Cell Marking

For some purposes, it is useful to follow cells after they have been introduced into a human patient. Often, the transplanted cells cannot be distinguished from preexisting cells in the subject; although cells can be marked with radioisotopes or dyes, a genetic tag is a better marker for long-term assessment of the persistence and distribution of the transplanted cells.

The first virus-mediated gene transfer trial in humans involved marking lymphocytes with a retroviral vector to study their persistence and distribution in patients (Rosenberg et al. 1990a). T lymphocytes from excised tumor masses were grown in culture, transduced with an MLV-based retroviral vector carrying a neo gene, and reinfused in large numbers in an attempt to destroy additional tumors still present in the patient. The marker gene persisted in cells in the circulation and in tumor deposits for several months (Rosenberg et al. 1990b). Of importance for later gene transfer trials in humans, the gene-marking procedure showed no obvious toxic effects; however, all patients were terminal cancer patients and thus long-term toxicities could not be adequately addressed.

HIV antigen-specific cytotoxic T lymphocytes used in an attempt to kill HIV-infected cells in patients have also been marked with a retroviral vector to study the persistence and distribution of cells following their infusion into patients (Riddell et al. 1992). Although this technique allowed detection of the infused cells over the short term, five of six patients developed cytotoxic-T-lymphocyte responses against cells carrying the marker gene (Riddell et al. 1996).

Retrovirus marking has been used to detect malignant cells that may still be present in bone marrow used for autologous transplantation (Rill et al. 1992). In this procedure, which is used in the treatment of several forms of cancer, marrow is harvested prior to what would be an otherwise lethal chemotherapeutic treatment designed to eliminate cancerous cells. Following chemotherapy, the marrow is reinfused into the patient. Cancer can reoccur either from cells in the patient that escaped the effects of the chemotherapy or from cancerous cells that contaminated the reinfused marrow. Cancerous cells in the marrow can be marked by exposure of the marrow to a retroviral vector prior to infusion. If the retroviral marker is present in the cancer cells that arise following treatment, then the reinfused marrow was the source of these cells. Recent studies show that recurrent cancer can be composed of multiple independently marked cancer cell clones derived from the infused marrow (Rill et al. 1994). These results strongly support the use of purification steps to eliminate cancerous cells in the marrow cell infusion.

Marking human marrow used for autologous transplantation with retroviral vectors has been used to study the contribution of the infused marrow to long-term hematopoiesis in humans and to examine gene transfer rates in human hematopoietic cells. These studies have shown that the transduced cells persist following autologous transplantation in humans (Brenner et al. 1993; Dunbar et al. 1995). The percentage of G418-resistant bone marrow colony-forming cells was high in one study, about 5–10% in five of five patients 1 year after transplantation (Brenner et al. 1993); in the other study, only three of nine patients showed persistence of the vector in hematopoietic cells at levels between 0.01% and 0.1% (Dunbar et al. 1995).

Genetic Disease

Since rates of gene repair by homologous recombination are currently too low to be of practical utility, gene therapy relies on the addition of genes to correct inherited genetic defects. The first government-approved trial of gene therapy to correct genetic disease targeted a severe combined immunodeficiency due to defects in the enzyme adenosine deaminase (ADA) (Blaese et al. 1990). ADA-deficient individuals have severely reduced levels of T and B lymphocytes, and without treatment, they die in childhood from infection. For treatment of ADA deficiency, lymphocytes were harvested from blood by leukapheresis, transduced with a retroviral vector expressing ADA and Npt, and grown to large numbers in culture. About 10¹⁰ cells were then reinfused. This treatment was repeated at 1.5-month intervals due to the limited life span of lymphocytes. The results of this trial (Blaese et al. 1995) indicate a sustained increase in ADA in circulating cells in one of two children treated and correction of some of the immune defects characteristic of ADA deficiency in both children. Importantly, in one patient, the levels of vector-encoded ADA remained high in circulating T cells during a 2-year period without additional T-cell infusions, and about half of the circulating cells carried the retroviral vector. These results show that long-term persistence of vector-expressing T cells is possible.

Ideally, one would like to transduce hematopoietic stem cells that continuously give rise to lymphocytes to provide long-term treatment of ADA deficiency, and trials to test this possibility are under way (Hoogerbrugge et al. 1992; Blaese et al. 1993; Bordignon 1993). However, animal studies have indicated the difficulty of transducing stem cells (Schuening et al. 1991; van Beusechem et al. 1992, 1994). Recent experiments targeting hematopoietic stem cells from cord blood of neonates with ADA deficiency show that these cells are suitable targets for efficient gene transfer (Kohn et al. 1995).

