Gene therapy vector optimization: Efficient expression of hrGFP using a novel short polyadenylation signal derived from the soluble isoform of Neuropilin–1

Abstract

Abstract: : Purpose: The use of large gene products such as soluble VEGF receptors for the treatment of ocular neovascular disease are limited by the carrying capacity of vectors. To increase the gene carrying capacity we aim to test the efficacy of a very short polyadenylation (pA) signal. The soluble form of Neuropilin–1 (sNRP–1) utilizes a splice donor site read through at exon 11 that introduces three intronic amino acids, a stop codon, and a 15 base pair polyA signal. Our goal was to compare the expression efficiency of hrGFP using this very short sNRP–1 pA signal with that of the much larger SV40 pA signal for future use in ocular gene therapy vectors. Methods: Three pUC18 vectors were constructed containing either CMV–hrGFP–SV40–pA, CMV–hrGFP–sNRP–1–pA, or CMV–hrGFP–no–pA inserts. 15µg of each vector was transfected into 293T cells and incubated for 30 hrs at 37°C 5% CO₂. Fluorescent cells were visualized and photographed using a fluorescent microscope. Total RNA was harvested from each group and RT–PCR was performed in order to generate cDNAs for sequence analysis. Sequencing was performed on a ABI 310 genetic analyzer to ascertain polyadenylation sites. Total protein was also isolated from each group and 50µg was loaded onto a SDS–PAGE gel. Semi–quantitative Western blots were performed using a hrGFP polyclonal antibody to compare expression levels. Results: All cells demonstrated robust green fluorescence with the exception of the no–pA control. RT–PCR demonstrated an approximate 200 bp difference between the hrGFP–sNRP–1(pA) cDNA and the hrGFP–SV40(pA) cDNA. Sequencing confirmed that pA had occurred where expected and that the short signal from sNRP–1 acted as a strong signal with no aberrant interference from possible cryptic polyadenylation sites. Western blot analysis revealed that the protein levels between the sNRP–1 and SV40 pA constructs were practically identical. The no–pA vector was incapable of producing message transcripts and thus yielded no protein. Conclusions: Many gene therapy vectors such as AAV are limited by their packaging constraints on insert gene size. Manipulating vectors to harbor larger genes is beneficial and allows for a broader theraputic range. Most vectors today contain large bulky polyadenylation signals that may not be necessary. We have demonstrated that the extremely small sNRP–1 pA signal is efficient in terminating mRNA capable of expressing protein in the pUC18 vector. Future aims are to elucidate mRNA stability and to clone this sequence into an AAV vector and test efficacy in vitro and in vivo.

Keywords: gene/expression • gene transfer/gene therapy • transcription