Designing Our Descendants: The Promises and Perils of Genetic Modifications

4 Scientific Methodologies to Facilitate Inheritable Genetic Modifications in Humans

c h a p t e r f o u r Scientific Methodologies to Facilitate Inheritable Genetic Modifications in Humans Bhavani G. Pathak, Ph.D. The methodologies described in this chapter are being developed primarily to facilitate somatic cell therapy or improve transplant technologies in humans. However, when fully developed, they may be used to facilitate inheritable genetic modifications in humans. The scientific hurdles facing human germ-line intervention are similar to those in somatic gene transfer research and include methods for (1) introduction of the corrected gene into the target cell, (2) integration of the gene into the recipient genome, and (3) proper expression of the introduced gene. The different cell types in humans conducive to germ-line intervention are germ cells and their precursors; ova, sperm, and spermatogonial stem cells, respectively; zygotes, fertilized eggs at the single-cell stage;and pluripotent cells,like embryonic stem cells that have the potential to develop into various cells of the adult. Methods for Introducing DNA into Cells Viral Vectors Viruses have an innate ability to infect cells, a feature manipulated in their use as vectors for gene therapy. Viral vectors used currently in human gene transfer trials consist of the following types: retroviral, used in approximately 60 percent of clinical trials; adenoviral, used in approximately 10 percent of clinical trials; other viral vectors, used in approximately 10 percent of clinical trials.The other 20 percent of vectors used in such trials consist of nonviral vectors .1 The following section describes each of these systems and briefly highlights their advantages and disadvantages. Retroviral Vectors Retroviral vectors used in gene transfer research have been modified primarily to accomplish two goals: to infect host cells but not replicate in them, thereby preventing their spread to other cells of the body, and to accept exogenous DNA (i.e., a corrected copy of a defective gene) in lieu of their own DNA without affecting ability to infect and integrate into the host genome. Murine Moloney leukemia virus (MuLV), a retroviral vector used traditionally in gene transfer experiments, has been manipulated to accept up to 8 kb2 of exogenous DNA. MuLV-based vectors recognize and bind to specific receptors on the target cell and subsequently internalize their nuclear material. Cells lacking this receptor are not receptive to infection by the viral particle. Although attempts have been made to modify this recognition by attaching specific ligands3 to the surface of the viral particle such that it will recognize a particular target cell, they have been largely unsuccessful. This is because manipulation of the viral surface proteins appears to affect its ability to inject genetic material into the target cell. Retroviral vectors infect only dividing cells, and this poses a major drawback in the use of these vectors for gene therapy since it limits the types of target cells that can be used. Certain retroviruses such as lentiviruses (e.g., HIV-1) can infect nondividing cells, but their use raises issues of safety because of the possibility of recombination with endogenous cellular sequences that may result in the production of a pathogenic virus.4 Currently,attempts are being made to create hybrid vectors that combine useful features of various retroviruses in the hope that a better vector for gene therapy may be created. Another limitation of retroviral vectors is their inability to accommodate more than 8 kb of DNA. This size limitation usually prevents the inclusion of the exogenous gene’s own regulatory elements,which may be large or have spatial requirements that cannot be accommodated by the vector. As a result, exogenous DNA expression is often driven by viral elements present in the vector (elements in the long terminal repeats, or LTRs), and this could pose a problem with correct expression of the gene.This drawback is of particular signi ficance in germ-line interventions because of the potential to affect multiple 56 Bhavani G. Pathak generations.Additionally,retroviral vectors integrate randomly in the genome, and could disrupt endogenous gene expression near the site of integration. Therefore, their use for directed germ-line manipulation may not be ideal, since altered gene expression could adversely affect embryogenesis.5 Adenoviral Vectors Adenoviral vectors are derived from DNA viruses and are currently used in gene transfer trials related to, for example, cystic fibrosis and cancer treatment. These viruses are large, and vectors derived from them can...