Gene therapy: science fiction or reality?

Gene therapy has been proposed to be the modern medical cure for multiple diseases such as cancer, genetic disorders, as well as a potentially potent mediator of behavior. In the early days of gene therapy, the primary purpose was to develop therapies that would cure monogenetic diseases. However, with rapid advances in DNA technologies, specifically in the discovery of genes associated with disease pathologies, gene therapy strategies have expanded to include more complex diseases such as cancer, cardiovascular disease, and metabolic disorders. Furthermore, due to new developments in gene therapy vehicles, novel gene therapy strategies have begun to encompass not only curative therapies but also palliative therapies. Currently, new roads in this research have also initiated debates as to whether behaviors such as promiscuity (1) and alcoholism (2) should be included into the candidate disorders for gene therapy application. Thus, in addition to extending or improving life, gene therapy may also allow manipulation of the distinct differences between nature and nurture and thus not only affect disorders of the body, but potentially those of the mind as well.

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Transgenes for Gene Therapy Delivery

To accomplish the myriad of goals that gene therapy is now presented with, we must first define what to deliver and how to deliver it for each disease. Genetic information that is introduced to a cell or tissue via gene therapy is called a transgene. The vehicle that carries this genetic information is described as a gene therapy vector. For the most part, the therapeutic effect of a gene therapy transgene occurs via three distinct mechanisms, which depend on the type of transgene delivered. The first mechanism is mutation compensation. In this case, a mutated or missing gene is replaced with a fully functioning gene. Early gene therapy studies focused mainly on mutation compensation for use in the correction of p53 status in cancer cells or the restoration of the cystic fibrosis transmembrane conductance regulator gene in the epithelium of the lung. Thus, in these experiments, the gene is replaced via a gene therapy vector and the therapeutic effect seen is a result of the restoration of proper gene expression and function.

Another type of transgene that is commonly used is a purely therapeutic gene. In this example, the specific gene may not be missing or mutated in the target cells, but increased expression of this gene leads to a reduction in the disease pathology or symptoms. In this scenario, the genetic disease is not the result of a single genetic change but usually a myriad of genetic changes that may or may not be known. This type of gene therapy is used for many autoimmune diseases. These diseases are the result of the dysfunction of multiple genes that allow the body to see its own substrates as foreign invaders and thus the immune system attacks. However, the introduction of a single gene or blocking of a single enzyme may be able to reduce the attack of the immune system on the body and provide a therapeutic or palliative effect. In this regard, the exact mutations or genetic changes that occurred to cause the disease may not be necessarily known. Another example for therapeutic gene delivery is to increase endogenous levels of a gene product that may or may not be already present. For example, the introduction of the vascular endothelial growth factor (VEGF) gene, a stimulator of blood vessel growth, may allow the rerouting of circulation in ischemic heart disease or other circulation disorders. Although VEGF is not defective in the organism, the expression of additional VEGF may be sufficient to allow the body to reroute blood flow to an ischemic area and improve symptoms.

Finally, the last mechanism, suicide gene therapy, utilizes transgenes known as "suicide genes," which selectively kill the cells they are expressed in, and are thus primarily used for cancer treatment. These "suicide genes" such as thymidine kinase from herpes simplex virus (HSV-tk) and cytosine deaminase (CD), are the most commonly used. However, others are pursuing the use of proapoptotic genes. However, HSV-tk and CD have the distinct advantage of the use of a prodrug that these suicide genes convert to a toxic byproduct that kills the cells. When levels of cell toxicity get too high, the administration of the prodrug can be reduced or removed to spare normal tissue damage.

Gene Therapy Vectors

Although the choice of the transgene within the gene therapy vector is vital to a positive response, there are also many variations of potential gene therapy vectors that can be employed to deliver these genes, each having their own positive and negative aspects. However, the goals are the same, regardless of the vector origin. First, the ideal gene therapy vector must not only deliver optimal levels of the transgene, but it must also provide an appropriate duration of transgene expression for the therapeutic effect to be achieved. This usually means that the vector must also have limited immunogenicity, so that it has time to express the transgene to achieve an effect before immune system clearance. Second, the ideal gene therapy vector must also be able to target a specific cell type and limit expression in normal tissues, especially the liver where toxicity can result from accumulation of the vector or high levels of transgene expression. Finally, complex issues such as unexpected or unpredictable genome integration of the transgene should be addressed so that secondary diseases, like leukemia as seen in the SCID trials, are not caused from the treatment itself. For ease of discussion, we will discuss a few promising gene therapy vectors and categorize them as either viral or nonviral vectors.

