Gene Therapy of Human Disease

Table of Contents 1. Introduction 2. Methods of Transfer 2.1. Naked DNA transfer 2.2. Viral gene transfer 2.2.1. Adenoviruses 2.2.2. Retroviruses 2.2.3. Lentiviruses 2.2.4. Adeno-associated viruses 2.2.5. Herpes simplex viruses 2.2.6. Other viruses 2.3. Nonviral gene transfer 2.3.1. Chemical and physical methods 2.3.2. Liposomal gene transfer 2.3.3. Protein/peptide gene transfer 3. Disease Targets for Gene Therapy 3.1. Genetically determined protein deficiencies 3.1.1. Gaucher disease 3.1.2. Adenosine deaminase deficiency 3.1.3. γc Cytokine receptor deficiency 3.1.4. Cystic fibrosis 3.2. Gene therapy for acquired diseases 3.2.1. Immune stimulation in the treatment of cancer 3.2.2. Activation of ganciclovir by herpes thymidine kinase 3.2.3. Tumor suppressor genes 3.2.4. Acquired immunodeficiency syndrome (AIDS) 3.2.5. Rheumatoid arthritis 3.2.6. Cardiovascular disease 4. Conclusions 1. Introduction The treatment of genetic diseases has seemed to many a daunting challenge. What, after all, can be done if the immutable basic blueprints of the body have a serious imperfection? In reality, even in the middle of the last century it was possible to improve greatly the quality of life and, indeed, to save the lives of patients with some such genetic diseases. The successful approaches included dietary manipulation as in phenylketonuria or galactosemia, surgery to correct various deformities, and sometimes avoidance of inciting factors in the environment, as in acute intermittent porphyria. However, until the latter part of the century just past, the possibility of actually changing the faulty genetic blueprints was beyond the imagination of the realistic medical scientist. In 1972 Friedmann and Roblin (68) argued persuasively that defective genes could be replaced by those with the correct sequence. In the following 30 years a great deal of effort has been expended to bring such a therapeutic endeavor to fruition, thus far with limited success. 2. Methods of Transfer Although it has been possible to achieve expression of genes delivered to the cytoplasm (28,37,100,119) the most common and useful strategy is to deliver the gene of interest to the nucleus. There are 4 extracellular barriers to such delivery: 1) opsonins, 2) phagocytes, 3) extracellular matrices, and 4) degradative enzymes (59). An opsonin is a factor that attaches to foreign material and renders it more susceptible to ingestion by phagocytes. Thus, opsonins can inactivate a gene delivery system by attachment, leading to the inactivation of the gene and/or its carrier. Phagocytes are cells that can inactivate a gene delivery system by engulfment or digestion. The extracellular matrix represents a physical barrier of polymerized protein and carbohydrates present between cells protecting target cells from relatively large DNA carrier systems. Finally, the extracellular fluid contains DNases that can rapidly digest unprotected DNA. Once all these barriers are overcome, the gene delivery system attaches to the plasma membrane where it faces cellular barriers. The first of these is the plasma membrane. Then the gene must be protected from nucleases in the cytoplasm and overcome the possibility of endosomal entrapment. The nucleic acid must enter the nucleus where the gene of interest can proceed to be transcribed and translated, and finally the protein traffics to the cellular location where it has a function. The ideal gene delivery vector is nontoxic, nonimmunogenic, easy to produce in large quantities, and efficient in protecting and delivering DNA into cells, preferably with a specificity for a particular cell type. This ideal vector remains to be discovered (112,185). Properties of existing transfection agents are compared in Table 1.TABLE 1: Properties of transfection agents*2.1. Naked DNA transfer The principal obstacle to cellular DNA uptake is charge (56). In an aqueous solution, such as the milieu that bathes cells in the body, DNA has a net negative charge. DNA tends to be repelled from cell membranes, because they, too, are negatively charged. There are a few exceptions where cells appear to be able to assimilate naked DNA; this includes the successful target protein expression after direct muscular injections in mice (22,38,56,181). While the mechanism of this type of gene transfer is unclear, a small amount of tissue damage or increased pressure at the injection site may play a role (56). A few other types of cells and tissues can be transfected by the direct injection of naked DNA (70); these include the thyroid gland (154), certain tumor types (172), liver cells (84), skin (82), and myocardial cells (153). Other types of cells are quite resistant to transfection unless a carrier is used. Naked DNA, larger in size than oligonucleotides, is not readily endocytosed and must therefore be packaged into a delivery