Enhanced Gene Expression Conferred by Stepwise Modification of a Nonprimate Lentiviral Vector

Abstract

The practical application of gene transfer as a treatment for genetic diseases such as cystic fibrosis or hemophilia has been hindered, in part, by low efficiencies of vector delivery and transgene expression. We demonstrated that a feline immunodeficiency virus (FIV)-based lentiviral vector pseudotyped with the envelope glycoprotein from the baculovirus Autographa californica (GP64) efficiently transduces and persistently expresses a reporter gene in respiratory epithelium in the absence of agents that disrupt cellular tight junction integrity. GP64-pseudotyped FIV also efficiently transduced murine hepatocytes after tail vein delivery. To improve the FIV-based vector, we tested the contribution of a series of modifications to luciferase expression in vitro and in vivo. These modifications included the addition of spleen necrosis virus U5 (SNV U5) and mutation of the major splice donor and gag start codon located in the packaging region of the FIV transgene plasmid. After vector modification, we observed significantly enhanced expression of luciferase in respiratory epithelia after nasal application and in the liver after tail vein delivery. In addition, we observed significantly enhanced human factor VIII production after tail vein delivery. These sequential modifications provide an improved FIV lentivirus platform for gene therapy applications and may be applied to other retroviral vectors.

Lentivirus Vector Can Be Readministered to Nasal Epithelia without Blocking Immune Responses

“…The priming dose(s) of FIV carried a firefly Luc transgene without a promoter. No Luc expression was observed following the delivery of this vector either in vitro (data not shown) or in vivo (38). The test dose was the identical lentivirus vector, with an RSV promoter driving Luc.…”

“…The FIV vector system utilized in this study (21,43) expressed either mouse erythropoietin (mEPO), nucleus-targeted ␤-galactosidase (␤-Gal), or firefly Luc. Pseudotyped FIV vector particles were generated by transient transfection and concentrated 250-fold by centrifugation, and their titers were determined by real-time PCR as previously described (38). mEPO cDNA was obtained from Open Biosystems (clone identification no., 8734014; accession no., BC119265), the sequence was confirmed, and the cDNA was cloned into the FIV3.3RSV(Rous sarcoma virus) backbone (38).…”

Codon optimization of human factor VIII cDNAs leads to high-level expression

“…23,29 This construct has been incorporated into plasmid vectors where it has conferred therapeutic levels of expression. [30][31][32] A human BDD FVIII construct containing the first 226 amino acids of the B domain, including 6 N-linked asparagine glycosylation sites has been incorporated into many gene transfer vectors, including plasmid, 33 LVs, 34 and ␥-retroviral vectors 35 and is more efficiently secreted both in vitro 17,[35][36][37] and in vivo. 17,37 The goal of this study was to investigate the effect of FVIII expression cassettes with various B domain constructs.…”

“…Previous in vivo studies have demonstrated expression of therapeutic levels of FVIII in vivo in adult hemophilia A mice after systemic injection of vector, 34,[40][41][42] transplantation of transduced bone marrow cells, 33,35 transplantation of transduced bone marrow cells with targeted platelet-specific expression, 43,44 and transplantation of transduced blood outgrowth endothelial cells. 45 However, FVIII expression levels mediated from many of these approaches have been low (1%-5% normal human) and expression transient because of formation of neutralizing antibodies.…”

Comparison of Factor VIII Transgenes Bioengineered for Improved Expression in Gene Therapy of Hemophilia A

“…fVIII expression has been observed in mice after administration of transduced cells expressing BDD hfVIII (Dwarki et al, 1995;Moayeri et al, 2005), BDD hfVIII containing L303E=F309S (Moayeri et al, 2004;Kang et al, 2005), partial BDD hfVIII (Cerullo et al, 2007), and partial BDD hfVIII containing F309S (Sinn et al, 2007). We previously demonstrated that BDD porcine fVIII (pfVIII) is expressed at 10-to 14-fold greater levels than BDD hfVIII in vitro and that this increase in production is due to highly efficient secretion (Doering et al, 2002(Doering et al, , 2004.…”