Viral Gene Therapy Vectors

One of the most versatile and easily manipulated viral vectors for gene delivery is the adenoviral vector. There are over 50 identified adenovirus serotypes which are associated with 6 species. In most cases, adenoviral infection causes a mild upper respiratory tract infection, although infection can also occur in the gastrointestinal tract and in the eye. Human adenovirus serotype 5 has been used in multiple clinical trials with little side effects and has generally been considered to be safe. However, though not common, adenovirus can induce an overwhelming toxic immune response as seen in the Gelsinger case. (3) Nevertheless, it is still generally accepted that adenovirus is very safe for human gene therapy use. Adenovirus has the ability to infect a broad range of tissues, but this is widely dependent on the presence of the coxsackie and adenovirus receptor (CAR) and alpha 5 integrins. However, some cancer cells and other tissues that are of interest from a gene therapy prospective do not have high levels of CAR to facilitate infection. (4) In this regard, our lab and others have sought to alter the viral specificity to induce infection in specific tissues and limit expression in nontarget tissues. To do this, the proteins found on the capsid of adenovirus, such as the protein knob, which interacts with CAR on the cell surface, can be modified to change the viral specificity. Knob protein, which projects out the end of fiber shafts from the adenovirus capsid, can be altered to include knob proteins from other adenovirus serotypes, (5-16) single chain antibodies (17) or other motifs to increase infectivity. (18-23) Thus, the virus can infect cells independent of the native receptor CAR and also be retargeted to specific tissues. In addition to this, our laboratory has sought to restrict transgene expression via the use of tissue specific promoters which allow the transgene to be transcribed only in select tissues. Using tissue specific promoters, we can further refine the transgene expression to our target cells and limit the exogenous expression of the transgene in nontarget tissues, such as the liver, which lead to toxicity. (24-28) Taken together, we seek to create new generation vectors that are highly specific to target cells and have low toxicity. Other areas of development regarding the improvement of adenoviral vectors include modulation of capsid proteins to evade immune system response and clearance, (29,30) increase duration of gene expression via genetic modification, (31) as well as the incorporation of imaging motifs to allow for monitoring of the virus administration and action. (32-35)

Another commonly studied virus for gene therapy is adenoassociated virus (AAV). One advantage of AAV is that it naturally expresses transgenes for a longer time frame and also has multiple serotypes to allow for infection into a large variety of tissues. However, one disadvantage of AAV is that productions of AAV gene therapy vectors require the use of a helper virus for generation and assembly. This makes the AAV production system more complicated and subject to issues associated with high titer production and purity. Additional disadvantages of AAV include its limited transgene capacity (less than 5 kb) and its induction of a strong immune system response. However, AAV, similar to adenovirus, has a broad tissue tropism because it also uses integrins in addition to heparin sulfate proteoglycans (as a primary receptor), and fibroblast growth factor 1 (as a coreceptor) for infection. For example, AAV serotype 2 infects lung epithelium which is refractory to many types of viral infection. Due to this, AAV-2 is being studied in phase two clinical trials for cystic fibrosis with promising results. (36,37)

In contrast to adenovirus and AAV vectors, which produce exogenous transgene expression in most instances, retroviral vectors integrate the transgene DNA into the host genome and are thus a promising candidate for curative treatment. Although retrovirus gives an obvious advantage in terms of long-term expression of the transgene, there is the risk of undesirable side effects due to permanent integration into the genome. These risks have been realized in the SCID trials, in which several of the children that received therapy via use of retroviral vectors developed leukemia. In these children, the retrovirus inserted DNA into their host genome, and this insertional mutagenesis was manifested as a proliferation of T-cells. However, despite setbacks such as these, the use of retroviral vectors for gene therapy is still promising as we learn more about how retrovirus insert and traffic cDNA and how to control their genome integration. In this regard, we do know that retroviral vectors, so named because they copy their DNA into the host genome via reverse transcriptase, contain two copies of their RNA viral genome surrounded by a cell membrane-like viral envelope. These envelopes fuse with the host cell membrane or are endocytosed, which is dependent on retroviral type. However, the core viral RNA is then transcribed to cDNA using the reverse transcription complex which contains the reverse transcriptase mentioned above. The cDNA produced is then translocated to the nucleus where it can integrate into the host cell genome. It is at this critical step that more studies are necessary to harness the power of retroviral infection and increase clinical safety. However, in addition to random site integration issues, retroviral vectors must also circumvent numerous barriers to their infectivity and expression efficiency. Most of these barriers are natural host cell defenses such as cellular restriction factors which obstruct the passage and intracellular trafficking of retroviral vectors. As studies reveal more information regarding the mechanism of these cellular restrictions, advances in circumventing these barriers can be achieved. Despite these limitations, advancements in targeting and the ability of retroviruses such as lentivirus to infect nondividing and dividing cells keep it at the forefront of viral gene therapy delivery systems. (38-40)

