SAFE & EFFECTIVE LONG-TERM
GENE THERAPY TECHNOLOGY

INTRODUCTION
Conventional biotechnology implements the use of recombinant proteins to treat patients suffering from various pathologies. Gene therapy proposes the use of nucleic acids (DNA) to treat patients rather than the use of proteins. There are many differences between both therapeutic platforms. One central difference between both is that gene therapy promises a single definitive curative clinical event in which the cells within the patient will be responsible of producing the therapeutic protein for a prolong period of time (years) or perhaps even for the rest of the patients life. On the contrary, the treatment of patients with recombinant proteins requires the constant injection of the therapeutic protein into the patient for the duration of the patients life.







GENE THERAPY
The field of "gene therapy" utilizes various methods and techniques that introduce wild type "normal" functioning genes into the cells of a patient. These cells will then produce the "normal" protein, thus correcting the underlying pathology caused by the absence of the protein or its defective structure. Gene therapy can also be of use in pathologies in which the long term presence of a determined protein intervenes in a specific aspect of the pathologic process. Introduction of the therapeutic gene can be done either directly into the patients cells (in vivo gene therapy) or cells from the patient can be extracted, transduced in a laboratory and then re-introduced into the patient (ex vivo gene therapy).







LIVER BASED GENE THERAPY
Given various anatomical properties and its central role in the production of most soluble proteins in the blood, the liver is an important target organ for gene therapy as the secreting platform for the therapeutic protein.

Pathologies such as certain forms of diabetes or hemophilia would be resolved if the gene coding the wild type functional protein would be delivered into hepatocytes.



In turn, these cells would synthesize and secrete the protein into the circulation (B) correcting the deficiency and resolving the pathology.

As a result, a variety of methods and techniques have been developed with the goal of permanently transferring genes into the liver.

However, the data reported by various scientific groups strongly suggests that the observed transient nature of foreign gene expression observed in the liver is due to a severe immunological response directed to the transduced cells which in turn triggers the removal of the hepatocytes carrying the therapeutic gene. This immune response has been divided into two central components: a) the activation of a strong inflammatory response and b) the activation of programmed cell death, (apoptosis). (Andre Lieber, & Mark Kay J Virol 1998, 72(11): 9267)

In this regard, a variety of strategies have thus been developed with the aim of ameliorating or attenuating the immune response directed to the transduced cells thus extending the expression time of the transgene. Immune suppression with various drugs such as cyclophosphamide, cyclosporine, azathioprin and prednisone has shown relative improvements extending transgene expression to several weeks. Other strategies include blocking the CD40 –CD40L interaction using CTLA4 immunoglobulin Fc (CTLA4-Ig). Another strategy to prolong transgene expression has been described; since tumor necrosis factor alpha (TNFa) is a central mediator of inflammation in gene therapy, a soluble form of the TNF receptor has been employed as well as anti-inflammatory cytokines such as IL-10. In addition, the transient nature of foreign gene expression is also due to the activation of programmed cell death, a process called apoptosis. In the context of gene therapy, efforts to control apoptosis have focused on diverting the signals involved in its activation mainly through TNFr, Fas and TRAIL and Bcl-2.

In addition to pharmacological strategies, another method for extending foreign gene expression has been through the use of viral vectors that do not induce such a strong immune response. In this regard, gutless adenovirus vectors have been used as well as AAV virus. These vectors have taken gene expression from a couple of weeks to a few weeks, perhaps even months.

Despite this important progress, there is still a fundamental need to safely accomplish prolonged/sustained expression of the therapeutic gene using the liver as a platform for the secretion of the therapeutic protein.

KEY FINDINGS


GBT’s proprietary gene therapy technology accomplishes the following key findings:

1) GBT’s gene therapy technology completely avoids viremia.

2) Avoiding viremia thwarts the unwanted presence of vector in peripheral non target organs. Of particular importance is the avoidance of vector genomes in the germ cells of the testes and ovaries.

