41 Ocular gene therapy

KEY FEATURES

Gene therapy involves the therapeutic alteration of gene expression to correct an inborn or acquired error, or to modify pathologic cellular behavior. There are two main classes of gene therapy – germline and somatic. Various types of viral vectors are employed in gene therapy. Current diseases being evaluated for therapy include malignancy, inherited retinal disorders, and retinal vascular diseases such as neovascular macular degeneration.

insert genes into cells.5 It was at this point that the proof of principle was illustrated with stable introduction of the bacterial HPRT gene analog into deficient mammalian cells.6 It was also during this period that it was postulated, based on the insight gained regarding polyomaviruses, that it may be possible to manipulate other viruses; amid a genetic revolution this gave way a decade later to the development of retroviral vectors and then to several other viral-mediated gene transfer techniques, several of which are used today and are currently the mainstay of gene therapy applications.

INTRODUCTION TO GENE THERAPY

Germline gene therapy is the replacement of genes in which an offspring would inherit a new trait. This type of gene modification is still in the early stages of human medical intervention but examples do exist, such as in cows modified to have elevated levels of milk production or the capability to secrete human hormones, or “knockin” and “knockout” mouse models, used for decades to elucidate gene function. The second, currently more medically viable and potentially less controversial, is somatic gene therapy. Somatic gene therapy is the introduction of genetic material directly into the body after development. There are three categories of somatic gene transfer: in vivo, in situ, and ex vivo. In vivo is the introduction of genes directly to the body via the blood stream; for example, in situ involves targeting a specific organ for gene transfer, such as the eye. By contrast, in ex vivo gene transfer a particular class of cells are removed from a patient, modified, and then replaced back into the patient in order to treat an abnormality or regain a particular function.

The notion of gene therapy arose in the 1960s, and with the advent of stable cell lines and advancing techniques of DNA isolation and manipulation, that idea became more of a reality. Early techniques involved primarily the use of bacteria, such as Escherichia coli, and utilized the fact that their genomes are comprised of circular DNA which can be shared with other bacteria in a process known as conjugation. This type of gene transfer is what gives bacteria the ability to share resistance genes among colonies. Microbial molecular biology soon gave rise to advances in techniques such as gene cloning and circular plasmid DNA construction. These techniques offered insights into microbial genetics and in addition led scientists to ponder whether these concepts could be applied to human maladies.

One of the first human diseases to be investigated for gene therapy was the hypoxanthine guanine phosphoribosyl transferase (HPRT) deficiency associated with Lesch–Nyhan syndrome. Researchers studying this disease were able to demonstrate that exogenous DNA was capable of uptake and expression into mammalian cells. Unfortunately, the methods available at the time were unable to produce stable expression efficiently in these cells.1 It was not until the late 1960s through early 1970s that more viable methods came into development. A major discovery arose when researchers were able to elucidate key events in the ability of polyomavirus to transform cells, integrate, and stably express their DNA.2–4 This, in addition to chemical transformation methods (i.e., calcium phosphate), enhanced researchers’ ability to

CURRENT VIRAL VECTORS

The viral vectors predominantly in use today fall into two categories: DNA viral-based and RNA viral-based. Each group has various attributes contributing to advantages or disadvantages depending on the desired therapeutic outcome. The two most commonly utilized DNA viruses are the adenovirus and the adeno-associated virus (AAV). The use of adenoviruses for gene transfer purposes was first demonstrated in 1985, by transducing human cells with thymidine kinase (TK) gene.7 Since then, their application has been documented in numerous gene therapy protocols. Wild-type adenovirus is classified as a nonenveloped iscosahedral viral particle with an average size of 80 nm. Housed within the viral capsid or protein shell is a 36-kb double-stranded DNA genome. Viral replication consists of a two-phase event termed early and late phase. During the early phase, the viral proteins expressed take control of cell cycle machinery, aid in evading immune response signaling, and initiate viral replication. The late-phase viral protein production is associated with viral assembly and packaging, at which point particle buildup and cell lysis occur. Recombinant adenoviral vectors are engineered to be replication-deficient by the removal of the earlyphase expressing genes. By manipulating the native viral genome to consist only of cis-acting elements such as inverted terminal repeats (ITRs), these vectors are considered to be “gutless” (Figure 41.1). This requires that their assembly be performed in a helper-dependent manner. This is facilitated in trans with the use of either co-transfection techniques or packaging cell lines, which provide the key structural components required for full viral particle assembly. Adenoviral vectors are capable of harboring large transgene inserts, can infect cells independent of cell cycle stage, an important consideration of ocular gene therapy due to the G0 state of the eye, and offer a high degree of transgene expression. The main drawback for the use of adenoviral vectors is that in some cases severe immune-mediated responses have been observed. This, and the fact that adenoviruses lack the capability to integrate, makes their transgene expression very transient, usually persisting for 10–50 days on average.