Nonviral Gene Therapy Vectors

In addition to the use of multiple types of viral vectors for gene delivery, there are many studies aimed at developing nonviral delivery systems. These delivery systems usually deliver the DNA via a physical method, such as naked DNA delivered by ultrasound or electroporation, or they utilize endocytosis-mediated mechanisms. However, both of these systems have limited efficiency whether due to their method of entry, or due to DNA degradation in the endosomes or lysosomes of the target cells. In trials for cancer gene therapy, cationic liposomes have shown some success in humans despite their limited infectivity compared with viral gene therapy vehicles. Cationic liposomes use cationic molecules such as the adenovirus mu protein or protamine sulfate to protect the DNA from degradation. However, cationic liposomes are still limited by their decreased ability to deliver the transgene to the nucleus of the cells for expression. (41) Current studies aimed at further engineering of these liposomes have included machinery to transport the DNA to the nucleus efficiently. In addition to liposomes, new studies of nonviral vector delivery include the use of polymers to deliver DNA to cells. The large and negatively charged DNA is condensed inside the positively charged polymer to facilitate endocytosis. Although this system is also limited by lack of nuclear delivery, acceptable infection efficiencies are achieved. In addition, there have been many advances in targeting by incorporating ligands into the polymers; thus, many studies are also using receptor-mediated endocytosis via transferrin (42) and folate. (43,44) However, in the realm of nonviral delivery, the most exciting developments have been in the creation of biodegradable polymers, which increase the transfection efficiency by slowly releasing DNA around cells as the polymer degrades, thus providing longer term expression of the transgene and higher transfection efficiency overall. Gains have also been made due to the introduction of polyethylene glycol (PEG) or PEG-like polymers, which increase efficiency via immune system evasion.

These recent advances in gene therapy, and others, give hope to millions of patients that gene delivery vectors with low toxicity and high efficacy are on their way. As mentioned previously, in regards to adenovirus and other viral and nonviral delivery systems, significant gains are being made to improve targeting, duration of expression, and immune system evasion. Thus, the future of gene therapy is by no means bleak. In fact, recent studies have indicated that we are on the cusp of discovering clinically efficacious gene therapy agents that embody these goals. On the horizon is the use of AAV as a treatment for genetic blindness caused by Leber congenital amaurosis (LCA). Recent reports have demonstrated restoration of the retinal pigment epithelium--specific protein 65 kDa (RPE65) into the retina of RPE65-null dogs and rats. (45-48) In these studies, gene delivery was specific, durable, and non-target organ toxicity was nonexistent. Due to the nature of the retina, there was also a limited immune response to the vector administration. The spectacular results of these studies, and a few others, have validated the quest for ideal gene therapy vectors and inspire us to continue our pursuit of the ideal vector which we hope will move the field of

gene therapy from the subject of science fiction to the reality of human clinical use.

References

1. Lim MM, Wang Z, Olazabal DE, et al. Enhanced partner preference in a promiscuous species by manipulating the expression of a single gene. Nature 2004;429:754-757.

2. Thanos PK, Rivera SN, Weaver K, et al. Dopamine D2R DNA transfer in dopamine D2 receptor-deficient mice: effects on ethanol drinking. Life Sci 2005;77:130-139.

3. Couzin J, Kaiser J. Gene therapy. As Gelsinger case ends, gene therapy suffers another blow. Science 2005;307:1028.

4. Douglas JT, Kim M, Sumerel LA, et al. Efficient oncolysis by a replicating adenovirus (ad) in vivo is critically dependent on tumor expression of primary ad receptors. Cancer Res 2001;61:813-817.

5. Bauerschmitz GJ, Guse K, Kanerva A, et al. Triple-targeted oncolytic adenoviruses featuring the cox2 promoter, E1A transcomplementation, and serotype chimerism for enhanced selectivity for ovarian cancer cells. Mol Ther 2006;14:164-174.

6. Borovjagin AV, Krendelchtchikov A, Ramesh N, et al. Complex mosaicism is a novel approach to infectivity enhancement of adenovirus type 5-based vectors. Cancer Gene Ther 2005;12:475-486.

7. Glasgow JN, Kremer EJ, Hemminki A, et al. An adenovirus vector with a chimeric fiber derived from canine adenovirus type 2 displays novel tropism. Virology 2004;324:103-116.