vehicle, or vector (171), capable of efficient entry into cells (23,56). Naked DNA does not provoke specific immune responses; however, DNA containing dinucleotide sequences comprising cytosine followed by guanine (CpG sequences) are unmethylated when produced in bacteria and, when flanked by 2 purines on 1 side and 2 pyrimidines on the other, they provoke the innate immune response (82,104). Naked DNA can be delivered without the use of needles with a gene gun or the Jet gun (Intraject, Weston Medical, Cambridge, UK, http://www.weston-medical.com). The gene gun uses a high pressure helium stream to deliver DNA coated onto gold particles into the cytoplasm, while the Jet gun uses liquid under pressure for delivery into interstitial spaces (82). 2.2. Viral gene transfer Viruses have evolved specialized mechanisms for cellular binding and intracellular delivery, and capitalizing on these mechanisms has been a favored approach in attempting to achieve highly efficient transfer of genes to mammalian cells. Viral gene delivery has been analyzed using a number of vectors including adenoviral, retroviral, lentiviral, adenoassociated viral, and herpes simplex virus vectors. 2.2.1. Adenoviruses: Adenoviruses are highly effective vectors for transient gene transfer to different cell types (95). These are double-stranded DNA viruses whose capsid is composed of hexons and pentons, namely the penton base and fiber monomers (15). These viruses are relatively easy to produce in large quantities (67), and offer a large capacity for foreign genes, up to 38 kb (67,91). First-generation adenoviral vectors retain all essential adenoviral genes except E1A and E1B, the early functions required for the activation of most other viral genes during a productive infection (91). Second-generation adenoviral vectors are characterized by the additional inactivation of the E2 functions of DNA-binding protein or viral DNA polymerase, or by the inactivation of the E4 gene that has oncogenic potential and key regulatory functions (91). Vectors with deletions of both the E1 and E3 genes can accommodate transgenes of up to 8 kb in size; E3 is dispensable for virus propagation in cell culture (91). Adenoviral gene delivery leads to the episomal persistence of the target gene of interest within the viral vector, and it triggers a potent host immune response that makes this type of virus-mediated gene delivery unsuitable for repeated administration (67). The immune response to viral proteins expressed from adenovectors and transgene products leads to clearance of transfected cells (91). This immune response is humoral with neutralizing antibody to viral capsid proteins (7), and cellular with a cytotoxic T lymphocyte response against transfected cells (146). The host immune response consists of inflammation, production of neutralizing antibodies, and a virusspecific cytotoxic T lymphocyte response (67). The recent death of a volunteer in an adenoviral gene therapy experiment has prompted a review of the safety and efficacy of adenoviral vectors (110,111). The patient was a young man with an inherited defect of ammonia metabolism, namely ornithine-transcarbamylase (OTC) deficiency. He died in September 1999, 4 days after a genetically altered adenovirus had been infused into his liver (110,112). Over the past few years, several groups have created fully “gutless” or third-generation adenoviral vectors by removing all viral genes and replacing them with substitutes produced by helper viruses (91,112). In some reports virtually no toxicity was observed when such vectors were given to mice at high doses (112); nonhuman primate trials with gutless viruses have not yet been reported (112). In addition, encapsulated adenoviral minichromosomes with genomes of approximately 13 kb have been developed for gene transfer; as in the case of “gutless” adenoviral vectors, adenoviral minichromosomes require helper viruses and several serial passages for production (164). It is currently difficult to produce high-titer batches of gutless adenovirus and to eliminate contamination by live helper virus (91,112). There is also some suggestion that empty capsids of adenovirus are immunogenic; this possibility requires further exploration before future clinical trials (112). 