Improved gene delivery formulations and expression systems for enhanced transfection efficiency

Effective gene therapy requires efficient delivery of DNA to transfected cells followed by high levels of gene expression. Our laboratory has developed two new delivery formulations LPDI containing the DNA condensing agent protamine sulfate and reconstitued chylomicrons containing a hydrophobic lipidDNA complex. LPDI can produce higher levels of gene expression than the corresponding liposome/DNA complexes alone both in vitro and in vivo. Reconstituted chylomicrons also induce high gene expression in the liver through intraportal injection. To increase overall gene expression of delivered DNA, an improved cytoplasmic expression system has been developed using a unique T7 RNA polymerase autogene pCMVIT7-T7pol. High levels of reporter gene expression were detected in the presence of this new autogene. Zntruduction: Many advancements have been made over the past several years in the fields of non-viral gene therapy to enhance gene delivery and expression to cells both in vitro and in vivo. This had mainly been due to the development of formulations such as cationic liposomes for delivery of transgenes to cells. Cationic liposomes are good delivery formulations due to such charateristics as nonimmunogenicity, minimal toxicity and simplicity of large scale production and use (ref. 1-3). However, these first generation cationic liposomes are serum sensitive, less efficient in cell transfections and are not targetable to become cell specific. To overcome these drawbacks, several new delivery formulations such as LPDI (ref. 4) and reconstituted chylomicrons have been developed in our laboratory. The current, improved version of LPDI consists of a cationic peptide, protamine sulfate, which condenses the DNA to form a DNNpolycation complex before the addition of cationic liposomes such as DOTAP to increase transfection efficiency both in vitro and in vivo (ref. 5-6). On the other hand, a hydrophobic lipid (TC-chol)DNA complex can be incorporated into reconstituted chylomicrons by emulsifying with a mixture of trigylcerides, phospholipid and cholesterol to reduce serum sensitivity (ref. 7). The latter formulation also could be targeted to specific tissues for gene delivery. Another major obstacle in gene therapy is inefficient expression of transfected DNA due to low nuclear transport where transcription machinery resides (ref. 8-9). Since the addition of a nuclear localization signal had limited success (ref. lo), a cytoplasmic expression system was developed to express DNA in the cytoplasm of transfected cells. ExogenousT7 RNA polymerase enzyme when delivered with a reporter gene driven by the T7 bacteriophage promoter can induce cytoplasmic reporter gene expression due to the lack of a nuclear localization signal in T7 RNA polymerase (ref. 11-13). This had led to the development of several T7 RNA polymerase autogenes made up of a T7 RNA polymerase gene driven by the T7 promoter to continuously supply transfected cells with high levels of endogenous T7 RNA polymerase (ref. 14-17). A novel T7 RNA polymerase autogene, pCMV/T7-T7pol, developed in our laboratory has been shown to induce higher, more sustained cytoplasmic reporter gene expression than previous autogenes or that found with a nuclear expression system driven by the cytomegalovirus promoter CMV and does not require exogenous T7 RNA polymerase enzyme.