Introduction to Gene and Stem-Cell Therapy

Introduction

Motor neuron diseases, such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA), are devastating neurodegenerative diseases that are progressive and fatal. There is no cure and the current standard of care is merely palliative. For example, riluzole is the only FDA-approved treatment for ALS and extends life for 3–6 months. Due to the inability of conventional therapies to have an impact on these diseases, research in gene and stem cell therapies have come into focus.

Gene and stem cell therapies have the potential to make a significant impact on the treatment of ALS and SMA. For this potential to be realized the optimal gene therapy vector or stem cell needs to be selected. The various options have their pros and cons, which will be discussed in this chapter. It is necessary to take the time to assess and match the disease with the appropriate therapy.

Gene Therapy

Gene therapy proposes to replace a defective gene with the correct copy or, to introduce a therapeutic gene that will support and improve the disease environment. Vectors, both viral and nonviral, are used to deliver the therapeutic gene to the target tissue and cells.

Nonviral Vectors

Nonviral vectors consist of a nucleic acid, either naked or complexed with a carrier that aids its passage into the target tissue. They have the advantage of a low risk factor due to the absence of a viral component. Also, there is unlikely to be an immune response caused by the vector, which increases their safety profile. Their capacity for delivering nucleic acid is large and they are easier to synthesize than viral vectors. However, their efficacy is low as they lack the efficient means to reach the nucleus that viral vectors possess. This limits their usefulness as a tool for treating diseases that are systemic or affect more than one organ type. Advances in developing compounds that package the nucleic acid, to prevent degradation and enhance delivery, has resulted in improvements in the effect of nonviral gene therapy. These developments include cationic polymers, some of which have the ability to be retrogradely transported. This would be useful for peripheral neuronal delivery. Some proteins have also been incorporated into nucleic acid carriers due to their ability to bind to cell-surface receptors. For example, tetanus toxin has the ability to target neuronal cells. Fragments of the tetanus toxin have the ability to bind to receptors, be taken up into cells and be transported in a manner similar to the full length toxin. The fusion of tetanus-like peptides with nucleic acid may assist targeting specific cell populations of interest in ALS and SMA. Another option for targeting of nonviral vectors are synthetic nanoparticles. Nanoparticles can be made from a variety of different materials such as lipids and polymers. For example, silica nanoparticles have been demonstrated to result in targeted expression in neuronal cells. This expression was on a par with expression from a herpes simplex virus (HSV) vector and had the advantage of having none of the vector-associated toxicity. It is interesting to note that the majority of the data for nonviral vectors in neuronal cells comes from in vitro studies. Further research is required in vivo to thoroughly evaluate the potential of these nonviral vector options.

Viral Vectors

Viral vectors use the natural ability of viruses to infect host cells and then use the cells’ machinery to express the transgene. They have the genes that allow them to replicate removed from their genome, which renders them safer to use in a gene therapy. Viral vectors can carry either DNA or RNA, can integrate their genetic material into the host genome or exist episomally, and have different tropisms for different tissue and cell types. They also vary in the size of the transgene they can encode. All of these factors have to be taken into account when matching a viral vector with the appropriate disease. The most common viral vectors used include Adenovirus (Ad), Adeno-associated virus (AAV), Lentivirus (LV) and HSV ( Fig. 7.1 ).

Adenovirus

Ad is a nonenveloped, double-stranded DNA virus and has the ability to transduce both dividing and nondividing cells. It most commonly causes respiratory infections. When Ad infects cells, it binds to cell-surface receptors via proteins on its capsid coat. Most Ad serotypes bind to the coxsackie and adenovirus receptor. The virus is internalized by endocytosis prompted by interaction between the viral capsid and integrin receptors on the cell surface. After cell entry, the virus is released from the endosome, interacts with cytoplasmic dynein and microtubules, and is moved toward the nucleus. The Ad capsid docks with the nuclear pore complex protein. The nuclear protein histone H1 protein attaches to the viral capsid and microtubule kinesin-1 disrupts nuclear-pore-complex-docked capsids and the nuclear pore complex. This allows for access of the viral genome to the nucleus. Ad does not integrate into the host genome but exists as a linear episome in the cell nucleus. The transcription and assembly of the progeny virus takes place in the cell nucleus. The progeny virus particles are released from the cell by virus-induced cell lysis.

The development of the Ad vector has gone through several iterations as genes responsible for replication have been deleted from the original viral genome. The first-generation Ad vectors had deletions in the E1 region and/or in the E3 region of the viral genome. The E1 region is responsible for encoding proteins necessary for early gene expression. The E3 region is involved in replication and packaging of the virus. Removal of these genes leaves a transgene capacity of approximately 8 kb. However, as this vector still expressed viral genes, it was found to elicit an immune response and was cytotoxic to cells. This limited the expression of the transgene and, consequently, its usefulness as a vector. The second-generation Ad vectors had more of the viral genome deleted. The E2 and E4 regions were removed along with the original E1 and E3 regions. The deletion of additional regions of the viral genome resulted in a reduced immune response but did not eliminate the problem completely. A side effect of the additional deletions left the vector unable to replicate by itself and it required a helper virus or a stably transfected cell line, expressing the deleted regions, to replicate. The third generation of Ad vectors had most of the viral genome deleted and have also been called gutted Ad vectors. They contain the minimal genes for virus production and packaging and have a capacity of approximately 36 kb.

Ad was first used in clinical trials in cystic fibrosis patients delivering the cystic fibrosis transmembrane receptor gene to the lung epithelium. Subsequent studies demonstrated that the highly immunogenic response elicited by Ad vector administration restricted the duration of transgene expression to approximately 2 weeks. Repeated administration of the Ad vector resulted in reduced expression of the transgene with each additional administration. Advances in Ad vector development toward third-generation Ad vectors have reduced the host immune response but not eliminated it entirely. This has had the effect of extending the expression of the transgene, with expression in nonhuman primate liver detected for up to 2 years. However, to the best of our knowledge, third-generation Ad vectors have not been tested in clinical trials.

The immune response and the resulting limited gene expression have focused use of Ad vectors on cancer treatment. This includes treatment of cancers of the central nervous system (CNS). For cancer treatment, the short-term expression of Ad is an advantage. A number of different strategies have been employed that utilize Ad vectors. They have been used to deliver cytotoxic genes directly to tumors. Another approach has been to deliver a gene that converts a prodrug to its active cytotoxic form. Ad has also been used to express immune-related genes to activate or draw immune cells in the vicinity of the tumor. Another option for Ad vector use is in the area of vaccine development. This has been achieved by raising antigens that recognize the transgene or by inserting the antigen into the capsid. This has been employed to develop vaccines against bioterrorism agents such as anthrax and also against agents such as cocaine.

Ad as viral vector has the capacity to be useful in specific areas such as cancer therapy. However, with its immunogenic profile, it is not a suitable choice for either systemic or organ-targeted gene therapy.

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