Lentiviral vectors containing the human immunodeficiency virus type-1 central polypurine tract can efficiently transduce nondividing hepatocytes and antigen-presenting cells in vivo

Abstract

High-titer self-inactivating human immunodeficiency virus type-1 (HIV-1)-based vectors expressing the green fluorescent protein reporter gene that contained the central polypurine and termination tract and the woodchuck hepatitis virus posttranscriptional regulatory element were constructed. Transduction efficiency and biodistribution were determined, following systemic administration of these improved lentiviral vectors. In adult severe combined immunodeficiency (SCID) mice, efficient stable gene transfer was achieved in the liver (8.0% ؎ 6.0%) and spleen (24% ؎ 3%). Most transduced hepatocytes and nonhepatocytes were nondividing, thereby obviating the need to induce liver cell proliferation. In vivo gene transfer with this improved lentiviral vector was relatively safe since liver enzyme concentration in the plasma was only moderately and transiently elevated. In addition, nondividing major histocompatibility complex class II-positive splenic antigen-presenting cells (APCs) were efficiently transduced in SCID and normal mice. Furthermore, B cells were efficiently transduced, whereas T cells were refractory to lentiviral transduction in vivo. However, in neonatal recipients, lentiviral transduction was more widespread and included not only hepatocytes and splenic APCs but also cardiomyocytes. The present study suggests potential uses of improved lentiviral vectors for gene therapy of genetic blood disorders resulting from serum protein deficiencies, such as hemophilia, and hepatic disease. However, the use of liver-specific promoters may be warranted to circumvent inadvertent transgene expression in APCs. In addition, these improved lentiviral vectors could potentially be useful for genetic vaccination and treatment of perinatal cardiac disorders. IntroductionHepatocytes are attractive target cells for gene therapy of genetic blood diseases, particularly hemophilia, and hepatic disorders (eg, familial hypercholesterolemia). Providing a long-term cure of these diseases requires not only that the hepatocytes be efficiently transduced, but also that the vectors remain transcriptionally active and give rise to therapeutic levels of functional protein. We have recently demonstrated that hemophilia A could be cured in newborn hemophilic factor VIII (FVIII)-deficient mice by intravenous injection of Moloney murine leukemia virus (MoMLV)-based oncoretroviral vectors encoding human FVIII. 1 In adult mice however, most hepatocytes are refractory to gene transfer performed with the use of MoMLV-based oncoretroviral vectors since disassembly of the nuclear membrane during cell division is required to allow the oncoretroviral preintegration complex to enter the nucleus. 2 Hence, it would be desirable to develop gene delivery strategies that could overcome the requirement for hepatic cell division and be used in adults.Macrophages and dendritic cells are also important target cells for gene therapy. By virtue of their potent antigen-presenting properties, they could be used to develop improved genetic vaccination strategies...

Liver gene therapy: advances and hurdles

“…[42][43][44] However, it turned out as well that immune response against the transgene product was elicited when large batches of lentivectors were administered to animals, thus precluding long-term expression of the transgene. 45 It was therefore required to use liver-specific promoters to circumvent induction of immune response after in vivo delivery of lentiviral vectors. 46 …”

Functional transfer of CD40L gene in human B-cell precursor ALL blasts by second-generation SIN lentivectors

“…15,19,[62][63][64] Moreover, our vectors contain two cis-acting elements (cPPT and Wpre) which should have optimized the expression of the transgene in the transduced B-ALL cells. [8][9][10][11]24,25 Their direct role, however, cannot be implied by these data, even if our previous data suggest an improved activity of the cPPT element in B-cell lines. 12 Finally, only few reports to date have addressed the issue of transducing leukemic BCP-ALL cells.…”

“…[4][5][6] Among the current technologies for gene transfer of leukemia cells, the latest generation of HIV-1-based lentiviral vectors represent the most efficient tool for their reported ability to transduce with stability, a wide variety of cell types in significant percentages. [7][8][9][10][11][12][13][14][15][16][17] B-cell precursor (BCP) blasts are very fragile and difficult to maintain alive in culture conditions. 18 The best available results so far for gene transduction have been obtained with second-generation lentiviral vectors monitoring the expression of the CD80 transgene up to 48 h. 5 More recently, second-generation lentiviral vectors have been further modified by deleting a portion of the U3 LTR region in the so-called self-inactivating vector (SIN) with improved safety, 8,9,15,19,20 and by the introduction of two cis-acting elements, one derived from the pol sequence, called central polypurine tract sequence (cPPT), 11,21,22 and the other, termed Wpre, obtained from the genome of the woodchuck hepatitis virus, both believed to increase transgene expression.…”

“…[7][8][9][10][11][12][13][14][15][16][17] B-cell precursor (BCP) blasts are very fragile and difficult to maintain alive in culture conditions. 18 The best available results so far for gene transduction have been obtained with second-generation lentiviral vectors monitoring the expression of the CD80 transgene up to 48 h. 5 More recently, second-generation lentiviral vectors have been further modified by deleting a portion of the U3 LTR region in the so-called self-inactivating vector (SIN) with improved safety, 8,9,15,19,20 and by the introduction of two cis-acting elements, one derived from the pol sequence, called central polypurine tract sequence (cPPT), 11,21,22 and the other, termed Wpre, obtained from the genome of the woodchuck hepatitis virus, both believed to increase transgene expression. [23][24][25] As previously demonstrated by our group 26 and others, [27][28][29][30][31][32] CD40 triggering by the CD40L molecule is capable of inducing a number of morphological and functional changes of BCP-ALL blasts, including the upregulation of the surface markers CD40, CD86, CD80, MHC class I and II, CD54 and CD58, and the secretion of chemoattractants MDC and TARC.…”

“…All vectors carry the GFP marker gene and were kindly provided by Dr Naldini, Torino, Italy, and are described elsewhere. [10][11][12][13][14][15][16][17][18][19][20][21][22][23] Viral vector particles were generated by cotransfection of the VSV containing plasmid pMD-G, the packaging construct pCMVdR8.74 and the indicated transfer constructs into 293T cells, followed by determination of concentration of particles by ultracentrifugation, as previously described. 65 Viral titers were evaluated by infecting a known number of REH cells with different volumes of viral vector preparations in a 200 ml volume.…”