Applying the PDR principle to AIDS

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  • J. theor. Biol. (1988) 130, 469-480

    Applying the PDR Principle to AIDS

    JOHN C. SANFORD

    Department of Horticultural Sciences, Cornell University, Hedrick Hall, Geneva, NY 14456, U.S.A.

    (Received 2 February 1987, and in revised form 9 October t987)

    The principle of pathogen-derived resistance (the PDR principle) has been put forward as a broadly-applicable conceptual tool for use in designing genes which will confer resistance to pathogens. This paper reveals an example of how the PDR principle may be applied in the field of human medicine. Specifically it is shown how the PDR principle can be employed in designing a series of genes which should be capable of protecting human blood cells from the retrovirus causing the AIDS disease. Prospects are discussed for using such genes in gene therapy treatment of people infected with this virus.

    Introduction

    The pathogen-derived resistance (PDR) principle (Sanford & Johnston, 1985), in its simplest form, can be stated as follows:

    "Nucleotide sequences derived from a pathogen can be used in the genetic modification of its host, such that the host becomes resistant to that pathogen."

    The concept of pathogen-derived resistance adds a new dimension to our under- standing of host-pathogen relations, and promises to be a useful tool in designing genes conferring resistance to pathogens. The genetic engineering of pathogen- derived resistance has already been demonstrated in several model viral systems. Sanford & Johnston (1985), analyzed the QB bacteriophage and predicted that the QB bacteriophage host (E. coil) could be made resistant to QB infection by at least three different PDR mechanisms. It was predicted that QB resistance could be engineered by: 1) cloning and expressing a QB regulatory protein (the coat protein) into the host; 2) subcloning a portion of the QB replicase gene, such that the host would express a protein fragment consisting of the RNA binding domain of the replicase; 3) expressing viral antisense RNA in the host (mRNA complementary to part of the viral genome). Two of these PDR mechanisms have already been proven valid. QB coat protein conditions high levels of resistance to QB infection when expressed in the host, with plaque number being reduced roughly 1000-fold (Grumet et aL, 1987a, b). A single antisense RNA construct had measurable but modest anti-viral effects in the QB system, seen mostly as reduced plaque size (Johnston & San ford, unpublished data). In the closely related SP bacteriophage system, Coleman et al. (1985), have demonstrated that certain antisense RNAs can have very significant anti-viral effects, as seen by reduced plaque number and plaque size. In addition, Abel et al. (1986) have shown that viral coat protein confers a degree of resistance to tobacco mosaic virus, when expressed in tobacco. Other recent examples of

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  • 470 J.C. SANFORD

    parasite-derived resistance include resistance to cucumber mosaic virus deriving from viral satellite eDNA (Baulcombe et al. 1986), and resistance to alfalfa mosaic virus deriving from the viral coat gene (Loesch-Fries et al. 1987). Taken collectively, such findings indicate that parasite-derived resistance (under the right circumstances) can be quite readily achieved, and can be realized through a variety of molecular mechanisms.

    Applying PDR to Medicine

    The general PDR concept was conceived and developed primarily for use in the field of agriculture, where genetic resistance is of great importance. In the field of human medicine, altering the genotype of the host has never been a credible method of fighting infectious disease. However, it is now believed that human somatic gene therapy will be possible in the relatively near future (Andersen, 1984), creating the prospect that PDR might be applied within the field of medicine. Two practical considerations would appear to limit PDR applications in medicine. First, the drastic and permanent nature of somatic gene therapy would seldom be justified. Where the human immune system failed to fight off infection, more orthodox medical treatments such as vaccination or chemotherapy would normally be practiced. Second, genetic protection would not be feasible in most cases, since most human tissues are not presently amenable to being genetically engineered. Emerging gene therapy techniques are only effective for the genetic modification of bone marrow and blood cells. Because of this limitation, the projected use of somatic gene therapy has generally been assumed to be limited to the correction of rare hereditary gene defects, where such defects center in bone marrow or blood cells (Andersen, 1984).

    Despite these limitations, there are certain persistent and life-threatening pathogens of the blood for which conventional defenses appear inadequate and where use of the PDR principle might be both feasible and justified. Most noteworthy of these diseases is the acquired immune deficiency syndrome (AIDS) (Wong-Staal & Gallo, 1985; Weiss et aL, 1986). This disease is caused by a retrovirus which has been called HTLV III, LAV, ARV, or most recently, HIV (Coffin et al., 1986).