8. Kanerva A, Mikheeva GV, Krasnykh V, et al. Targeting adenovirus to the serotype 3 receptor increases gene transfer efficiency to ovarian cancer cells. Clin Cancer Res 2002;8:275-280.

9. Kawakami Y, Li H, Lam JT, et al. Substitution of the adenovirus serotype 5 knob with a serotype 3 knob enhances multiple steps in virus replication. Cancer Res 2003;63:1262-1269.

10. Krasnykh V, Dmitriev I, Mikheeva G, et al. Characterization of an adenovirus vector containing a heterologous peptide epitope in the HI loop of the fiber knob. J Virol 1998;72:1844-1852.

11. Le LP, Rivera AA, Glasgow JN, et al. Infectivity enhancement for adenoviral transduction of canine osteosarcoma cells. Gene Ther 2006;13:389-399.

12. Short JJ, Pereboev AV, Kawakami Y, et al. Adenovirus serotype 3 utilizes CD80 (B7.1) and CD86 (B7.2) as cellular attachment receptors. Virology 2004;322:349-359.

13. Stoff-Khalili MA, Rivera AA, Glasgow JN, et al. A human adenoviral vector with a chimeric fiber from canine adenovirus type 1 results in novel expanded tropism for cancer gene therapy. Gene Ther 2005;12:1696-1706.

14. Stoff-Khalili MA, Stoff A, Rivera AA, et al. Gene transfer to carcinoma of the breast with fiber-modified adenoviral vectors in a tissue slice model system. Cancer Biol Ther 2005;4:1203-1210.

15. Takayama K, Reynolds PN, Short JJ, et al. A mosaic adenovirus possessing serotype Ad5 and serotype Ad3 knobs exhibits expanded tropism. Virology 2003;309:282-293.

16. Uil TG, Seki T, Dmitriev I, et al. Generation of an adenoviral vector containing an addition of a heterologous ligand to the serotype 3 fiber knob. Cancer Gene Ther 2003;10:121-124.

17. Hedley SJ, Auf der Maur A, Hohn S, et al. An adenovirus vector with a chimeric fiber incorporating stabilized single chain antibody achieves targeted gene delivery. Gene Ther 2006;13:88-94.

18. Bilbao G, Contreras JL, Dmitriev I, et al. Genetically modified adenovirus vector containing an RGD peptide in the HI loop of the fiber knob improves gene transfer to nonhuman primate isolated pancreatic islets. Am J Transplant 2002;2:237-243.

19. Contreras JL, Wu H, Smyth CA, et al. Double genetic modification of adenovirus fiber with RGD polylysine motifs significantly enhances gene transfer to isolated human pancreatic islets. Transplantation 2003;76:252-261.

20. Garcia-Castro J, Segovia JC, Garcia-Sanchez F, et al. Selective transduction of murine myelomonocytic leukemia cells (WEHI-3B) with regular and RGD-adenoviral vectors. Mol Ther 2001;3:70-77.

21. Reynolds P, Dmitriev I, Curiel D. Insertion of an RGD motif into the HI loop of adenovirus fiber protein alters the distribution of transgene expression of the systemically administered vector. Gene Ther 1999;6:1336-1339.

22. Wang M, Hemminki A, Siegal GP, et al. Adenoviruses with an RGD-4C modification of the fiber knob elicit a neutralizing antibody response but continue to allow enhanced gene delivery. Gynecol Oncol 2005;96:341-348.

23. Wu H, Seki T, Dmitriev I, et al. Double modification of adenovirus fiber with RGD and polylysine motifs improves coxsackievirus-adenovirus receptor-independent gene transfer efficiency. Hum Gene Ther 2002;13:1647-1653.

24. Reynolds PN, Nicklin SA, Kaliberova L, et al. Combined transductional and transcriptional targeting improves the specificity of transgene expression in vivo. Nat Biotechnol 2001;19:838-842.

25. Adachi Y, Reynolds PN, Yamamoto M, et al. Midkine promoter-based adenoviral vector gene delivery for pediatric solid tumors. Cancer Res 2000;60:4305-4310.

26. Yamamoto M, Alemany R, Adachi Y, et al. Characterization of the cyclooxygenase-2 promoter in an adenoviral vector and its application for the mitigation of toxicity in suicide gene therapy of gastrointestinal cancers. Mol Ther 2001;3:385-394.

27. Zhu ZB, Makhija SK, Lu B, et al. Transcriptional targeting of tumors with a novel tumor-specific survivin promoter. Cancer Gene Ther 2004;11:256-262.

