochemical and biophysical levels. Subsequent processing of the joint molecule to yield a corrected and re-paired DNA target will be investigated using both wild-type and repair deficient human cell-free extracts. In preliminary work, we have demonstrated that a wild-type extract catalyzes chimera-mediated correction of mutant antibiotic resistance genes help elucidate the correction pathway will be carried out using the extract as a source of enzymatic activity. Assays will involve both genetic and biochemical readouts. In parallel, large numbers of chimeric oligonucleotides that systemically differ in structure will be screened for activity. Preliminary results indicate that the all DNA strand directs the correction, while the strand containing RNA stabilizes the chimera-target complex. These modified chimeras, which increase the efficiency of the reaction in a cell-free extract, also elevate the frequency in cultured cells. Hence, biochemical results translate into whole cells
Keywords: biotechnology, gene therapy, oligonucleotide, synthetic nucleotide DNA, DNA repair, RNA, fusion gene, intermolecular interaction, site directed mutagenesis cell free system
Project start date: 2000-06-01
Project end date: 2003-04-30
5R01DK056134-02 (2001): $225000
1R01DK056134-01A1 (2000): $244500
NEW GENE THERAPY FOR CONNECTIVE TISSUE DISEASES
Eric B Kmiec, Professor
Pharmacologythomas Jefferson University
201 South 11th St
philadelphia, Pa 191075587
Grant 1R01AR044092-01 from National Institute Of Arthritis And Musculoskeletal And Skin Diseases IRG: MEDB
Project start date: 1996-04-01
Project end date: 1999-03-31
1R01AR044092-01 (1996): $138602