Gene Therapy: Overcoming Physiological Barriers

Callie Deng

Johns Hopkins University

Publication Date: January 15, 2015

At first glance, the idea behind gene-based therapeutics appears to be a straightforward one. Given the vast majority of diseases are caused by genetic mutation, gene-based therapy aims to reverse the damage through targeted delivery and expression of therapeutic DNA. One of the most common approaches involves the insertion of DNA that functionally replaces or supplements the impaired gene. To achieve this, DNA is isolated and packaged into a molecular vector that targets specific cells, such as tumor cells. Gene therapy can be done in vivo, via vector injection into the body, or ex vivo by isolating target cells and treating them before reintroducing the modified cells back into the patient’s body. In both cases, designing a vector capable of effective transfection is critical.

However, this task is every bit an engineering problem as it is a biological one; for effective uptake and integration of DNA to occur, the vector must overcome a host of physiological barriers, without causing cell damage. This can be achieved using either viral or non-viral vectors, but with varied results. Viral vectors are currently one of the most common and effective methods of gene delivery, accounting for approximately 66% of gene therapy clinical trials in 2012 (Ginn et al., 2013). In essence, this approach takes advantage of the readily available machinery that viruses employ to infect cells and replicate their own genetic material (Wang et al., 2012). These viral vectors are modified to eliminate the aberrant self-replicating nature of viruses that would otherwise be toxic to the human body. There are a wide variety of viruses with distinct advantages and disadvantages in terms of cell targeting, genome-integration, and immune response. For example, retroviruses randomly integrate their own genome into the target cell’s genome, which risks disrupting the function of another gene. On the other hand, adenoviruses (not to be confused with adeno-associated viruses) do not integrate their DNA into the target cell’s genome, which means that any corrective gene expression will be lost after a few weeks.

Some of the earliest clinical trials for utilizing viral vectors for gene therapy date back to the late 1990s and early 2000s, in studies on patients with X-linked severe combined immunodeficiency (SCID) (Qasim and Gennery, 2014). SCID is caused by a deficiency in the γc cytokine receptor, which blocks T and NK lymphocyte differentiation, survival, and growth early on. Researchers performed an ex vivo infection of CD34+ cells with a γc -retroviral vector on two patients. After a 10-month period, researchers observed that the normal-functioning γc transgene had been incorporated into the T and NK lymphocytes. Following treatment, both patients had lymphocyte counts and function comparable to their age-matched controls, indicating that the therapy had fully corrected the disease (Cavazzana-Calvo et al., 2000). Gene therapy has seen similar success in the treatment of Adenosine deaminase deficiency.

Though viral-vectors are still widely preferred for their effective transfection, non-viral vectors have seen an increase in their development and clinical usage in recent years. One of the major drawbacks of viral-vectors is its limited capacity to carry genetic material. Synthetic non-viral vectors can be engineered to accommodate much longer stretches of DNA to be incorporated into cells. But without the naturally evolved mechanisms of their viral counterparts, non-viral vectors have to be engineered such that they can overcome multiple intracellular and extracellular barriers on their own. Ideally, gene vectors need to be able to 1) assemble with the DNA cargo, 2) target cells of interest, 3) escape endosomes and transport to the nucleus, 4) evade phagocytosis, and 5) avoid nonspecific interaction and aggregation to toxic levels (Wang et al., 2012). The difficulty lies in orchestrating all of these functions in a single particle such that the structural design made to achieve one function does not later hinder the execution of another function.

Take for instance a vector that employs the chemical poly(ethylene glycol) (PEG), which is necessary for both steric stabilization and avoiding clearance by phagocytes. While these properties are useful when the vector circulates in the blood compartment, once the vector is internalized by a cell, PEG is counterproductive. The presence of PEG prevents interaction with the endosomal membrane, making it impossible for the vector to escape enzymatic degradation. The vector and its therapeutic DNA never make it to the nucleus. A solution to this challenge is to synthesize materials with “bio-responsive association/dissociation” properties (Wang et al., 2012). One such example is the incorporation of a pH-sensitive molecule that links PEG to the rest of the vector. In the acidic environment inside an endosome where PEG is counterproductive, the pH-sensitive linker hydrolyzes, thus cleaving off the PEG.

Another challenge for both viral and non-viral vectors is achieving targeted, long-lasting integration of therapeutic genetic material. Although DNA is commonly used, other genetic materials, such as siRNA and shRNA, also show therapeutic potential. Small interfering RNA (siRNA), binds its target mRNA to temporarily silence aberrant gene expression, so its vector only needs to enter the cell cytoplasm before the siRNA can take effect. Small hairpin-shaped RNA (shRNA) can be expressed on plasmid DNA to also trigger the interfering RNA pathway. Between the two, shRNA invokes more stable gene knockdown (Arthanari et al., 2010; Dai et al., 2009).

Most recently, gene therapy has shown promise in treating inherited disease via a “gene-repair” approach. Traditionally, viral-vector gene therapy has involved semi-random integration of a functional gene into the target cell’s genome. However, Genovese et al. proposed engineering a nuclease that excised the mutated gene and incorporating the functional gene through natural homologous recombination. A significant benefit of this gene-repair approach is that it precisely integrates the functional gene within the genome, thus avoiding disruption of other important genes that could lead to toxic side effects. The researchers transfected hematopoietic stem cells ex-vivo and following the “gene-repair” method, transferred the cells back into immunodeficient mice. The gene-corrected cells were present in the mice for up to 18 weeks after treatment (Fischer, 2014).

Gene therapy has significant potential as a treatment for both inherited and acquired diseases. The recent push for developing anti-HIV gene therapy in stem cells (Zhen and Kitchen, 2013) is testament of its evolving scope in medicine. Perhaps one of the reasons gene therapy is so appealing is because it addresses the root of disease: an error in gene function. Its success with inherited immune disorders gives reason to believe that similar successes can be achieved for other ailments. However, as it is, there are numerous physiological barriers that stand between the therapy and its effective application. Further research is necessary to incrementally, but thoroughly address each of the remaining biological obstacles to ensure effective, targeted gene delivery.


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