Inherited diseases, occurring in solid tissues in the body, are often caused by simple recessive mutations, derived from two carrier parents with a normal phenotype. In X-linked recessive diseases, hemizygous males with a mutant allele will be affected, while the carrier female will not. Gene Therapy (GT) for recessive diseases has typically used a DNA-based gene system to deliver a normal allele in a mammalian expression vector or via allogenic cell transplant. However, numerous problems have been identified with these gene complementing systems arising from poor gene expression control, poor distribution of transfecting vector, and host immune responses to the viral vectors or cells. These adverse events as well as our own long-standing interest in DNA mismatch repair (MR), have led us to develop a new approach to gene therapy. In this alternative therapy, the existing mutant allele is targeted with a specifically designed synthetic oligonucleotide, which pairs with and induces correction of the chromosomal target via (MR). This alternative gene therapy can be called gene editing. In preliminary studies, we have begun to target the Sickle allele in a-globin found in CD34+ hematopoietic stem cells from patients with this recessive disease Sickle Cell Anemia (SCA) to produce a population of hematopoietic cells from diseased patients, which contain a normal 6-globin allele. This translational research is aimed at an IND for treatment of SCA patients via stem cell transplantation with their own edited cells. In this proposal we will apply this new technology to address inherited mutations in solid tissues. We will focus on X-linked recessive dystrophinopathies in muscle of animal models Duchenne dystrophy (DMD). This disease affects 1:3300 live born human males with severe, progressive muscle wasting, and is fatal by age 20-24 years of age. All attempts at complementing GT via viral and/or allogenic cell transplantation of either muscle or bone marrow precursor cells have provided no significant clinical improvement in patients. However, we have published a pilot study that demonstrated the feasibility of our gene editing in a canine model of DMD and the effects were long-term (11 months) albeit, subclinical. We therefore propose to follow up on these studies using the latest advances developed for the Sickle cell therapy paradigm. All studies in this Phase 1 proposal will relay on the ex vivo MR selection developed in yeast for SCA.
