ELSEVIER Advanced Drug Delivery Reviews 17 (1995) 369-371

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Inhibition of HIV-l with intracellular immunization with RNA decoys

Clay Smith*, Seong-Wook Lee, Eli Gilboa Duke University, Durham, NC 27706, USA

Accepted June 1995

Contents

1. RNA decoy inhibition of HIV-1 ..................................................... 2. Development and analysis of potent minimal RRE decoys ............................. 3. Gene transfer of RNA decoys into CD4+ T-cells and hematopoietic stem cells. ......... 4. Adeno-associated viral vectors expressing RNA decoys ............................... 5. Futuredirections ..................................................................

References ...........................................................................

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1. RNA decoy inhibition of HIV-l

A number of gene therapy strategies for inhib- iting HIV replication have been developed and tested in-vitro [1,2]. One gene therapy approach to HIV infection, termed intracellular immuniza- tion, involves genetically modifying HIV infec- table cells with vectors which stably expresses a gene product which inhibits HIV gene expression and replication [3]. One form of intracellular immunization is termed the RNA decoy strategy. RNA decoys exploit unique regulatory circuits operating in the course of HIV replication. Tat and rev are two key regulatory gene products which activate HIV gene expression by binding to specific regions of the nascent viral RNA, termed the trans-activation response (TAR) ele- ment and the rev response element (RRE), respectively [4]. The RNA decoy strategy is

* Corresponding author.

based on expressing short RNA transcripts cor- responding to TAR or RRE which will compete for the binding of tat or rev to their physiological targets on the viral RNA [.5]. Inhibition of tat blocks virtually all HIV transcription while inhi- bition of rev blocks expression of the late HIV genes including gag, pol, and env which are important in viral assembly. Unlike antisense RNA or ribozymes, RNA decoys may be less likely to be affected by HIVvariability. RRE and TAR are highly conserved sequences and muta- tions in tat or rev which affect RNA decoy binding will also affect binding to the authentic HIV RNA sequences. A specific concern associ- ated with the use of TAR or RRE decoys however, is that cellular factors may bind to these molecules resulting in cellular dysfunction. While we and others have previously shown that cells expressing TAR-containing transcripts confer varying degrees of resistance to HIV replication, attempts to dissociate TAR decoy function from cellular factor binding by intro-

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ducing mutations in the loop-sequence of TAR have been unsuccessful [6].

2. Development and analysis of potent minimal RRE decoys

In contrast to TAR decoys, it may be possible to define a RRE decoy which does not bind cellular factors. In early studies, we and others found that expression of a 45 nt transcript encompassing RRE stem-loops IIA and IIB significantly inhibited HIV-l replication in im- mortalized T-cell lines [7]. Subsequently, map- ping and in-vitro binding studies determined that the primary binding site of rev to RRE is a 13 nt purine rich “bubble” at the base of the stem loop II B [8]. As far as we know, no cellular factors have been identified which bind to this region. Based on this observation we developed a series of RRE decoys containing the 13 nt long minimal rev binding domain [9]. Expression of these minimal RRE decoys was driven by tRNA based expression cassettes which were designed to yield higher intracellular concentrations of RRE de- coys than previous expression systems [.5]. After screening a number of tRNA-minimal RRE decoy constructs, we identified two, termed RRE4 and RRES, which were more potent inhibitors of acute HIV infection in isolated cell lines than previously described TAR or RRE decoys. In addition, rev dependent CAT expres- sion was decreased in CEM cells by minimal RRE decoys confirming that inhibition of HIV was specifically mediated by blockade of rev function. In order to evaluate the efficacy of RRE decoys in a more realistic fashion than comparison of individual clonal cell lines, poly- clonal populations of transduced CEM cells were infected with HIV Using a flow cytometric meth- od for quantitating intracellular p24 expression, RRE5 but not RRE4, was found to be capable of significantly inhibiting intracellular expression of ~24. A possible explanation for the greater potency of RRES compared to RRE4 was in- ferred from computerized predictions of RNA decoy secondary structures. These analyses indi- cated that the minimal rev binding domain of

RRES is thermodynamically more stable than that of RRE4 or other minimal RRE decoys.