Familial hypercholesterolemia is due to defects in low-density lipoprotein (LDL) receptors. This disease provided another early target for gene therapy (Wilson et al. 1992). Treatment involved removing about 20% of the liver. The hepatocytes were disaggregated and cultured for 3 days. During cultivation, the cells were exposed to a retroviral vector carrying the LDL receptor, resulting in transfer of the receptor to about 30% of the cells. The hepatocytes were then reinfused into the liver. A small but significant decrease in serum cholesterol level (10–15%) was observed in the first patient treated (Grossman et al. 1994), but given the very high level of cholesterol in patients with familial hypercholesterolemia, this decrease presumably will have little clinical effect. On the other hand, levels of hepatic gene transfer achieved in this study may be adequate for treatment of other diseases that require lower levels of a therapeutic protein.

Cancer

A major emphasis in current gene therapy trials is the attempt to treat cancer by stimulating the immune response against the cancer cells. In one approach, genes that encode new antigens are transferred into some of the cancer cells to stimulate an immune response to these new antigens and also to minor antigens expressed by all of the cancer cells (Nabel et al. 1993). An alternative approach involves the production of immunostimulatory cytokines within tumors by a transfer of the genes either into the tumor cells or into cells such as fibroblasts that can be easily injected into the tumor (Tepper and Mule 1994). In both cases, the genes are typically transferred into tumor cells in vitro, and the cells are lethally irradiated prior to reintroduction into human patients to prevent the growth of the modified cells. Retroviral vectors have the advantage for this application in that they efficiently transduce primary cells, which are often difficult to grow, and they cause the transduced gene to be expressed at high levels. However, alternative approaches to gene transfer, such as direct injection of DNA or DNA/lipid mixtures (lipofection) into tumors, are being tested. None of these treatments have yet been shown to be effective.

Clinical trials of gene therapy for treatment of brain tumors are also under way (Culver et al. 1992; Oldfield et al. 1993). In this procedure, a herpes simplex virus thymidine kinase (HSV-tk) gene is transferred to brain tumor cells by direct injection of vector-producing packaging cells into the tumors. The patient is then treated with ganciclovir, a nucleoside analog of 2′-deoxyguanosine that is metabolized to a toxic nucleotide analog by the HSV-tk gene but not by the cellular thymidine kinase enzyme. Since murine retroviral vectors appear to transduce only dividing cells, the tumor cells should be transduced but not the normal nondividing cells of the brain. In addition, ganciclovir treatment kills other replicating tumor cells near the transduced tumor cells by a mechanism called the bystander effect, which probably involves transfer of the nucleotide analog produced from ganciclovir to nearby cells. Because the normal brain cells do not divide, they do not incorporate the nucleotide analog into their DNA. The clinical value of this treatment is not yet clear.

AIDS

A number of gene therapy approaches have been proposed for the treatment of HIV infection; retroviral vectors provide a method that can be used for gene delivery to the appropriate cell types, including CD4-positive T cells that are the targets for infection or the progenitor cells, including hematopoietic stem cells, that give rise to T cells. Techniques intended to inhibit HIV replication in T lymphocytes by the transfer of Rev dominant-negative mutant proteins and anti-HIV ribozymes have been approved for testing in humans by the U.S. Recombinant DNA Advisory Committee. But perhaps the best strategy would involve transfer of genes that would protect cells from infection by HIV. Such protection might be provided by synthesis of peptides that block HIV binding and entry at the level of the cell surface receptor for HIV. There is precedent for this approach. Mice and chickens that express fragments or complete retroviral Env proteins are protected against infection by the corresponding avian or murine leukemia virus (see Chapter 3. However, in both the mice and the chickens, the Env proteins are derived from endogenous virus that cannot, and do not, provoke an immune response. Expression of the HIV-1 Env protein in any but the most severely immunocompromised individual would likely provoke an immune response to the transduced cells; in acquired immunodeficiency syndrome (AIDS) patients, HIV-infected cells are turned over rapidly and continuously.

Another approach involves antisense RNAs or ribozymes that block reverse transcription or integration of HIV. In one experiment of this type, infection by avian viruses (RSV and an avian vector carrying the neo gene) was shown to be blocked by expression of antisense RNA corresponding to the polypurine region of the viruses (To et al. 1986). However, as has already been discussed, the general applicability of antisense RNA is questionable, and the general applicability of specific ribozymes has yet to be demonstrated.

