Potential use of retrovirus vectors against HIV-1: Construction of ribozymes/DNA-enzymes to interfere with HIV-1 replication

Ph D Students
Samitabh Chakraborti

HIV-1 pathogenesis is multifactorial and involve complex interactions between host and viral genes. We wish to identify some of those genes and selectively interfere with their functions. This goal will be achieved by using nucleic acid based approaches to interfere with the replication of HIV-1. Our approach is significantly different than protein based approaches that usually elicit undesirable immune responses. We wish to exploit the use of novel interfering genes and retrovirus vectors to obtain stable gene expression in the host cells that are exploited by HIV, mainly T lymphocytes and macrophages.

HIV-1 uses the HIV-1 coreceptors, the chemokine receptors along with CD4 receptor protein to gain entry and initiate infection in host cells. This is accomplished by the close interaction of HIV-1 envelope protein, chemokine receptors and CD4 protein. This results in membrane fusion and entry of the virus. Several virus genes are expressed that contribute to the pathology of the disease. Structural and regulatory proteins are produced that are essential for the virus replication cycle. In particular, HIV-1 Tat and Rev play an important role in governing transcription and replication of HIV-1. Since T lymphocytes and macrophages are the two major host cells for HIV-1 replication, it would be ideal to introduce novel interfering genes into hematopoietic stem cells.

DNA-enzyme cleavage sites in HIV-1 gag and nef regions

Since the genome of HIV-1 accumulates mutations at a rapid pace, any antiviral approach must target many regions simultaneously. We had for the first time reported bioefficacy of a multitarget DNA-enzyme against the X gene of hepatitis B virus (Goila and Banerjea, 2001). This will prevent or delay the appearance of escape mutants. Therefore, additional target sites have to be identified that are cleaved more efficiently. We report the identification of two DNA-enzyme cleavage sites in the most conserved HIV-1 p24 gag and Nef regions. In order to test the efficacy of these DNA-enzymes, initially the HIV-1 Gag and Nef genes were cloned into pcDNA vector such that their expression was under the control of CMV/T7 promoter. These recombinant plasmids were used to generate in vitro transcripts using T7 RNA polymerase. DNA-enzymes along with their mutant controls (disabled) were synthesized chemically. All the DNA-enzymes showed sequence specific cleavage activities. Interestingly, one of the two DNA-enzymes from each category, showed significantly more cleavage in the presence of lower amounts of Mg++. This information is important because we and others have earlier shown that DNA-enzymes that show sequence specific cleavage activity in the physiological levels of Mg++ (intracellular concentrations being in the range of 1 - 2 mM), are generally more bio-efficacious. DNA-enzyme-1422 (against HIV-1 gag) and DNA-enzyme-8844 (against Nef) (the number denotes the nucleotide position in pNL4-3 HIV-1 DNA) were found to be more potent in inhibiting HIV-1 gene expression. When cotransfected with the Gag DNA-enzyme, about four fold reduction in HIV-1 p24 production was observed. It is important to mention that this difference is observed when only a single round of replication has taken place because HeLa cells lack the appropriate HIV-1 receptors (CCR5 or CXCR4) needed for the virus to spread, and therefore, this data is significant. On the contrary, Nef-DNA-enzymes failed to interfere with the replication of laboratory adapted HIV-1. This is expected because Nef is not known to exert any significant effect in the in vitro replication of HIV-1. Nef, however, is known to exert global changes, the most interesting being its ability to down modulate CD4. We tested our most effective Nef DNA-enzyme (8844) for the down modulation of CD4 in a cell fusion dependent reporter gene assay and found out that this DNA-enzyme was at least 4 fold more effective in down modulating CD4 protein than the Nef DNA-enzyme (Dz-8822) that cleaved the substrate RNA poorly in the presence of lower concentrations of MgCl2. In summary we have identified a potent DNA-enzyme against HIV-1 gag that could be exploited for achieving specific suppression of HIV-1 and potentially could be combined with other antiviral approaches. This gene is the most conserved among all the genetic subtypes including genetic subtype C that is the most prevalent strain of HIV-1 in India.

Potential use of small interfering RNAs, lentivirus vector and hematopoietic stem cells as an antiviral approach against HIV-1

Human CD34+ cells were isolated from human fetal liver using the CD34 islation kit and purified on a magnetic column. The purity was checked by staining with CD34-FITC antibody and FACS analysis. The purity was usually in the range of 90% or more. They were then transduced with lentivirus vector that contained the reporter gene GFP under CMV promoter and pol III promoter directed small interfering RNA (siRNA) against HIV-1 Rev. Macrophages were derived from the these cells by culturing them in the presence of GM-CSF and M-CSF. Thymocytes were derived after placing these transduced cells into the thy/liv grafts of SCID-hu mice. Both macrophges and T-cells showed impressive inhibition against R5 and X4-tropic isolates of HIV-1.

Publications

Original peer-reviewed articles

1.     Dash BC and Banerjea AC (2004) Sequence specific cleavage activities of DNA-enzymes targeted against HIV-1 Gag and Nef regions. Oligonucleotides 14:41-47.

2.     †Anderson J, Banerjea A and Akkina R (2003). Bispecific short hairpin siRNA constructs targeted to CD4, CXCR4, and CCR5 confer HIV-1 resistance. Oligonucleotides 13:303-312 (†on deputation/work done elsewhere).

3.     †Banerjea A, Li M, Bauer G, Remling L, Lee N-S, Rossi J and Akkina R (2003). Potent inhibition of HIV-1 by lentiviral vector transduced siRNAs in primary human T-lymphocytes differentiated in SCID-hu mice and CD34+ progenitor cell derived macrophages. Mol Ther 8:62-71 (†on deputation/work done elsewhere).

4.     *Chakraborti S and Banerjea AC (2003). Inhibition of HIV-1 gene expression by novel DNA enzymes targeted to cleave HIV-1 TAR RNA: Potential effectiveness against all HIV-1 isolates. Mol Ther 7:817-826 (*in press last year, since published).

5.     *Chakraborti S and Banerjea AC (2003) Identification of cleavage sites in the HIV-1 TAR RNA by 10-23 and 8-17 catalytic motif containing DNA enzymes. Biomacromolecules 4:568-571 (*in press last year, since published).

6.     †Anderson J, Banerjea A, Planelles V and Akkina R. (2003) Potent suppression of infection by a stem-loop structured anti-CXCR4 siRNA. AIDS Res Human Retroviruses 19:699-706 (†on deputation/work done elsewhere).

7.     †Akkina R, Banerjea A, Bai J, Anderson J, Ming-Jie Li and Rossi J (2003) siRNA, ribozymes, and RNA decoys in modeling stem cell based gene therapy for HIV/AIDS. Anticancer Res 23:1997-2006 (†on deputation/work done elsewhere).

Reviews/Proceedings

1.     Chakraborti S, Sriram B and Banerjea AC (2003) In vitro selected RNA-cleaving DNA-enzymes from combinatorial libraries. In: Methods in molecular biology-ribozymes and siRNA protocols. (Ed: Mouldy Sioud) Humana Press, Totowa, New Jersey, USA, 279-290.