Gene Therapy Strategies & HIV-1 Life-Cycle | Human Genetics

Let us make an in-depth study of the gene therapy strategies designed to interfere with the HIV-1 life-cycle.

Basically Inhibition has been Envisaged at Three Major Levels:

(i) Blocking HIV-1 infection:

HIV-1 normal­ly infects T lymphocytes by binding of the viral gp120 envelope protein to the CD4 receptor on the cell membrane. Transfer of a gene encoding a soluble form of the CD4 antigen (sCD4) into T lymphocytes or hemopoietic cells and subsequent expression will result in circulating sCD4.

If the levels of circulating sCD4 are suffi­ciently high, binding of sCD4 to the gp 120 protein of HIV-1 viruses could be imagined to inhibit infection of T lympho­cytes without compromising T lympho­cyte function.

(ii) Inhibition at the RNA level:

The produc­tion of HIV-1 RNA can be selectively inhib­ited by standard antisense/ribozyme approaches, and also by the use of RNA decoys. The latter strategy exploits unique regulatory circuits which operate during HIV replication. Two key HIV regulatory gene products are tat and rev which bind to specific regions of the nascent viral RNA, known as TAR and RRE, respectively (Fig. 23.15). Artificial expression of short RNA sequences corresponding to TAR or RRE will generate a source of decoy sequences which can compete for binding of tat and rev, and possibly thereby inhibit binding of these proteins to their physiological target sequences.

(iii) Inhibition at the protein level:

There are numerous different strategies. One strategy involves designing intracellular antibodies, against HIV-1 proteins, such as the envelope proteins. Another involves introducing genes that encode dominant- negative mutant HIV proteins which can bind to and inactivate HIV proteins (trans- dominant proteins). For example, trans- dominant mutant forms of the gag proteins have been shown to be effective in limiting HIV-1 replication, possibly by interfering with multimerization and assembly of the viral core.

The Ethics of Human Gene Therapy:

All current gene therapy trials involve treatment for somatic tissues (somatic gene therapy). Somatic gene therapy, in principle, has not raised many ethical concerns other than its possible application in enhancement genetic engineering (any treatment involving- genetic modification of an individual’s cells in order to enhance some trait, such as height, without attempting to treat disease). Clearly, every effort must be made to ensure the safety of the patients, especially since the technologies being used for somatic gene therapy are far from perfect.

However, confining the treatment to somatic cells means that the consequences of the treat­ment are restricted to the individual patient who has consented to this procedure. Many, therefore, view the ethics of somatic gene therapy to be at least as acceptable as, say, organ transplantation, and feel that ethical approval is appropriate for carefully assessed proposals.

Patients who are selected for such treatments have severely debilitating, and often life-threatening, disease for which no effective conventional therapy is available. As a result, despite the obvious imperfections of the technology, it may even be considered to b” unethical to refuse such treatment.

Germline gene therapy, involving the genetic modification of germ line cells (e.g. in the early zygote), is considered to be entirely different. It has been successfully practiced on animals (e.g. to correct β-thalassemia in mice). However, thus far, it has not been sanctioned for the treatment of human disorders, and approval is unlikely to be given in the near future, if ever.

Human germ line gene therapy has not been practiced because of ethical con­cerns and limitations of the technology for germ line manipulation:

The lack of enthusiasm for the practice of germ line gene therapy can be ascribed to three major reasons:

The Imperfect Technology for Genetic Modification of the Germline:

Germ line gene therapy requires modification of the genetic material of chromosomes (most easily by chromosomal integration of an introduced gene). However, vector systems for accomplishing this do not allow accurate con­trol over the integration site or event.

In somatic gene therapy, the only major concern about lack of control over the fate of the trans­ferred genes is the prospect that one or more cells undergoes neoplastic transformation.

However, in germ line gene therapy, genetic modification has implications not just for a sin­gle cell. In addition to cancer, accidental inser­tion of a recombinant retrovirus within an important gene could result in a novel inherit­ed pathogenic mutation, for example.

The Questionable Ethics of Germline Modification:

Genetic modification of human germ line cells may have consequences not just for the individual whose cells were originally altered, but also for all individuals who inherit the genetic modification in subsequent genera­tions. Germ line gene therapy would inevitably mean denial of the rights of these individuals to any choice about whether their genetic constitution should have been modified in the first place.

Some, however, have considered that the technology of germ line modification will inevitably improve in the future to an acceptably high level and, provided there are adequate regula­tions and safeguards, there should then be no ethical objections.

Others perceive that, in addition to the question of the rights of indi­viduals in the future, this technology will inevitably lead to a slippery slope towards genetic enhancement. This would entail a program of positive eugenics, whereby planned genetic modification of the germ line could involve artificial selection for genes that are thought to confer advantageous traits. Inevitably, even if this were judged to be acceptable in principle, the question is raised of who decides what traits are advantageous.

The horrifying nature of post negative eugen­ics programs (most recently in Nazi Germany, and in many states of the USA where com­pulsory sterilization of individuals adjudged to be feeble-minded was practiced well into the present century) serves as a reminder to many of the potential Pandora’s box of ills that could be released if ever human germ line gene therapy were to be attempted.

The Questionable Need for Germline Gene Therapy:

Germ line genetic modification may be considered as a possible way of avoiding what would otherwise be the certain inheritance of a known harmful mutation. However how often does this situation arise and how easy would it be to intervene?

A 100% chance of inheriting a harmful mutation could most likely occur in two ways:

One is when an affected woman is homoplasmic for a harmful mutation in the mito­chondrial genome and wishes to have a child. The trouble here is that, because of the multi­ple mitochondrial DNA molecules involved, we still have very little to offer in the way of providing gene therapy for such disorders.

A second situation concerns inheritance of mutations in the nuclear genome. To have a 100% risk of inheriting a harmful mutation would require mating between a man and a woman both of whom have the same recessively inherited disease, an extremely rare occurrence. Instead, the vast majority of muta­tions in the nuclear genome are inherited with at most a 50% risk (for dominantly inherited disorders) or a 25% risk (for recessively inher­ited disorders).

In vitro fertilization provides the most accessible way of modifying the germ line. However, if the chance that any one zygote is normal is as high as 50 or 75%, gene transfer into an unscreened fertilized egg which may well be normal would be unacceptable: the procedure would inevitably carry some risk, even if the safety of the techniques for germ line gene transfer improves markedly in the future.

Thus, screening using sensitive PCR-based techniques would be required to identify a fertilized egg with the harmful muta­tion. Inevitably, the same procedure can be used to identify fertilized eggs that lack the harmful mutation. Since in vitro fertilization generally involves the production of several fertilized eggs, it would be much simpler to screen for normal eggs and select these for implantation, rather than to attempt genetic modification of fertilized eggs identified as carrying the harm­ful mutation.