3) One hundred percent (100%) of the administered vector genomes become transcriptionally active. This fact enables the possibility of using logarithms less amount of vector without compromising therapeutic protein output. On the contrary, when conventional methods of vector delivery are used, most of the administered vector load is lost in the circulation. Hence, therapeutic protein output is very low. Gene therapy groups throughout the globe try to compensate for the lost genomes by infusing astronomically high quantities of vector loads into patients and animal models of disease with well documented undesirable toxicological effects.

4) Since 100% of the administered vector genomes become transcriptionally active, pharmacological linear correlation between vector dose and therapeutic protein output is reproducibly achievable. Furthermore, GBT’s proprietary gene therapy technology achieves safe and effective control over therapeutic serum levels of any desired protein, either intracellular or secretable. On the contrary, when conventional methods of vector delivery are utilized, most of the administered vector load is lost in the circulation. Hence, control over how many genomes will be transcriptionally active is impossible. This in turn results in the well documented phenomenon known in the field of gene therapy as "non linear pharmacological kinetics". This translates into the undesirable fact that no matter how much more vector you administer into a patient or an animal disease model, there is no control over therapeutic protein output. Hence, achieving safe and effective reproducible control over therapeutic serum levels of a desired protein has not been reproducibly documented.

5) When GBT’s gene therapy technology was performed on rats and pigs using high dosages (>1010) of 1st generation (E1 deleted) and 3rd third generation (E1-E3 deleted) recombinant Adenovirus (both known to be highly immunogenic), no cytokine storm was observed. We hypothesize that GBT’s technology thwarts the well documented Adenoviral activated cytokine storm through two possible mechanisms; 1) the NFkB signal pathway is not activated or 2) the NFkB signal pathway prioritizes the anti-apoptotic HIF-1a pathway.

6) When GBT’s gene therapy technology was performed on rats and pigs using high dosages (>1010) of 1st generation (E1 deleted) and 3rd third generation (E1-E3 deleted) recombinant Adenovirus, no apoptosis (programmed cell death) was observed. We hypothesize that the well documented Adenoviral activated NFkB pro-apoptotic pathway was not activated.

7) Furthermore, when GBT’s technology was performed on rats and pigs using high dosages (>1010) of third generation (E1-E3 deleted) recombinant Adenovirus, no liver inflammation was observed histopathologically. We hypothesize that the combination of no cytokine storm, in addition to the avoidance of viremia, blocks the precise immunological signaling required to trigger Adenoviral induced liver inflammation.

8) Interestingly, when GBT’s gene therapy technology was performed on rats and pigs using high dosages (>1010) of 1st generation (E1 deleted) and 3rd third generation (E1-E3 deleted) recombinant Adenovirus, no significant liver damage was documented when measuring serum liver damage enzymes AST and ALT. We hypothesize that this is the reflection of no liver inflammation and no detectable histological damage. The low levels of ALT and AST document are related to the procedure required for properly execute GBT’s technology which resolved within 7 days of the procedure.

9) Importantly, GBT’s gene therapy technology achieved sustained transgene expression. When GBT’s gene therapy technology was performed on rats and pigs using 1st generation (E1 deleted) and 3rd third generation (E1-E3 deleted) recombinant Adenovirus as vectors, prolonged gene expression was observed; one year in rats, 2 months in pigs (so far).- We hypothesize that since no immunological activation was observed, transduced hepatocytes did not induce an immune response to be remove them from the pool of liver cells.

PLATFORM TECHNOLOGY
Potential areas of therapeutic use:
  • Hemophilia
  • Rheumatoid Arthritis
  • Diabetes
  • Chronic Pain
  • HIV / AIDS
  • Alpha-1-antitrypsin deficiency
  • Cancer
  • Multiple Sclerosis
  • Inborn Errors of Metabolism
Any pathology that can be treated by prolonged gene expression of a soluble or intracellular protein.