Biological Agents for Delivery of Therapeutic Genes

 

N. R. Reshma and U. Gayathri*

Department of Biotechnology, Udaya Scholl of Engineering, Nagercoil.

 

ABSTRACT:

 

 

INTRODUCTION:

Cancer is the most common cause of death in developed countries, which are generally responsible for fatalities. Conventional therapies for cancer such as chemotherapy and radiotherapy are characterized by poor survival rates due to multiple factors including tumor development of drug-resistance and their lack of tumor specificity, resulting in undesirable side effects on healthy cells and therefore limitations on therapeutic dose. Gene therapy is a realistic prospect for the treatment of all cancers, and involves the delivery of genetic information to the patient to facilitate the production of therapeutic proteins, focusing on the treatment of monogenetic disorders such as cystic fibrosis, severe combined immunodeficiency (SCID) and muscular dystrophy. Therapeutic agents utilized in gene therapy strategies are typically encoded by DNA and produced within target cells, such as cancer cells.

 

The process generally involves introduction of DNA to a cell (transfection), which encodes for a protein and the necessary genetic elements required for expression of the gene of interest to effect successful protein production.

Therapeutic agents utilized in gene therapy strategies are typically encoded by DNA and produced within target cells, such as cancer cells. The process generally involves introduction of DNA to a cell (transfection), which encodes for a protein and the necessary genetic elements required for expression of the gene of interest to effect successful protein production. Efficient delivery of the therapeutic gene to the target tissue or cell is the most significant hurdle for successful gene therapy.

 

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Gene Delivery Vectors:

Gene Delivery Vectors carry DNA into cells involves cationic polymers, cationic peptides and cationic lipids (liposomes). Mechanical or physical techniques include the application of energy waves to cells to create transient pores in the cell membrane, thereby permitting entry of plasmid without killing the cell. Mammalian cell ‘poration’ systems include electroporation and sonoporation. Pathogenic viruses possess an innate ability to effectively Gene delivery systems can be grouped into non-biological or biological. The use of chemical means to invade human cells and express their genes within the cell. Gene therapists have harnessed the ability of viruses to package DNA, transfer it to a cell, and produce proteins within it. Viral vectors have an advantage in that their superb efficiency as delivery vehicles has evolved naturally. In terms of practicality of usage, there are associated difficulties in production, size restrictions on transgenes in some viral vectors, anti-vector immunologic responses limit their usage to a single application, while several human cancers are devoid of viral receptors and thus not transducable by viral vectors.  From a safety point of view, many viruses are toxic and elicit systemic inflammatory reactions and clinical trials have highlighted safety fears over viral vector integration (which has resulted in cases of leukemia) and fatal immune responses.

 

Bacteria as gene therapy vectors:

Like viruses, the innate biological properties of bacteria permit efficient DNA delivery to cells or tissues. The concept of exploiting bacterial species as biological gene vectors has existed for some time, and the use of bacteria to deliver therapeutics offers many advantages over other gene delivery approaches. Bacteria fall within the ‘non-viral’ class of delivery systems, investigated primarily for safety reasons, yet the biological nature of bacterial vectors means that many of the inherently beneficial traits of viral vectors are retained. Bacterial presence in excised patient tumors was first reported in the 1950s while the correlation between bacterial infection and tumor regression dates back to the nineteenth century when regression of certain tumors were noted in patients with streptococcal and clostridial infections.

 

Tumor targeting following systemic administration:

The main goal of cancer treatment is to focus therapy on tumors without harming healthy cells. When a patient’s cancer has spread, then ideally, a treatment should be administered throughout the patient’s body (e.g., intravenous administration) to target all potential tumors present, including secondary tumors at early stages of development. The ideal anti-cancer therapy would selectively eradicate tumor cells, whilst inimizing side effects to normal tissue. To successfully address these issues, it is necessary to identify “unique” tumor attributes that separate them from normal surrounding tissue. Many conventional modalities utilize the accelerated cell turnover of tumors to achieve a therapeutic response; however, only 3–5% of tumor cells are in the growth fraction, limiting efficacy. Therefore, other tumor-specific factors may present more optimal targets, such as exploiting the knowledge that 95% of tumors are hypoxic to some degree.

The first bacteria observed to have an effect on cancer cells belong to the Clostridium genus. In 1813, regression was observed in tumors of patients who developed concomitant “gas gangrene” caused by C. perfringens.

