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A Simple Human Immunodeficiency Virus Vector System for Selective Infection of CD4+ Cells and Inducible Expression of Foreign Genes

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A Simple Human Immunodeficiency Virus Vector System for Selective Infection of CD4+ Cells and Inducible Expression of Foreign Genes EXPERIMENTAL and MOLECULAR MEDICINE, Vol. 29, No 2, 103-110, June 1997 A simple human immunodeficiency virus vector system for selective infection of CD4+ cells and inducible expression of foreign genes Yeon-Soo Kim1 These results provide a rationale for the use of HIV- 1 retroviral vector system in the design of gene 1 Institute for Cancer Research, College of Medicine, therapy of HIV infection. Yonsei University, Seoul 120-752, Korea Ke y w o r d s : HIV-1, retroviral vector, AIDS, CD4, Tat, gene Accepted 10 May 1997 therapy Introduction Abbreviations: ADA, adenosine deaminase; AIDS, acquired immunodeficiency The acquired immunodeficiency sysdrome (AIDS), caused syndrome; CAT, chloramphenicol acetyltransferase; HIV, human immunodeficiency by human immunodeficiency virus (HIV), is a chronic β virus; HSV, herpes simplex virus; X-gal, 5-bromo-4-chloro-3-indolyl -D-galactoside disease primarily characterized by a decrease in CD4+ T lymphocytes. Since the discovery of HIV in 1983, significant progress has been made toward the discovery and development of anti-HIV drugs. In addition, consi- derable progress has been made in the development of treatments for various opportunistic infections. Although Abstract these treatments can delay desease progression, side effects associated with their use and the development The alteration of T lymphocyte functions as a conse- of viral resistance to these drugs may inevitably lead to quence of human immunodeficiency virus (HIV) treatment failure. For these reasons, alternate therapies infection is a potential target for the genetic treatment for HIV infection are needed. of the acquired immunodeficiency syndrome (AIDS). Recent advances in the application of gene therapy to One approach to the gene therapy for AIDS is to block several diseases (Spangrude et al., 1988; Drumm et al., the replication of HIV–1. Tat–dependent expression 1990; Nabel et al., 1990; Rosenberg et al., 1990) have of forein gene and selective infection of CD4+ cells stimulated interest in the therapeutic potential of nucleic by retroviral vector might be useful for abrogating acids expressed endogenously by cells. The genetic the production of HIV–1 from cells. As part of studies correction of T lymphocytes in the first gene therapy to examine the feasibility of this concept, I constructed experiment with adenosine deaminase (ADA) deficiency tat+ and tat - HIV–1 proviral vectors that express all has shown that “sick” T lymphocytes can be made HIV–1 genes except for env and/or tat gene. When “healthy” (Blease and Culver 1992). Since the immuno- tat+ or tat- HIV-1 particles were used for infection of deficiency results when the HIV makes T lymphocytes HeLa T4 cells containing the endogenous - g a l a c - sick, genetically altering HIV-infected T lymphocytes tosidase (lacZ) gene under the control of the HIV-1 may be a useful therapy. Although several approaches promoter and transactivation response element have succeeded in producing anti-HIV activity in cultured sequences, only the tat+ HIV-1 particles trans- cells by transfection with vectors containing diphtheria activated the lacZ gene expression. This activation toxin A gene, HIV-specific ribozyme, herpes simplex of lacZ expression following HIV infection of Tat- virustype 1 thymidine kinase (HSV1-TK), interferon gene, cells that stably contained but did not express the or antisense gene (Harrison et al., 1991a; Caruso and lacZ construct was determined to be an efficient Klatzmann 1992; Rossi et al., 1992; Lisziewicz et al., process. I also constructed simple HIV-1 vectors that 1995; Su et al., 1995), the difficulty in obtaining sufficient express the lacZ gene in a Tat-dependent manner or expression of the desired gene in a sufficient number of the hygromycin B phosphostransferase gene (Hygr) cells in vivo has remained an obstacle. under the control of the SV40 early promoter. The Murine retroviral vectors also could be used for Tat-dependent vector conferring the lacZ+ ph e n o t y p e constitutive expression of potentially therapeutic genes was assayed by β-gal staining after infection of Tat+ in cells of HIV-infected individuals. Because the receptor or Tat- cells. The activation of lacZ expression was of the commonly used amphotropic murine leukemia observed only in tat+ cells. Another simple HIV-1 virus is present on a variety of cell types, these vectors vector containing the Hygr gene was used for retroviral provide a potential way for introducing genes into early production from HeLa cells expressing the HIV-1 lineage hematopoietic cells as well as mature T lympho- env gene and infection of CD4+ or CD4- cells, but