The second class of DNA viral vectors is AAV. The use of AAV for gene transfer has rapidly grown within the last decade and is considered by many to be one of the safest viral-based gene transfer methods available. AAV particles are small single-stranded DNA viruses composed of a 4.7-kb genome. These icosahedral-shaped virions range in size from 20 to 25 nm. Their genome consists of two open reading frames, which code for the Rep and Cap genes, with ITRs on each side of the genome. The Rep genes code for replication-dependent proteins

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VIRAL VECTOR-ASSOCIATED RISKS

Safety and ethical concerns have always been associated with gene therapy. The potential risks associated with recombinant DNA research prompted the organization of the Asilomar conference of 1975, in which Paul Berg and colleagues laid down the foundation for biosafety with regard to this field of science.16 A few years later, the National Institutes of Health (NIH) developed guidelines for the use of gene therapy in human subjects. These guidelines later led to the formation of various committees, such as the Recombinant DNA Advisory Committee, in charge of ensuring proper ethics and provisions for human safety. Several events warranted action towards the formation of these committees. One of the earliest experiments to raise public attention was the action by Dr. Martin Cline of UCLA in 1980. In an effort to treat two individuals with the condition beta-thalassemia, a rare hemoglobin disorder, Cline disregarded university internal review and performed the experiments overseas. As a result, he faced harsh criticism by multiple groups, was stripped of all NIH funding and was banned from performing any recombinant DNA experiments.17,18

Probably the most notable and publicised complications involved two human clinical trials: one for the treatment of severe combined immunodeficiency (SCID), the other involving a patient being treated for ornithine transcarbamylase (OTC) deficiency. SCID, also referred to as “bubble boy disease,” was of interest to gene therapy pioneers as they recognized this disease as a prime candidate for a gene replacement strategy. Since SCID patients lack the T-cell enzyme adenosine deaminase (ADA), a protein crucial to the immune response, replacement of a functional ADA gene was felt likely to be curative.19 Despite encouraging early results, some patients treated with ADA genecontaining retroviruses developed leukemia. It was later discovered that the integrative viral vector had positioned the ADA gene proximal to a “tumor suppressor” region of the genome in some cells, thus immortalizing those cells.20 Thus, at least some of the dangers associated with retroviral integration were made clear.

A second complication arose in 1995 when a patient with OTC deficiency was treated with adenoviral particles containing a functional copy of the human OTC gene. OTC deficiency results in an inability to break down ammonia byproducts in the blood formed during protein synthesis. One patient who received a large dose of adenoviral OTC developed an overwhelming immune response and died.21 This tragedy resulted in an immediate temporary suspension of adenoviral use for gene therapy by the NIH. It also triggered the development of stricter guidelines for gene therapy usage in human subjects.22

VIRAL VERSUS NONVIRAL VECTORS

Although viral gene therapy vectors offer the greatest degree of sustained therapeutic gene expression seen to date, several other nonviral gene transfer modalities exist. These consist of two main categories: chemical and physical gene transfer. Chemical gene transfer often employs the use of lipid or polymer-based vehicles to transport DNA to cells where transient expression in these cells is possible. The lipidbased vehicles, or more specifically cationic liposomes, are formed when aqueous plasmid DNA is mixed with hydrophobic phospho­ lipids, similar to eukaryotic cell membranes, to form micelle-like structures with the DNA of interest packaged internally. Cationic polymers are formed from either natural or synthetic small-molecular-weight peptides covalently bound to create complex branching structures. Various chemical functional groups lend charge and physical characteristics enveloping the DNA and thus producing a reliable vector delivery vehicle. Access to the intercellular space is achieved by liposomal or polymer cell fusion and is internalized by endocytosis. Once intercellular, these vehicles are packaged into an endosome where the plasmid DNA is liberated, shuttled to the nucleus, and transcribed. Once transcribed, the message is processed in the cytoplasm and translated to protein as usual.

