Development of an HSV-based Model System to Identify Events Critical for Embryonic Myogenesis

Muscular dystrophy is a devastating disease in which no treatment or cure exists. A promising therapy for muscular dystrophy is the transplantation of cells that are able to contribute to existing muscle fibers or generate new muscle fibers. While several cell populations have been shown to demonstrate this phenomenon in mouse models, the mechanism by which these cells are able to differentiate into myogenic cells is largely unknown. The goal of this research was to (i) create HSV vectors that are useful for expressing muscle development genes in developing embryos in culture (ii) design strategies to produce and characterize large and diverse HSV vector libraries of expressed genes from different cells or tissues and (iii) design methods by which this expression vector library can be screened for expressed gene functions that participate in skeletal muscle lineage determination. The outcome of these studies were intended to provide a method for exploiting the high level infectivity and growth of HSV in embryonic stem (ES) cells and germ layer derivatives, the virus ability to accommodate large DNA inserts, and the ability to create vector libraries potentially useful in selection of lineage determining genes based on complementation of virus growth. The engineering and characterization of a replication defective HSV vector that is useful for the delivery of genes to embryonic stem cells and their derivatives was described. The deletion of ICP4, ICP22 and the conversion of the immediate early genes ICP0 and ICP27 to the early gene class by promoter exchange generated a vector, JDββHE. JDββHE was found to be non-toxic to ES cells and vector infection of ES cells did not interfere with germ layers formation. This vector was engineered to be dependent on a single IE gene ICP4 for replication. Gene expression from the JDββHE HSV vector, including the expression of eGFP and the early myogenic gene Pax3, was demonstrated in infected ES cells and cells of the embryoid body in a robust and transient manner. In order to discover genes which contribute to muscle differentiation, a cDNA library was constructed within HSV viral vectors in order to be used in the identification of novel gene functions which play a role in the activation early myogenic promoters. For this purpose, the HSV genome was modified to contain BAC elements as well as the Gateway recombination system, which facilitated efficient incorporation of cDNAs into the vector genome. HSV cDNA vectors expressed gene products at the transcriptional and protein level. Functional analysis of the cDNA inserts demonstrated that the library consisted of at least 15,000 unique genes. Some of these genes have been shown to participate in biological functions related to muscle differentiation, such as Id1 and Cand2.Finally, a conditional replication strategy was developed by which the recently produced HSV cDNA library could be used to identify genes that activate early myogenic promoters. This study exploited the characteristic of the JDββHE vector in which viral replication can be restored upon introduction of the essential viral gene ICP4. By placing this essential viral gene under the control of the early myogenic promoter Pax3, viral replication was found to be dependent on activation of this cellular promoter in a Pax3-expressing rhabdomyosarcoma cell line. In a similar manner, HSV cDNA library vectors that express a Pax3 activator will be able to replicate in the presence of the conditional ICP4 construct, thereby capturing the relevant gene product by the release of viral progeny into the supernatant. The future use of this strategy to identify gene products that specifically activate an early myogenic promoter would lead to the testing of these gene products for their ability to induce downstream myogenic gene expression and differentiation. In this manner, a better understanding of early muscle differentiation might be achieved and applied to the use of stem cells in transplantation studies using muscular dystrophy models.