 

Lactic Acid Bacteria:

A class of bacteria, commonly referred to as Lactic Acid Bacteria (LAB), as classified by their shared ability to produce lactic acid, have been utilized naturally in food and dairy fermentations for centuries, and as such are ingested regularly by humans. Members include the Lactococcus and Bifidobacterium genera. Certain strains are known to colonize the gut (commensal bacteria) living symbiotically with humans, aiding in digestion and gut health. Pre-clinical studies have demonstrated the ability of some such bacteria to accumulate and grow specifically within tumors following intravenous injection. For example, bifidobacteria are native, harmless residents of the human gut (commensals), and certain strains have been shown to have health-promoting or probiotic benefits, and have a lengthy track record of safety in humans in the context of orally administered probiotics as food supplements. First time that ingestion of a non-pathogenic bacterium (Bifidobacterium breve) in large numbers can result in its egress from the gut to the blood supply, and traffic throughout the body, accumulating specifically within cancerous tissue (Cronin et al., 2010). The term bacterial translocation refers to trafficking of bacteria from the gastrointestinal tract (GIT), and investigations into this phenomenon are normally confined to pathogenic bacterial sepsis associated with various conditions (Berg, 1999). The ability of microorganisms to translocate, survive, and proliferate in extra-intestinal tissues involves complex interactions between the host defense mechanisms and the bacterium’s ability to invade host tissues. Although the importance of host immune function and the bacterium’s intestinal population size have been implicated as significant contributory factors, the precise mechanisms involved remains unknown.

 

Genetic engineering:

Because LAB are key manufacturing components in the food industry (cheese, yoghurt, etc.), their improvement in terms of robustness or production of new flavors has resulted in this group of bacteria being among the most studied with respect to genetic manipulation. Decades of research have made exquisite genetic engineering tools available for manipulating these bacteria. Through engineering of secreting constructs, these bacteria can mediate high-level production of soluble agents within tumor masses. We engineered our Bifidobacterium breve vector (and a number of other strains) to robustly express different genes of interest to produce desired proteins specifically within the tumors. Reporter gene (light emitting) tagging of bacteria permits their real-time visualization in vivo (Yu et al., 2004). We have engineered an imaging system for bifidobacteria that permits their detection in a variety of murine tumor models in real time by luminescence imaging. Live imaging of tumor colonizing bacteria facilitates tracking of the vector during treatment, post administration. This is advantageous for early phase clinical trials. The nature of the bacterial lux system is such that no exogenous substrate is required for detection, since all genes required for enzyme and substrate are transcribed from the 5-gene cassette (Riedel et al., 2007), and therefore is uniquely suited for imaging in the clinic with existing CCD equipment. Indeed there may even be potential for such a system to be used in a diagnostic setting, with imaging of bacteria revealing the location of metastatic tumors

 

Benefits of LAB:

i)Efficacy: These vectors are replication competent, resulting in increasing levels post delivery. Furthermore, bacteria carry multiple copies of plasmid per cell, resulting in high levels of gene delivery and expression. As a cell therapy, DNA entry to tumor cells is not required as is the case with traditional gene therapies.

ii) Safety: Food-grade bacteria have been consumed throughout history without associated pathologies. A safety property unique to bacterial vectors is their sensitivity to clinically available antibiotic treatments, enabling their control post-administration, an invaluable property for safe gene therapy.

iii) Lack of immunogenicity: A number of clinical trials involving IV administration of bacteria for tumor cancer therapy have been initiated, featuring pathogenic genera including Clostridium and Salmonella. While the toxicity observed with these vectors outweighed therapeutic benefits, the studies nonetheless pave the way for future more optimal, less toxic bacterial alternatives. Unlike current biological vectors, LAB vectors are not derived from pathogens, resulting in no requirement for genetic modification to render them safer.

iv) Cost/Ease of production: There exists a wealth of scientific, technological, and industrial experience with these bacteria. In terms of large-scale manufacture, viral vector particle manufacture is an extremely cumbersome, time-consuming, and expensive process. Traditional GMP-grade naked plasmid DNA isolation is less expensive, but often requires combination with expensive chemical vector (liposomes, cationic polymers, etc.), or delivery equipment (electroporation, etc.).