28. Zhu ZB, Makhija SK, Lu B, et al. Transcriptional targeting of adenoviral vector through the CXCR4 tumor-specific promoter. Gene Ther 2004;11:645-648.

29. Scott JM, Chamberlain JS. Gutted adenoviral vectors for gene transfer to muscle. Methods Mol Biol 2003;219:19-28.

30. Wu H, Han T, Belousova N, et al. Identification of sites in adenovirus hexon for foreign peptide incorporation. J Virol 2005;79:3382-3390.

31. Yant SR, Ehrhardt A, Mikkelsen JG, et al. Transposition from a gutless adeno-transposon vector stabilizes transgene expression in vivo. Nat Biotechnol 2002;20:999-1005.

32. Lee CT, Lee YJ, Kwon SY, et al. In vivo imaging of adenovirus transduction and enhanced therapeutic efficacy of combination therapy with conditionally replicating adenovirus and adenovirus-p27. Cancer Res 2006;66:372-377.

33. Raben D, Buchsbaum DJ, Khazaeli MB, et al. Enhancement of radio-labeled antibody binding and tumor localization through adenoviral transduction of the human carcinoembryonic antigen gene. Gene Ther 1996;3:567-580.

34. Rogers BE, Rosenfeld ME, Khazaeli MB, et al. Localization of iodine-125-mIP-Des-Met14-bombesin (7-13)NH2 in ovarian carcinoma induced to express the gastrin releasing peptide receptor by adenoviral vector-mediated gene transfer. J Nucl Med 1997;38:1221-1229.

35. Zinn KR, Douglas JT, Smyth CA, et al. Imaging and tissue biodistribution of 99mTc-labeled adenovirus knob (serotype 5). Gene Ther 1998;5:798-808.

36. Wagner JA, Nepomuceno IB, Messner AH, et al. A phase II, double-blind, randomized, placebo-controlled clinical trial of tgAAVCF using maxillary sinus delivery in patients with cystic fibrosis with antrostomies. Hum Gene Ther 2002;13:1349-1359.

37. Moss RB, Rodman D, Spencer LT, et al. Repeated adeno-associated virus serotype 2 aerosol-mediated cystic fibrosis transmembrane regulator gene transfer to the lungs of patients with cystic fibrosis: a multi-center, double-blind, placebo-controlled trial. Chest 2004;125:509-521.

38. Goff SP. Retrovirus restriction factors. Mol Cell 2004;16:849-859.

39. Bieniasz PD. Restriction factors: a defense against retroviral infection. Trends Microbiol 2003;11:286-291.

40. Bieniasz PD. Intrinsic immunity: a front-line defense against viral attack. Nat Immunol 2004;5:1109-1115.

41. Kaneda Y, Tabata Y. Non-viral vectors for cancer therapy. Cancer Sci 2006;97:348-354.

42. Qian ZM, Li H, Sun H, et al. Targeted drug delivery via the transferrin receptor-mediated endocytosis pathway. Pharmacol Rev 2002;54:561-587.

43. Sudimack J, Lee RJ. Targeted drug delivery via the folate receptor. Adv Drug Deliv Rev 2000;41:147-162.

44. Sudimack JJ, Adams D, Rotaru J, et al. Folate receptor-mediated liposomal delivery of a lipophilic boron agent to tumor cells in vitro for neutron capture therapy. Pharm Res 2002;19:1502-1508.

45. Bainbridge JW, Mistry A, Schlichtenbrede FC, et al. Stable rAAV-mediated transduction of rod and cone photoreceptors in the canine retina. Gene Ther 2003;10:1336-1344.

46. Dinculescu A, Glushakova L, Min SH, et al. Adeno-associated virus-vectored gene therapy for retinal disease. Hum Gene Ther 2005;16:649-663.

47. Acland GM, Aguirre GD, Bennett J, et al. Long-term restoration of rod and cone vision by single dose rAAV-mediated gene transfer to the retina in a canine model of childhood blindness. Mol Ther 2005;12:1072-1082.

48. Jacobson SG, Acland GM, Aguirre GD, et al. Safety of recombinant adeno-associated virus type 2-RPE65 vector delivered by ocular sub-retinal injection. Mol Ther 2006;13:1074-1084.

Meredith A. Preuss, PhD, and David T. Curiel, MD, PhD

From the Division of Human Gene Therapy, Departments of Medicine, Surgery, Pathology and the Gene Therapy Center, University of Alabama Birmingham, Birmingham, AL.

Reprint requests to David T. Curiel, MD, PhD, Division of Human Gene Therapy, 901 19th Street, South, BMR2--502, Birmingham, Alabama 35294-2172. Email: [e-mail]