The second class of nonviral gene transfer involves the physical insertion of DNA into cells. This is achieved by a variety of means, with the end result being the cellular uptake of naked plasmid DNA. The simplest means to this end is by direct injection of DNA systemically, normally via the blood stream, where uptake is somewhat arbitrary but is usually confined to the lymphatic system. Other methods can rely on the delivery of DNA via high-pressure application of either air or fluid. A“bioballistic” approach employs gold nanoparticles that surround the DNA and are shot into an organ or tissue by high air pressure. An analogous approach, termed hydroporation, uses high-pressure fluid, similar to physiological blood pressure, delivered to the site of interest. Although these approaches have demonstrated efficacy, their use for ocular gene therapy is likely limited. Other physical methods of gene transfer exist; these include electroporation, magnetofection, and ultrasound. Electroporation is the use of finely tuned micropulses of electricity which create small pores in a cell’s membrane and aid in the absorption of DNA. This method has been demonstrated to achieve efficient and rapid import of DNA into the nuclear region and is a routine choice for in vitro gene transfer.23 Magnetofection is dependent upon magnetic nanoparticles which complex with DNA; these mixtures are then guided by strong magnetic fields to the area of interest. DNA uptake is far greater via this method compared to naked DNA injection alone,24 yet the implications of long-term exposure to these magnetic particle in vivo are not fully understood. Finally, ultrasound is an alternative to increasing cellular permeability. This method has been shown to increase DNA uptake severalfold and appears to be a relatively safe approach. The fact that ultrasound is routinely used for diagnostic imaging by ophthalmologists makes its potential use for gene transfer attractive.

Nonviral gene therapy techniques are intriguing for several reasons. They offer a relatively safe approach to the introduction of foreign DNA to various cell types and can usually be delivered to a specific tissue type. Their application is relatively noninvasive; therefore patient discomfort can be easily managed. The main disadvantage for their use is threefold. First, the expression of the therapeutic gene is completely transient and ranges from a few hours to a few days. Second, the amount of gene product produced via these methods is not uniform. Some techniques are only capable of delivering relatively small amounts of DNA, resulting in the potential of an insufficient dosage. The third disadvantage is the fact that nontargeted cells may also be transfected, which could result in undesired gene expression in bystander cells (Table 41.1).

STRATEGIES FOR RECESSIVE VERSUS DOMINANT DISEASE

The strategies for the treatment of recessive and dominant diseases vary greatly and are constantly evolving. In recessive disease conditions, the underlying pathology is often due to a defect or mutation in both copies of a single gene. The parents of affected individuals are usually phenotypically normal and are heterozygous for disease alleles. Only when their offspring inherit both copies of the abnormal alleles are phenotypic ramifications evident. Some of the most recognizable diseases affecting individuals worldwide are recessive in nature and include cystic fibrosis, hemophilia, albinism, and phenylketonuria. Numerous recessive diseases are theorized to be highly correctable by gene therapy applications. The basic notion is that the insertion of a nonmutated gene copy into appropriate cells or tissue could lead to therapeutic phenotype rescue. One disease that has shown great promise and success, especially from an ophthalmic point of view, has been the genetic treatment of Leber’s congenital amaurosis (LCA) in the Briard dog model and recently in human patients. Replacement of the RPE65 gene by an AAV vector to affected animals has been shown repeatedly to restore vision to a state similar to that of control animals.25 This treatment has displayed such promise that it is currently being tested in multiple multicenter phase I clinical trials.

Dominant disease usually results when one gene allele is mutated and this change is sufficient to cause disease. This can occur because

Diseases Retinal in Mechanisms and Drugs • 4 section

• 41 chapterTherapy Gene Ocular