 

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Mechanism of tumor-specific replication:

Hypoxia is caused by rapidly growing tumors with insufficient blood supply, and is a well established feature of solid tumors. It has been proposed that the anaerobic nature of hypoxic/necrotic regions within tumors promotes growth of anaerobic and facultatively anaerobic bacteria. Areas of necrosis may also provide nutrients such as purines to further promote the growth of bacteria. The involvement of bacterial chemotaxis towards chemo-attractant compounds Bacterial colonisation of tumors was initially attributed to the hypoxic nature of solid tumors present in necrotic regions (e.g., aspartate, serine, citrate, ribose or galactose) produced by quiescent cancer cells has also been suggested as a contributing factor. Bacterial colonisation of tumors was initially attributed to the hypoxic nature of solid tumors (low O₂ levels). Hypoxia is caused by rapidly growing tumors with insufficient blood supply, and is a well established feature of solid tumors. It has been proposed that the anaerobic nature of hypoxic/necrotic regions within tumors promotes growth of anaerobic and facultatively anaerobic bacteria. Areas of necrosis may also provide nutrients such as purines to further promote the growth of bacteria. The involvement of bacterial chemotaxis towards chemo-attractant compounds present in necrotic regions (e.g., aspartate, serine, citrate, ribose or galactose) produced by quiescent cancer cells has also been suggested as a contributing factor.

 

The irregular and leaky nature of tumor blood vessels may be an important factor in this phenomenon. As tumors develop, they promote the formation of new blood vessels (neo-angiogenesis). However, these newly formed vessels are highly disorganized with incomplete endothelial linings and blind ends, resulting in ‘leaky’ blood vessels and sluggish blood flow. This leaky tumor vasculature may allow circulating bacteria to enter tumor tissue and lodge locally. Indeed, Yu et al. demonstrated the ability of IV administered bacteria to home to and replicate within cutaneous wounds as they healed. The vasculature of healing wounds would closely resemble the neo-angiogenesis observed in tumors. Interestingly, bacteria were cleared from wounds in these experiments (presumably by the immune system) as they healed, unlike in tumors. It is also believed that tumors are an immunological sanctuary, where bacterial clearance mechanisms are inhibited. A variety of mechanisms are employed by cancerous cells to avoid detection by the immune system resulting in inadequate immune activity within tumors,potentially providing a sanctuary for bacteria to evade immune clearance. before such an approach becomes applicable. Oncolytic vectors can also be administered systemically to achieve tumor-targeted activity

 

Exploitation of tumor-specific bacterial growth:

The specific nature of bacterial colonisation of tumors, by taking advantage of their unique physiology, may be exploited to aid cancer treatment in several ways. Because of the high degree of specificity of bacterial accumulation within tumor masses, the activity of engineered bacteria is confined to cancer sites. In the case of non-invasive species, strains can be engineered to secrete therapeutic proteins locally within the tumor environment, external to tumor cells. This cell therapy approach is especially suitable for indirectly acting therapeutic strategies, such as anti-angiogenesis and immune therapy (described later). Invasive bacteria capable of delivering genes intracellularly to cancer cells may also be administered systemically with the aim of targeting bactofection to tumors.

 

Therapeutic Strategies:

Direct cell killing:

The most direct gene therapy strategy to treat tumors involves introducing a vector and gene to a malignant cell that directly induces death of that cell. There are a number of mechanisms by which this can be achieved, including the delivery of genes cytotoxic to the cell (pro-apoptotic genes or so-called ‘suicide genes’) or through oncolysis induced by the bacterial vector itself (as is observed with Clostridium and Salmonella).

 

Oncolytic vectors:.

 The oncolytic approach uses replication competent bacteria that are capable of spreading through the tumor tissue to infect neighbouring cells, with cancer cells killed as a result of infection. Therapeutic trials employing clostridial species mainly rely on the natural oncolytic activity of the vector to achieve tumor therapeutic responses. Following IV administration, clostridial spores germinate within tumors, killing cancer cells as they replicate, and have been shown to produce significant oncolytic effects in preclinical and clinical studies.Replication-competent Salmonella vectors have also been shown to be oncolytic.Zhao and co-workers demonstrated that the S. typhimurium A1 strain grows in the cytoplasm of prostate cancer cells and causes nuclear destruction and cell death.

 

Cytotoxic genes:.