3. Gene transfer of RNA decoys into CD4+ T-cells and hematopoietic stem cells

Gene therapy with minimal RRE decoys will involve transplantation of vector transduced CD4+ T-cells or totipotent hematopoietic stem cells. In order to efficiently quantitate gene transfer into primary cells, we developed a series of vectors that express a truncated version of the human nerve growth factor receptor (d-NGFR) under the transcriptional control of a variety of promoters. Transduced cells can be readily iden- tified and isolated by immunofluorescence stain- ing and fluorescence activated cell sorting (FACS). Using this system, we developed pro- cedures for achieving retroviral vector mediated gene transfer into 20-30% of peripheral blood CD4+ T-cells using clinically applicable superna- tant based gene transfer procedures. Co-culture retroviral mediated gene transfer conditions were optimized so that 40-50% of peripheral blood CD4+ T-cells could be transduced. Using these transduction procedures, we introduced minimal RRE decoy encoding retroviral vectors into CD4+ T-cells, sorted them based on d- NGFR positivity, and challenged the sorted cells with primary isolates of HIV-l. Preliminary evi- dence indicates that minimal RRE decoys protected peripheral blood CD4+ T-cells from infection with several different primary isolates of HIV-l.

4. Adeno-associated viral vectors expressing RNA decoys

In addition to retroviral vectors, we have begun to explore the utility of adeno-associated viral vectors for gene transfer into hematopoietic cells. Adeno-associated viral (AAV) vectors en- coding both TAR and minimal RRE decoys were stably transduced into the human T-cell line CEM-SS. These AAV vectors expressed TAR and RRE RNA decoys from tRNA ~01111 pro- moters at high levels without any apparent de-

C. Smirh el al. I Advanced Drug Delivery Reviews 17 (199-i) 369--771

leterious effects on cell growth or expression of CD4. DNA blot analysis indicated that RNA decoy encoding vectors were not rearranged and were integrated into the genomic DNA of select- ed cell lines. Vector DNA with the appropriate TAR and RRE sequences was isolated from transduced cell lines after prolonged growth in culture further confirming that the vector DNA was stable through multiple cell cycles. Cell lines expressing TAR and RRE decoys inhibited HIV gene expression and replication by 70-99% as determined by measurement of intracellular and extracellular HIV p24 expression. Adeno-associ- ated vectors encoding RNA decoys appear to be promising vectors for gene therapy of HIV infection and may be particularly useful in studies involving hematopoietic stem cells.

decoys. In order to reduce the viral burden that transplanted gene modified cells will have to cope with, we are exploring novel approaches to reducing the HIV viral burden including myelo- ablation and administration of sequential combi- nations of anti-retroviral agents. In the long run, it is likely that intracellular immunization will be only one facet of a multi-pronged approach to reducing viral burden and reconstituting the immune system in persons with HIV infection.

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Despite the promising results achieved in the in-vitro studies with the minimal RRE decoys, a number of issues will need to be addressed in order for this strategy to be therapeutically beneficial. It now clear that the HIV reservoir is extremely high in infected individuals [lo]. Even the most potent antiretroviral agents currently available do not decrease the viral burden by more than 2-3 orders of magnitude due to the rapid emergence of drug resistant quasispecies. Consequently, any transplanted gene modified cells will be faced with a large, vigorous, and dynamic HIV infection. It is unlikely that gene modified cells will have much of an impact on the spread of virus in this setting so that our current emphasis is on developing intracellular immuni- zation strategies which protect immunologically competent cells in the face of a robust HIV infection as opposed to merely inhibiting viral replication. In order to improve the potency of minimal RRE decoys, we are developing combi- nation vectors which encode minimal RRE de- coys along with ribozymes and improved TAR

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