Genetic engineering

Genetic engineering is the process by which a functional gene is introduced into a new tissue or organ in order for it to express a new characteristic or feature. Genetic engineering, in the form of 'gene therapy', reached the public media through attempts in the early 1990s to cure severe combined immunodeficiency disorder (SCID; otherwise known as 'bubble boy disease'). Investigators in type 1 diabetes, as in many other fields of medicine, rushed into this promising area; leading objectives were modification of islet cells to render them resistant to immune destruction prior to transplantation, altering various cell types to convert them into insulin-producing cells for later transplantation into the same individual, or altering bone marrow cells in such a way that they would improve therapeutic outcomes (such as prevention of late complications) following transplantation. Sadly, the reality of genetic engineering did not match its promise, and scientific research in this area declined dramatically in recent years relative to a decade ago. However, progress in other medical disciplines over the same time period has recently rekindled interest in this otherwise promising notion.

Introduction

A good portion of our understanding the pathological basis for human disease emanated from advances in the broad discipline of molecular biology. However, the benefits of molecular biology extend far beyond the perceived origins of disease and include uses in diagnostics, research tools, as well as therapy. Indeed, one of the more promising therapeutic applications for molecular biology is that of genetic engineering. Genetic engineering involves manipulation of an organism's genome whereby foreign DNA or synthetic genes are introduced into an organism or cell of interest.[1] An example of where such an approach benefited those with type 1 diabetes is that of insulin-producing bacteria; a form of genetic engineering where, in the 1980s, recombinant insulin replaced the hormone previously extracted from pancreases of animals. More recently, in an attempt to overcome the limitation of insufficient insulin due to a lack of beta cells within the body, investigators have sought to restore the this ability in type 1 diabetes patients via genetic reprogramming of various form of cells into insulin producing cells. This list would include turning both adult stem cells as well as embryonic stem cells (ESC) into insulin-producing cells. As an alternative to stem cell-based approaches, others have attempted to convert non-beta cells into functional insulin-producing surrogates; this approach, through the genetic introduction of specific transcription factors has produced cells such as hepatocytes, pancreatic exocrine and endocrine cells, or others. Yet another approach involves the introduction of the insulin gene into target tissues. Finally, the concept of improving existing beta (islet) cells by genetic engineering has also been attempted; this, involving the introduction of genes into islets (prior to their transplantation) that would render them resistant to immune destruction. While advances in each of these approaches have occurred over the years, a successful means for using genetic engineering to allow for avoidance of exogenous insulin replacement is not currently available, thereby remaining a hope for the future.

Using the insulin gene to generate insulin-producing cells

Since the advent of gene therapy, a number of investigations have noted the ability to cure diabetes (i.e. reverse hyperglycaemia in either induced or spontaneous animal models of disease) with introduction of an insulin or proinsulin gene into one of a variety of cell types. Examples of non-beta cells effectively altered to produce insulin include muscle, fibroblasts, neuroendocrine cells and hepatocytes (i.e. liver cells). The gene for this hormone is injected alone, however – being attached to one a series of regulating constituents (promotors) including the insulin, cytomegalovirus, phosphoenolpyruvate carboxykinase and L-pyruvate kinase, amongst others alongside of being shuttled into a cell by means of a virus (e.g. adenovirus, adeno-associated virus, etc.). Sadly, in most instances, a number of limitations have occurred that thus far have limited this approach for eventual application to human testing. Amongst these, the production of insulin was low and often transient, the kinetics of production did not match those akin to a normal beta cell response to glucose, and in the case of proinsulin, additional machinery (i.e. proteases) necessary to render modification to a biologically active form was lacking. To this end, amongst the aforementioned cell types tested, hepatocytes have been of most interest due to their possessing molecules capable of sensing glucose (e.g. glucokinase, glucose transporter 2). Yet, the kinetics of insulin production, thus far, is far from normal with such efforts. Beyond this, the immune system of animals treated with these viruses (that are vital for the introduction of the insulin gene) often respond against said virus, thereby limited the ability to 're-dose' an individual with the therapy allowing for long-term production when and if the ability for animal to produce insulin is lost. Hence, for the last few years, efforts in this area have slowed (in terms of progress). However, it is hoped that with new discoveries in viral vectors and knowledge gains in terms of the response of an insulin producing cell to insulin, that the future will find a way for this technique to have an impact on those with type 1 diabetes.

Reprogramming gut and pancreatic cells

As noted above, one means of increasing the efficiency of devising a cell capable of producing an effective insulin response to glucose stimulation is to use cells of similar embroyonic (i.e. developmental) origin. In the case of beta cells, obvious candidates would therefore include the gut and pancreas, including both exocrine as well as endocrine components. To this end, intestinal K cells, a constituent of the gut thought to possess constituents favouring glucose responsiveness, have been modified in mice injected with viral vectors possessing the human insulin under the control of the gastrointestinal inhibitory peptide promoter. These mice expressed and secreted insulin from intestinal K cells in a glucose responsive fashion. While promising in mice, improved methods of gene delivery are required to human application as the efficiency of this process is low and the process of cellular turnover may require repeated application. Turning to the pancreas, a process known as islet neogenesis, the budding of new islets from pancreatic progenitor cells located in or near pancreatic ducts, has long been assumed to be an active process in the pancreas. With this, it is not surprising that several studies have suggested that insulin-producing cells can be generated from adult pancreatic ductal tissues, exocrine acinar cells, as well as (normally) glucagon-producing alpha cells. Here too, each of these notions form promise but a variety of limitations, ranging from safety (including tumorgenicity), duration of production, kinetics of the insulin response, effective dosing, and more, have thus far posited these notions to the realm of future therapies for type 1 diabetes.

References

  1. ^ http://www.ornl.gov/sci/techresources/Human_Genome/medicine/genetherapy.shtml

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