Bacterial vectors can mediate expression of agents that are cytotoxic to the host cell. The extracellular domain of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent apoptotic agent in tumor cells, with minimal toxicity to normal cells. Attenuated S. typhimurium has been used to express TRAIL under the control of the prokaryotic radiation-inducible RecA promoter, with systemic administration of this vector resulting in xenograft tumor reduction.B. longum has also been utilized to express this agent within murine tumors resulting in significant regression.Indirectly cytotoxic genes have also shown promise. Prodrug activating genes (suicide genes) encode a protein that is capable of directly or indirectly causing cell death. While some suicide genes express products that are directly toxic for the cell, e.g., Diphtheria toxin or Pseudomonas exotoxin, the best known agents encode enzymes that convert non-toxic pro-drugs into highly toxic metabolites. Gene-directed enzyme prodrug therapy (GDEPT) is a two-step approach. In the first step, the transgene is delivered into the tumor, while in the second step, a prodrug is administered which is selectively activated by the expressed enzyme. The most widely used system is the thymidine kinase gene of the Herpes Simplex Virus (HSVtk) in combination with the prodrug ganciclovir.

 

Anti-angiogenic therapy:

Angiogenesis is the formation of new capillary blood vessels from existing microvessels.For cancer therapy, strategies based on the manipulation of angiogenesis are referred to as anti-angiogenic strategies and seek to prevent new vessel formation or to inactivate pre-existing vessels. Gene-based anti-angiogenic therapy holds the potential to provide long-term anti-angiogenic protein production, and can be readily used in conjunction with other strategies. Endostatin is an endogenous inhibitor of angiogenesis, first discovered in 1997.It suppresses endothelial cell proliferation and acts as a competitor of angiogenic inducers secreted by tumor cells, such as fibroblast growth factor and vascular endothelial growth factor.

 

Immune therapy:

Upregulating the immune system:.

Cancer immunotherapy approaches concentrate on killing the tumor cells through direct or indirect intervention of various effector cells of the immune system, which include antibody-producing B cells, CD8⁺ CTL, CD4⁺ helper T cells, and NK cells. Gene therapy can be employed to induce tumor or other cells to produce immune upregulating cytokines that can attract and enhance anti-tumor activity of various lymphocytes. S. typhimurium has been used in several murine trials examining immunotherapies, with significant tumor reduction resulting from local bacterial expression or tumor cell expression of the immune-stimulating molecules IL-18, CCL21, LIGHT or Fas ligand.Preclinical studies have also used bifidobacteria in combination therapy with cytokines such as granulocyte colony-stimulating factor (GCSF), resulting in superior anti-tumor effects.Interestingly, the immune response was primarily directed against tumor cells rather than the bacterial vector cells.

 

DNA vaccination:.

The goal of cancer vaccines is to break tolerance of the immune system to specific antigens known to be expressed mainly or exclusively by particular tumor cells. DNA vaccines expressing a defined tumor antigen have shown significant promise both preclinically and in clinical trials. Bacteria that target inductive cells of the immune system are highly attractive candidates for vaccine delivery and have been developed as live vehicles for inducing protective responses to a wide variety of antigens.Members of the Salmonella genus have been widely used as antigen carriers and several well-characterized safety attenuated strains are available.Salmonella is capable of triggering both humoural and antigen-specific T-helper and cytotoxic responses. S. typhimurium vectors deliver transgenes to the body via the monocyte cell population. After oral intake, the bacterial vector cells are phagocytosed by monocytes in the intestine. The monocytes differentiate and migrate to lymph nodes and the spleen. Finally, the attenuated auxotrophic S. typhimurium (unable to replicate in mammals) lyse and release plasmid into the cytoplasm of monocytes, followed by expression of the desired antigen and presentation to the immune system.

 

CONCLUSIONS:

Despite the potential for live bacterial delivery systems, it is clear that in many cases further work is required to limit potential adverse side-effects and to optimise delivery. Since cancer is a multi-faceted disease, combining therapies with different therapeutic targets can achieve synergistic effects. Other combinations examined preclinically involve augmentation of tumor bacterial colonisation by attempting to make the tumor environment more hypoxic to “attract” bacteria. ‘Biological containment’ strategies may aid in overcoming these issues, whereby the vector is engineered to survive in the host but not in the external environment where specific nutrients are limiting.  A safety property unique to bacterial vectors is their sensitivity to clinically available antibiotic treatments, enabling their control post-administration, an invaluable property for safe gene therapy. Overall, the development of live bacterial vectors with potential for delivery of therapeutic agents is an exciting area of research that is gaining acceptance by clinicians and regulatory authorities for its potential to deliver positive clinical outcomes. Whilst more needs to be done to improve the safety and efficacy of some systems, this is clearly a technological approach that will yield dividends in the coming years.

 

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