cytes. However, because of difficulties encountered both H y gr colony was observed only from CD4+ c e l l s . in isolating and effectively infecting hematopoietic stem 104 Exp. Mol. Med. cells and because the primary target for HIV infection BglII site(+20) in the R region of the HIV-1 5’LTR to an appears to be helper T cells, it is possible that a vector artificial S alI site (Sodroski and others 1985) located capable of specifically infecting mature T lymphocytes upstream the initiation codon of rev. TAR-luc and dTAR- might be useful. One method for targeting potentially luc contain the XhoI-BamHI and BglII-BamHI luciferase therapeutic genes to cells suscetible to HIV infection gene fragment of pGL2-Basic Vector (Promega, Madison, (CD4+ cells) would be to use an HIV vector. As known WI) at the S alI -B amHI and B g lI I -B a m HI sites in the well, retrovirus entry into cells is dependent on recognition pTat120B, respectively. of the cellular receptor by the viral envelope glycoprotein. A env-HIV-1 proviral clone YK161 was made in two By using an HIV vector, entry of the foreign gene into steps as follows: The infectious proviral clone pNL4-3 cells would occur only if the cells expressed the HIV (Adachi et al., 1986) was digested with StuI and NheI, receptor, the CD4 molecule (Maddon et al., 1986). the digested DNA was treated with Klenow fragment, In addition, since a number of the genes that might and the ends were religated (regenerating the NheI site) be used to interfere with HIV may be toxic to the cell, to produce clone pGB107. pGB107 thus contains a 42 6 - constitutive expression of these genes would be harmful. bp inframe deletion in the e n v gene and can produce Another method for expressing genes in specific cells is truncated gp120 and intact gp41. pGB107 was digested to use unique HIV c i s- and t r a n s-acting elements that with NheI (regenerated enzyme site), the digested DNA control gene expression from the HIV LTR. Efficient was treated with Klenow fragment, and the ends were gene expression from the HIV LTR is dependent on the religated, resulting in plasmid YK161. YK152-10 was viral protein Tat (Sodroski et al., 1985; Cullen, 1986). Tat constructed by introducing a stop codon in 11st amino is an RNA-binding protein; its target sequence, the Tat acid codons of t a t gene in the HIV-1 proviral clone activation response element (TAR), is a 59-nucleotide YK161. sequence present in the newly transcribed R region of the To construct YK1001, the HIV-1 proviral clone pNL4- 5' LTR (Rosen et al., 1985). Since Tat is normally found 3 (Adachi et al., 1986) was digested with SpeI and Xh oI only in cells infected with HIV, this type of vector might and treated with the Klenow fragment and the ends were provide a means by which foreign genes that interfere ligated to produce the intermediate clone YK1000. To with HIV replication could be expressed specifically in introduce the β-galactosidase gene (lacZ) into YK1000, HIV-infected cells. Because expression of the foreign the plasmid was digested with BssH II and treated with gene is dependent on Tat, this vector system provides a the Klenow fragment. The lacZ gene was obtained from way in which to introduce genes into cells where they LN βZ (Kim, T., unpublished data) by digestion the plasmid will be maintained but not expressed until induced. This with Hi n dIII and Sa lI, treating it with the Klenow fragment, strategy forms the basis for an “inducible” gene therapy and isolating the appropriate DNA fragment. This fragment against HIV. This control of gene expression might was then blunt end ligated into the B s sH I I - d i g e s t e d decrease potential side effects associated with expression YK1000 to produce the vector YK1001. To construct of the foreign gene in uninfected cells. YK1002, pNL4-3 was digested with N s iI and the ends In the study presented here, simple HIV vectors that were ligated to produce an intermediate clone. To introduce contain no intact viral open reading frames and that the hygromycin B phosphotransferase gene (Hygr) into expresses foreign report genes from the HIV LTR or the intermediate clone, the plasmid was digested with heterologous internal promoter were constructed. These Nh e I. The Hygr gene promoted from SV40 early promoter vectors were used as markers to characterize the Tat- was obtained from GB108 (Buchschacher and Panganiban inducible and CD4+-specific vector system and its 1992) by digesting the plasmid with NheI and isolating usefulness for delivery of gene expressed from the HIV the appropriate DNA fragment. This fragment was then LTR. ligated into the N heI-digested intermediate vector to produce the YK1002. The replication-defective HIV-1 helper construct MSM∆env50 (McBride, S. and Panga- Materials and Methods niban, A. T., unpublished data) contains a deletion in the env ORF but is capable of expressing other proteins Plasmid construction necessary for viral propagation.
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