By Kevin E. Noonan --
One of the perennial promises of biotechnology is the possibility that genetic diseases can be cured by replacing a defective gene with a normal copy of it. Gene therapy has been attempted on and off for about thirty years, although initial attempts were sporadic and in some cases of doubtful provenance. And the field suffered a serious setback when a patient died undergoing clinical gene therapy trials. Gene targets for therapeutic intervention have traditionally been related to diseases mediated by hematopoietic cells, since these are most easily manipulated ex vivo and returned to the body, but there have also been attempts to cure diseases of other somatic tissues. One traditional problem has been the choice of vector: since in only rare instances have attempts been made to target the replacement gene to the site of the genetic lesion, vectors for delivering exogenous genes have involved viral sequences. But viruses (particularly efficiently integrating viruses like retroviruses) raise the specter of oncogenic transformation that has retarded their clinical development. Alternatives, most commonly adenovirus or adeno-associated virus (AAV) (see graphic below), while having less oncogenic risk have been associated with immunological responses due to the prevalence of anti-adenovirus antibodies in humans as a result of naturally occurring infections.
All this may explain the excitement that has greeted the report by authors from University College London Cancer Institute, the Royal Free National Health Service Trust, Queen Mary School of Medicine, Basingstoke and North Hampshire NHS Foundation Trust, St. Jude's Research Hospital, the University of Chicago, Children's Hospital of Philadelphia, and the Howard Hughes Medical Institute. The report, published in the New England Journal of Medicine this week, reports the results of clinical trials of gene therapy involving human blood clotting Factor IX (at right). The disease, also called Hemophilia B, is like Hemophilia A an X-linked genetic disorder associated with uncontrolled bleeding, arthopathy and early death. It is only controlled by frequent intravenous injection of Factor IX protein. It suffers not only from expense and being palliative rather than providing a cure, but also because inhibitors can arise that reduce the effectiveness of the treatment. Until now, clinical trials of gene therapy for Factor IX have shown only transient expression of the transgene but also cytotoxic T-cell response against the cells (hepatocytes) transduced with the transgene-encoding vector.
The new study, with a total of six patients, differed from earlier attempts in three ways. First, the authors produced a "codon-optimized" expression cassette in which "complementary dimers" of the construct were packed in each virion. These vectors were found to express the transgene "at substantially higher levels" than the previously used single-stranded AAV vectors. Second, the virus capsids were "pseudotyped" with a capsid of serotype 8, which the authors report have a lower levels of native serum antibodies in the human population due to a lower prevalence of infection with the serotype 8 AAV (AAV8). Finally, the virus was administered through a peripheral vein rather than into liver tissue directly, which provided a safer route for patients having compromised blood-clotting capacity.
The virus was administered in three concentration levels: low (200 billion vector genomes/kg body weight); intermediate (600 billion vector genomes/kg body weight); and high (2 trillion billion vector genomes/kg body weight). Patients were screened for the absence of neutralizing antibodies to AAV8. All patients expressed less than 1% of normal amounts of Factor IX in the blood and all but one of them were receiving Factor IX protein therapy. Only three adverse effects were reported, two of which involved anemia and the third being associated with an unrelated surgery.
All three treatment groups showed positive effects of gene transfer. In the low dose group, one participant showed elevated (~2% of normal) Factor IX levels for 16 months after treatment and while prophylactic Factor IX treatment could be discontinued this patient required additional therapy associated with "accidental injury and elective surgery." The second participant in the low dose trial showed less immediate effects of the transgene (~1% of normal Factor IX levels); these levels rose to about 2% of normal 23 weeks (~6 months) after therapeutic administration of the virus. The intermediate dose group showed variable initial response (one patient showed less than 1% Factor IX while the other showed about 4% of normal Factor IX levels immediately after treatment), but over the course of the study these values stabilized to about 2-3% of normal Factor IX levels. Finally, one member of the high dose group showed an initial large effect (Factor IX levels at about 7% of normal) but these levels dropped suddenly to about 3% in a manner consistent with hepatocyte injury due to an immunological reaction against the viral capsid. The other member of the high-dose cohort showed persistent levels that were 8-12% of normal Factor IX values, although these levels were reduced after periods of intense physical activity.
Importantly, the study reports no humoral (antibody) immunological response to the Factor IX transgene product but did show a primary immune response against the AAV8 capsid. Also significant was that no T-cell mediated immune response against the transgene were detected. There was such a response, however, against the AAV8 viral vector, particularly in the higher dosages cohorts, although this resolved.
Skeptics will note that this report is based on a total of six patients, falling far beneath any success level that would be accepted by any regulatory agency for widespread use. The authors recognize this, but also note that "this gene-therapy approach, even with the associated risk of transient hepatic dysfunction, has the potential to convert the severe bleeding phenotype into a mild form of the disease or to reverse it entirely." That promise has driven gene therapy research for a generation and this success suggests that that promise may soon be fulfilled.
For additional information regarding other related topics, please see:
• "EMA Continues to Defer Approval of First Gene Therapy Application in Europe," July 26, 2011
• "Renova Therapeutics Begins Congestive Heart Failure Gene Therapy Trials," February 8, 2011
• "GSK Forms Alliance for Development of Gene Therapy Techniques for Rare Diseases," October 20, 2010
• "Gene Therapy Experiencing a Revival," May 3, 2010
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