    The development of an unorthodox and "permanent" treatment for the AIDS disease would appear highly justifiable, given the scope of the epidemic (Curran et al., 1985), the lethal nature of the disease, the permanent nature of the provirus (Weiss, 1985, Folks et al., 1986), and the questionable efficacy of more orthodox treatments. The development of anti-HIV genes and an effective gene therapy treatment for AIDS would appear to be feasible given the proven effectiveness of the PDR principle (Sanford & Johnston, 1985; Coleman et al., 1985; Abel et al. 1986; Baulcombe et al., 1986, Grumet et al., 1987a, b; Loesch-Fries et al., 1987), the well-characterized genetics of the HIV virus (Wong-Staal & Gallo, 1985), the apparent localization of the virus in cell types that can be genetically altered (Wong-Staal & Gallo, 1985; Barnes, 1986; Gartner et al., 1986), and the theoretical stability of PDR in the face of new viral strains (Sanford & Johnston, 1985). Perhaps the greatest uncertainty regarding the potential efficacy of a gene therapy cure for AIDS involves the localization of HIV within cells that can be genetically altered.

  • PDR PR INCIPLE AND A IDS 471

    While it is clear that HIV infects T4 lymphocytes and other cells in the bloodstream and macrophages in the brain, it is possible that HIV may also infect brain neuron cells (Navia et al., 1986a,b; Johnson & McArthur, 1986). Such cells are not presently amenable to genetic modification, therefore they are not themselves subject to gene therapy. However, by curing blood cell infection, possible brain cell infection can be prevented where it has not yet occurred. If brains cells are actually infected, it might be hoped that the curing of blood cell infection and restoration of the immune system might reverse such brain infection, or at least stop its spread.

    Numerous viral target sites and diverse mechanisms for implementing the PDR principle become evident upon analyzing the genomic structure and the reproductive mechanisms of HIV. The basic viral functions to be blocked by anti-HIV genes are reverse transcription, proviral transcription and translation, and assembly and export of viral particles. A series of anti-HIV mechanisms blocking these processes will be discussed in the following sections. In the interest of simplicity, anti-viral gene products will be emphasized, rather than genes. (The basic structure at the actual genes can largely be deduced from their products.) In all cases, the virus' own sequences and machinery will be turned against itself, which is the essence of the PDR principle.

    Gene Products Designed to Block Reverse Transcription

    All retroviruses, including HIV, must undergo a complex process of reverse transcription within a host cell before that cell can successfully be infected. Therefore, blocking reverse transcription is a logical first line of defense against HIV and other retroviruses.

    BLOCKING THE FOUR HYBRID IZAT ION STEPS OF REVERSE TRANSCRIPT ION

    There are four critical steps in the reverse transcription of HIV, which require nucleic acid hybridization (Gilboa et aL, 1979). If any of these hybridizations are blocked, the infection process will be aborted. Each of these hybridization steps can theoretically be blocked by pre-hybridization of the critical sites in the viral genome to complementary molecules coded for by genetically-engineered "resist- ance" genes in the host cell. The general process of using DNA or RNA which will hybridize and block viral sequences has been termed "hybridization interference" (Zamecnik et aL, 1986). In the present case hybridization interference will be described as a means for blocking reverse transcription, but it can also be used to block mRNA translation and viral packaging. Some of these mechanisms have been discussed previously by Zamecnik et al. (Stephenson & Zamecnik, 1978; Zamecnik & Stephenson, 1978; Zamecnik et al., 1986), but not from the perspective of identifying coding sequences for use in gene therapy.

    BLOCKING PRIMER BINDING

    A retrovirus exists as a single-stranded RNA at the time of infection. The first hybridization step which is critical for reverse transcription involves the annealing

  • 472 J.C. SANFORD

    of a primer complex (lysine tRNA from the host, complexed to the viral reverse transcriptase enzyme) to the primer binding site (PBS) in the central portion of the viral genome. Host-encoded RNA complementary to this general region and extend- ing beyond it (anti-PBS) should compete with the primer complex for this site, and once annealed to the site, should effectively block the initiation of reverse transcrip- tion from its proper starting point. The effectiveness of this block will depend on the relative numbers of primer complexes vs. anti-PBS molecules and their access to the site.