2.2.2. Retroviruses: Retroviruses represent a group of viruses whose RNA genome is reverse transcribed into genomic DNA in the infected cell. The basic retroviral genome contains 3 genes known as gag, pol, and env; these genes are flanked by elements known as long terminal repeats (LTRs) (171). LTRs define the beginning and the end of the viral genome and are required for the integration of the host genome; they also serve as enhancer-promoter sequences. Viral LTRs can control the transcription of a transgene, or specific enhancer-promoter elements can be engineered in with the transgene. The retroviral genome also contains a packaging sequence, Ψ, that enables the viral RNA to be distinguished from other RNAs in the cell (171). Retroviruses offer the potential advantage of integrating genes into host chromosomes for long-term stability in dividing cells. One potential disadvantage to this form of gene transfer is the random retroviral insertion that occurs into the host genome, leading to the potential activation or inactivation of genes critical to the normal functioning of the host (67,117). In addition, human complement rapidly inactivates retroviruses; this has lead to the design of modified retroviral vectors that prevent complement activation and complement-mediated elimination (146). Retroviruses can recombine with cellular or viral DNA or RNA to produce new oncogenic viruses or replicationcompetent retroviruses (146). Indeed, the reverse transcriptase-polymerase chain reaction analyses of T-cell lymphomas that developed 6 months after the engraftment of autologous stem cells in 3 of 10 rhesus macaques during a retroviral gene transfer trial revealed the presence of several different recombinant murine leukemia viruses (MuLV) (142). Specifically, the env gene of the helper packaging virus recombined with the LTR of the Moloney MuLVderived vector to form a retrovirus that does not exist in nature (142). The retroviral vector used in this study has been replaced by a new generation of vectors possessing shorter regions of homology with the helper virus; regions of homology as short as 8–10 nucleotides have been associated with breakouts of replication-competent retroviruses (142). Another important problem that is usually encountered when retroviruses are used as vector is their inactivation by the host. In transgenic animals microinjected DNA constructs give rise to more predictable expression than infection with recombinant retroviruses; these findings suggest that host cells have the ability to recognize and inactivate retroviral DNA (17). Eukaryotic genomes are protected by elaborate defense systems that prevent the expression of alien or abnormal transcription units. The insertion of retroviral DNA into a mammalian host can trigger transcriptional silencing of the inserted sequences via mechanisms that usually involve DNA methylation within regulatory regions (16). To enhance the applicability of retroviral gene transfer, efforts have been made to devise strategies to modify the host range of the retroviral envelope glycoproteins to achieve retroviral vectors capable of delivering their genes to target human cells with high specificity (151), so-called pseudotyping of the vector. These strategies can be characterized based on the nature of the molecular target, the targeting element, and the host range modification. The molecular target is usually either a receptor or a protease. It can be approached using soluble adaptor molecules, for example, streptavidin, that have 1 binding site that interacts with the retrovirus and a second that interacts with the targeted cell (151). Alternatively, the retroviral envelope glycoproteins can tolerate a variety of genetically encoded modifications by mutations that enhance or hinder specific structures, for example, receptor binding domains, making them more susceptible to retroviral infection (151). Another approach is to block virus infectivity by grafting a protease-cleavable blocking domain onto the viral coat protein. For example, vectors containing a factor Xa-cleavable EGF domain poorly infected EGF receptor-positive target cells until they were treated with factor Xa protease (151). Additional advances in retroviral targeting should become possible with the further elucidation of crystal structures of retroviral envelope glycoproteins and the development of strategies to select optimally targeted vectors from vector display libraries (151). The transduction of hematopoietic stem cells is enhanced by the presence of fibronectin or of peptides derived from fibronectin, such as that designated CH-296 (76,183). This effect is thought to be due to colocalization of fibronectin on retrovirus on the cell surface. 2.2.3. Lentiviruses: Lentiviral vectors are human immunodeficiency virus (HIV)-based retroviral vectors that have the advantage of integrating into the genome of both proliferating and nonproliferating cells (126). They can be modified to widen their host cell range to infect most dormant cells, such as neurons (155). However, as the most commonly known lentiviral vectors are HIV derived, the safety of these vectors must be tested extensively. Before human studies can be undertaken, these vectors must be shown to be unable to recombine or interact with another virus to produce an active HIV strain. The packaging cells used to produce high-titer virus currently contain HIV proteins, which presumably will be eliminated in the near future (155). HIV contains the gag, pol, and env genes. In addition, it carries genes for 6 accessory proteins termed tat, rev, vpr, vpu, nef, and vif(3,171). These accessory proteins have the potential to cause cell damage or disease; in fact, nef can produce an acquired immunodeficiency syndrome (AIDS)-like disease if expressed in transgenic mice (77,155). The HIV env gene product restricts the infection of HIV-based vectors to CD4+ cells. This gene has been replaced with env sequences from other RNA viruses that have a broader infection spectrum; for example, glycoprotein from vesicular stomatitis virus (VSV-G) binds to many cells, including neurons (155,171). Self-inactivating (SIN) viruses have a deletion inactivating the U3 region, which is duplicated and flanks the virus on both sides; this region contains active promoter elements that can drive the synthesis of viral RNA and downstream cellular RNA (155). The injection of lentiviral vectors has been shown to result in sustained expression for over 6 months in rodent brain, liver, muscle, eye, and pancreatic islet cells, without the inactivation of expression as is seen in prototypical retroviral vectors (171). 2.2.4. Adeno-associated viruses: Adeno-associated viruses (AAV) are single-stranded DNA parvoviruses that integrate their genes into human chromosomes of dividing and nondividing cells (44,171). They are not known to cause disease in the human population. Nonetheless, 80% of adults have circulating antibodies to AAV (171). AAV have 2 genes, cap and rep, that are located between inverted terminal repeats defining the beginning and the end of the virus, and containing the packaging sequence (171). The AAV inverted terminal repeats mediate the chromosomal integration of the host genome (105). The cap gene encodes the viral capsid or coat proteins, while the rep gene product is involved in viral integration and regulation (171). In the presence of the rep gene product, AAV vectors integrate selectively into a region of chromosome 19, near a breakpoint commonly observed in chronic lymphocytic leukemias; this site specificity seems to be lost when the viral genome of vectors has been replaced with the gene to be transferred (44,171). Helper viruses provide the additional genes that AAV require to replicate. Their gene therapy applications are limited because they can transfer only small foreign genes (93), and AAV vector preparation is laborious due to the toxic nature of the rep gene product and some of the adenoviral helper proteins (171). Helper viruses are no longer necessary to produce AAV vectors; this function can be performed with plasmids that essentially eliminate contaminating wildtype virus (30). AAV have been used successfully for long-term correction of Hemophilia B in a dog model with portal vein (159) or muscular (83) injections of an AAV vector expressing clotting factor IX, the factor deficient in Hemophilia B (106). A phase 1 trial of AAV CFTR in sinusitis has also been described. The maxillary sinuses were used as a model of cystic fibrosis because they have ion transport systems and microbiology similar to those of the lower respiratory tract. The results of this randomized, nonblinded dose escalation protocol revealed that successful gene transfer in this setting is dose-dependent, with DNA transfer detected in 2 of 10 patients for at least 40 days (177). 2.2.5. Herpes simplex viruses: Herpes simplex virus type 1 (HSV-1) is another vector that has been studied for its capacity to transfer genes to mammalian cells. It is a relatively large virus that contains more than 80 genes (171). Approximately half of the genes of HSV encode accessory functions implicated in the host virus life cycle, genes that can be deleted without consequence to viral replication in cell culture (61). HSV infects the cells of the nervous system, specifically neurons of sensory ganglia where it can establish a lifelong presence as an extrachromosomal element in a quiescent phase referred to as latency (61). Reactivation from latency of unmodified HSV results in viral replication and clinical signs of infection (61). In the latency phase of HSV, even in the case of virus mutants defective for and genes are and RNA are expressed (61). HSV mutants are toxic for neurons in and many viral and vector are rapidly after injection into the (61). has been made in the development of defective viral mutants with toxicity (61). The use of HSV as a vector for gene transfer is limited because many have been infected by this virus and to of HSV (171). vectors, those from which essentially all viral proteins are have relatively of virus particles vectors containing a deletion of the gene produce viral particles (171). 