Lysosomal storage diseases (LSDs) are a family of rare inherited diseases caused by a mutation in genes of lysosomal enzymes and proteins, resulting in excessive accumulation of metabolites and lack of nutrients for homeostasis. Individual LSDs have a low prevalence, but collectively they have a combined prevalence of 1:8000. Enzyme replacement therapy and bone marrow transplantation are two common treatments, but production of neutralizing antibodies and graft versus host disease hampers treatment. Gene therapy using lentivirus yields encouraging outcomes, but can induce tumorigenesis. Thus, novel treatments are needed. Lysosomal enzymes/proteins are found in extracellular vesicles (EV) that mediate intercellular communication. CRISPR gene editing of a patient's somatic cells will lead to production of functional enzymes/proteins in circulation via EV and/or hematopoietic cells and ameliorate symptoms.
Three aims are proposed to establish efficacious CRISPR treatment strategies and to elucidate the mechanism in which direct genome editing of somatic cells or transplantation of CRISPR-edited hematopoietic stem/hematopoietic stem progenitor cells can treat a mouse model of MPS VII.
Specific Aim 1 : Define Optimal Condition(s) and Off-target events of CRISPR in Treating Gusb/MPS VII Aim 1A: To validate the editing efficiency, synthesis and secretion of ?-Glu and off targeting events following genome editing.
Aim 1 B: Determine the best route of AAV2DJ delivery. The treatment efficacy of multiple administrations with AAV2DJ-Sa-CRISPR viral vectors will be analyzed. Mice will be subjected to 1) HRTII in vivo confocal imaging for reduction of corneal haze; 2) Survival rate determination; 3) In vivo 3D CT scan to determine liver size; 4) ?-Glu activity.
Aim 1 C: Intrastromal injection of AAV2DJ-Sa- CRISPR to examine the efficacy of gene editing in treating corneal haze.
Specific Aim 2 : To Determine the Efficacy of Gene Editing Therapy of Hematopoietic Stem and Stem Progenitor Cells (HSC/HSPC) for Gusb mice Lin-Sca1+ HSC/HSPC will be isolated from donor Gusb mice and subjected to CRISPR editing and expanded. The CRISPR-edited HSC/HSPC will then be transplanted to gamma-irradiated recipient mice via ROIV. The treatment efficacy will be assessed as described in Aim 1.
Specific Aim 3 : To Determine Efficacy of Homology Mediated End Joining-based CRISPR (HMEJ) for Gusb/MPS VII as a Proof of Principle for LSDs.
Aim 3 A: Gusb MEF will be used to validate the genome editing efficiency of a binary AAV consisting of AAV2DJ-SpCas9 and AAV2DJ-sgRNA/donor DNA template containing selective transgenes Aim 3B: will determine the efficacy of administration of the binary AAV2 vectors for Gusb mice.
Aim 3 C: Transplantation of CRISPR edited Gusb Lin-Sca1+ HSC/HSPC to recipient Gusb mice that will be examined as described in Specific Aim 1. The proposed studies will lead to the development of effective therapeutic strategies for MPS VII and other types of LSDs, which can ultimately be translated to the bedside.
Lysosomal storage diseases are a family of disorders caused by a mutation in lysosomal enzymes and proteins leading to the accumulation of metabolites and lack of nutrients in cells, which have a combined prevalence of 1:8000. Enzyme replacement therapy and bone marrow transplantation are two common treatment regimens, but these are compromised by the production of neutralizing antibodies and graft versus host disease. The proposed studies are to validate the efficacy of CRISPR gene editing for treating a mouse model of MPS VII (Gusb) by administration of AAV2 vector(s) via retro-orbital intravenous injection in adult mice and intrahepatic injection of neonates as well as allo-transplantation of CRISPR edited Gusb HSC/HSPC.