Barriers to Effective Gene Transduction

A key concern in the development of gene therapy is the immune response to vector-encoded proteins. A normal protein may be recognized as foreign by the immune system in an individual that lacks or makes an altered form of the protein. Marker proteins that facilitate vector production are also likely to generate immune responses. Indeed, cytotoxic T cells reactive against a vector-encoded protein have been found in patients receiving T cells modified with a vector that encodes a hygromycin phosphotransferase-HSV thymidine kinase fusion protein (Riddell et al. 1996). Surprisingly, immune responses against the neomycin phosphotransferase protein carried by many marker vectors have not been described. It is worth pointing out, for some of the possible uses of human gene therapy (e.g., the treatment of cancer), that the development or enhancement of the host's immune response is the goal, whereas in other protocols (e.g., gene replacement and the treatment of AIDS), it is important to avoid provoking the host's immune system. This distinction is an important issue in the design and choice of vectors for specific applications.

Another potential barrier to in vivo gene transduction in humans is the sensitivity of many retroviral vectors to inactivation by human complement (see above Principles of Retroviral Vector Design, Retroviral Packaging Cells). These problems can be overcome by using appropriate packaging cell lines (Cosset et al. 1995). In addition, although human serum is able to inactivate commonly used amphotropic vectors, extracellular fluid from the brain (cerebrospinal fluid) does not exhibit this activity (Russell et al. 1995).

Safety Issues in Gene Therapy

The primary issue regarding the safety of retroviral vectors for human use is the possibility of the generation of helper virus from vector-producing cell lines. Most of the human gene therapy studies under way use amphotropic MLV vectors, and early studies indicated that amphotropic helper virus was not a pathogen in nonhuman primates (Cornetta et al. 1990, 1991). However, infusion of bone marrow cells that had been infected with large quantities of amphotropic helper virus into monkeys after ablation of endogenous marrow cells by γ-irradiation resulted in lymphoma in some of the animals (Donahue et al. 1992; Vanin et al. 1994; Purcell et al. 1996). It is also true that even improved packaging cell line/vector combinations can still produce helper virus at low frequency. Of 57 large lots (total of 855 liters) of clinical grade vector stocks made by using PA317 packaging cells (Miller and Buttimore 1986) (Table 1) produced by Genetic Therapy Inc., one lot had evidence of low-level helper virus contamination. The virus was subsequently cloned and shown to be a recombinant between vector and helper virus DNA used to make the packaging cell line (Otto et al. 1994). In another example, replication-competent retroviral production by GP+envAM12 packaging cells (Markowitz et al. 1988b) (Table 1) containing a retroviral vector has been documented (Chong and Vile 1996), showing that even the latest-generation packaging cells having split viral protein-coding regions (Fig. 5) can produce helper virus. However, it should be emphasized that helper virus production in these examples was a very rare event, and presumably the frequency can be further reduced by careful elimination of homologous overlap between vector and helper virus sequences. In the examples above, homologous overlap existed at all of the junctions required for helper virus generation from the vector and the deleted helper virus sequences (see Fig. 5).

Other contaminants present in retroviral vector stocks also raise safety issues. Virus-like 30S (VL30) RNAs transcribed from endogenous retrovirus-like elements present in mouse and rat cells can be transmitted following infection of these cells with replication-competent helper virus (Sherwin et al. 1978; Besmer et al. 1979; Scolnick et al. 1979). Packaging cells can also encapsidate and transduce VL30 and other viral sequences (Rodland et al. 1987; Hatzoglou et al. 1990; Scadden et al. 1990; Ronfort et al. 1995; Purcell et al. 1996). The vast majority of these elements do not contain significant open reading frames for protein translation, but the effects of their transfer are not known. In addition, the genomes of cells used to make packaging cell lines do contain endogenous replication-competent retroviruses that can be activated by irradiation and chemical methods, but production of these viruses under normal conditions of vector harvest from packaging cells has not been detected (Scadden et al. 1990; Damian et al. 1996).

Insertional mutagenesis by retroviral vectors is often cited as a safety concern. This issue has been raised because proviral insertion can cause the inactivation of tumor suppressor genes or the activation of oncogenes (see Chapter 10. However, this concern can be raised for any gene transfer method that results in new DNA being inserted into random sites in the genome of the target cell. In situations where relatively few cells are modified, as in the case of gene transfer into rare hematopoietic stem cells, the total number of insertion sites will also be small, and the risks are expected to be very low. The risks are higher in cases where large numbers of cells are transduced, which increases the number of independent integration events, for example, in gene transfer to lymphocytes for treatment of ADA deficiency (Blaese et al. 1990) or in gene transfer to hepatocytes for treatment of familial hypercholesterolemia (Wilson et al. 1992). However, there is as yet no evidence that transduction by helper-free retroviral vectors has had any adverse effects in animals or humans, and only continuing experience will determine whether rare deleterious events occur in patients.