    BLOCKING THE "F IRST JUMP" OF REVERSE TRANSCRIPT ION

    The second nucleic acid hybridization required for successful HIV reverse tran- scription involves the "first jump" of the reverse transcriptase to a new template. Reverse transcription proceeds toward the 5' end of the virus, continuously degrading the RNA template as the cDNA is synthesized. When the enzyme reaches the 5' end of the RNA template, it apparently stops and backs up, leaving the single- stranded 3' end of the cDNA exposed. Because there is a redundant 98 base pair sequence at both ends of the RNA genome, which is called the R-sequence, the first 98 base pairs of the cDNA is complementary to the last 98 base pairs of the RNA genome. This allows nucleic acid hybridization, and circularization of the RNA-DNA complex. This is necessary for reverse transcription to continue.

    RNA which is either complementary to the R-region (thereby annealing to the viral RNA), or equivalent to the R-region (thereby annealing to the cDNA), should block circularization of the complex, and the infection is aborted.

    BLOCKING IN IT IAT ION OF PLUS-STRAND DNA SYNTHESIS

    The third nucleic acid hybridization required for successful reverse transcription involves initiation of plus-strand DNA synthesis. As reverse transcription proceeds past the R-region where the "first jump" has occurred, a region known as the "polypurine region" is transcribed into DNA. This DNA region is where plus-strand synthesis is initiated, beginning with the annealing of an RNA primer at this site. The reverse transcriptase enzyme recognizes this RNA/DNA complex, and causes a nick at a specific site in the primer, from where DNA synthesis begins (Resnik et aL, 1984; Smith et aL, 1984). Plus-strand DNA synthesis then proceeds in the opposite direction as reverse transcription, back trhough the R-region, making a complement of the original reverse transcription RNA primer. The hybridization of a primer to the polypurine region, and the subsequent initiation of DNA synthesis at the correct point, can theoretically be blocked by complementary RNA molecules spanning and extending beyond this region, but lacking perfect homology to the natural primer at the key nick recognition site. Even if such molecles act non- specifically as initiators of plus-strand synthesis, the DNA terminus will be shifted, eliminating the terminal repeats of the double-stranded DNA virus, which are essential for viral insertion into the host chromosome (Panganiban & Temen, 1983; Panganiban, 1985).

  • PDR PR INCIPLE AND A IDS 473

    BLOCKING THE " 'SECOND JUMP" OF REVERSE TRANSCRIPT ION

    The fourth nucleic acid hybridization required for successful HIV reverse tran- scription involves the "'second jump" of the reverse transcriptase to a new template. After the "first jump" has occurred, reverse transcription continues along the new template (from the 3' end of the viral genome), back towards the PBS site where the process first began. The RNA primer is displaced from the PBS region of the viral genome either by plus-strand synthesis or by reverse transcription, resulting in a break in the circular complex. Reverse transcription proceeds through the PBS region and at the end of the molecule it stops and backs up for a second time, leaving a single-stranded DNA complement of the PBS region exposed. Plus-strand DNA synthesis has produced a terminus complementary to this. Annealing of these ends results in re-circularization and allows for the continuation of the synthesis of viral DNA in both directions. This fourth nucleic acid hybridization can be blocked either by RNA complementary to the PBS region, or RNA equivalent to it. An RNA molecule complementary to the PBS region, as already discussed, also has the potential to interfere with the initiation of the reverse transcription process.

    BLOCKING REVERSE TRANSCRIPTION WITH FALSE PRIMERS AND FALSE TEMPLATES

    The reverse transcription process can in theory be sidetracked through the use of false priming at incorrect sites, and through the use of false templates which should simultaneously "disarm" primer complexes while producing anti-viral cDNA.

    Reverse transcription must begin in a specific place within the viral genome in order for the resulting cDNA to be infectious and have the correct termini. The initiation point for this process is controlled by the primer complex. This complex consists of a reverse transcriptase molecule bound to a tRNA molecule. The tRNA molecule, which is supplied by the host cell, has its 3' tail fully exposed when it is complexed to the enzyme, such that this tail can hybridize to the primer binding site (PBS) of the virus. In the case of HIV, it is lysine tRNA which is involved in the primer complex. It should be possible to create false primers by taking the natural lysine tRNA sequence and modifying it so that the "tail" (the last 18 base pairs at the 3' end) are complementary to some part of the HIV genome different...