ENZERNA BIOSCIENCES LLC — Department of Health and Human Services SBIR Phase I: 200

Description

DESCRIPTION provided by applicant Millions of individuals are affected by diseases caused by mutations in mitochondrial encoded genes which lead to a diverse set of clinical disorders ranging from visual impairment optic neuropathy to accelerated aging to hypertrophic cardiomyopathy Unfortunately there are no commercially available technologies that can enable investigators to dissect the function of these genes With several thousand mitochondrial genomes per cell combined with the impermeability of the mitochondria to nucleic acids it is not possible to create gene knockout mutations in all copies of the mitochondrial genome or to effectively ablate mitochondrial gene expression using RNA interference technology Because investigators cannot dissect the function of mitochondrial encoded genes it has been difficult to develop effective therapies for diseases caused by mutations in the mitochondrial genome In this proposal we outline a new enzyme mediated method to ablate mitochondrial DNA encoded RNA expression using Artificial Site Specific RNA Endonucleases ASREs which can be engineered to be transported into every mitochondrion where these ASREs can bind to and destroy specific mitochondrial RNAs The distinguishing feature of ASREs is the presence of an RNA binding domain PUF domain which consists of a series of amino acid modules that recognize one ribonucleotide that can be arranged in an array to recognize specific nucleotide sequences By combining ASRE ablation of a target gene phenocopy of a knockout mutation with the expression of corresponding human disease allele integrated into the nuclear genome unique cell culture models of human mitochondrial diseases can be generated To assess feasibility of this approach Enzerna Biosciences in collaboration with the company co founder and inventor of the technology Dr Zefeng Wang will create ASREs to ablate expression of the mitochondrial Complex I ND and ND genes using drug inducible expression systems in which the ASREs are integrated into defined transcriptionally active regions of the nuclear genome using homologous recombination based strategies Success will be indicated by the identification of ND or ND ASRE targets that mediate andgt reduction in RNA protein expression accompanied by andgt reduction in Complex I functional activity Second we will create cell lines that in which mito ND expression is ablated while a wild type wt ND or mutant ND ND G A gene associated with optic neuropathy is simultaneously expressed to replace the ablated mito ND protein Success will be indicated by rescue of ND protein expression in the mitochondria rescue of functional activity by the expression of the wt allele in the presence of ASRE mediated mito ND knockdown and inability of mutant ND protein expression to rescue Complex I functional activity In Phase once proof of concept is demonstrated we will generate a bank of cell lines in which the expression of each mitochondrial gene is ablated In the long term this bank of mitochondrial gene andquot knockoutandquot cell lines will open new opportunities to examine the molecular and cellular mechanisms of mitochondrial disease and provide valuable models for the development of novel therapeutic agents for intervention PUBLIC HEALTH RELEVANCE Although millions of individuals in the US and throughout the world are affected by diseases caused by mutations in mitochondrial encoded genes there are no technologies that enable investigators to dissect the function of these genes With over mitochondria per cell with each containing at least two genomes per mitochondrion combined with the impermeability of the mitochondria to nucleic acids there are no commercially available technologies to dissect the function of each gene of the mitochondrial genome As a result it has been difficult to develop effective therapies for diseases caused by mutations in the mitochondrial genome In this proposal we outline a new enzyme mediated method to ablate mitochondrial DNA encoded RNA expression using Artificial Site Specific RNA Endonucleases ASREs which can be engineered to be transported into every mitochondrion and bind to and destroy specific mitochondrial RNAs Combining ASRE ablation of target genes with the expression of human disease variant alleles unique cell culture models of human mitochondrial diseases associated with mitochondrial encoded genes can be generated which will open new opportunities to examine the molecular and cellular mechanisms of disease and provide valuable models for the development of novel therapeutic agents