2.2.6. Other viruses: The and viruses are viruses that are under as possible vectors The vectors derived from these viruses deliver their RNA genomes to the cytoplasm of target cells where they can produce large of protein from high of the gene of interest They not integrate into the genome, and thus produce foreign protein only (171). or based DNA vectors are under for (171). Their genome consists of kb of double-stranded DNA at the The viral system is for use in gene therapy because of its use as a its large and the of its molecular The virus contains a RNA polymerase, a transcription and and a polymerase, the expression of genes within the cytoplasm of mammalian cells is a double-stranded DNA virus under as a vector for gene It has a genome the terminal repeats that many other viral vectors, and it can both quiescent and dividing cells It has been tested in and it does not neutralizing antibody or cytotoxic immune against infected cells wildtype was an of early batches of the with reports of some human sequences Nonetheless, is oncogenic in and it into cellular genomes its safety must be before it can be used as a vector for human gene transfer 2.3. Nonviral gene transfer Nonviral gene transfer with vectors an of efficient gene delivery to result in lower of The of gene therapy is to the successful viral mechanisms for cellular barriers that block efficient expression of the target gene while the associated with gene The of a vector could include specific binding to the cell endosomal to the and, in some integration into the target cell The of gene delivery is the transfer of encapsulated plasmids from the to the nucleus (56). In this plasmids are endocytosed by cells into the endosomal The of this with its be to plasmids rapidly (56). is known to of and has been used in some gene therapy to endosomal of DNA 2.3.1. Chemical and physical have been developed to DNA with such as and the to DNA and it to be more readily up by cells. Chemical are for clinical gene methods such as and are also relatively for gene the of an that the cell DNA to enter the cytoplasm (82). for example, and the have been to overcome some of the barriers to efficient gene transfer of the generation of (146). In has an capacity that is thought to be a key element in its efficient gene transfer In addition, transfection by has been described. In this are coated with antibodies that to specific cell they can in the cells when the are by transfection of cells with DNA or other The are using a and the cells are to tissue culture for This has the advantage of targeting specific cell and it targeted cells to up a variety of different The that this of cells as determined by and that it results in than cells 2.3.2. Liposomal gene are composed of a membrane an aqueous The net charge of on the type and of the form with DNA at least in part via an with the negatively charge of DNA The in can DNA (59). Liposomal gene transfer has several including of of and the ability to large DNA While this of gene transfer has useful the of to DNA must be to the development of toxic the mechanism of DNA delivery and DNA remains to be fully In addition, have a limited of delivery and gene and they have with negatively target specific cells using such as antibodies to the of they can be as and (146). integrate viral envelope glycoproteins into the to mediate the cellular and entry of at the cell or within the endosomal (146). 2.3.3. Protein/peptide gene with DNA in protein and gene transfer, that is between the and and the negatively in the DNA The of and have been to DNA and to in cellular uptake of plasmids mechanisms (146). of DNA and are designated while DNA with of and are (59). DNA binding elements that are effective in gene transfer include and the protein and with repeated sequences chain to be critical to size and in gene transfer with 8 or as as those with or more DNA using a and produce large particles as determined by at that peptides with repeats of DNA and produced that in from to as the chain increased The of an as a potential followed by a to the of in a in size and of transfection as determined by production in cells as compared with There is to and control DNA and packaging with these as as the of the of such (59). Once DNA has been the of gene delivery can be enhanced by the of into the These may target specific cells via a cell gene delivery constructs contain a and a DNA-binding usually have been targeted using a number of different including EGF and an of gene transfer the cellular of for Other strategies to enhance gene delivery include with the following 1) to enhance 2) to endosomal and 3) agents to with factors that the after into the milieu (59). have random libraries to select peptides with potential gene therapy applications have 2 systems that viral for example, a and a endosomal have developed a of gene transfer by An of DNA transfection via DNA binding and was also tested in in a murine model The transfection of plasmids and chain in with

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