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Research
Hemophilia B (HB) is an inherited bleeding disorder that is linked to the X-chromosome. HB develops as a result of a defect in the gene that encodes coagulation factor IX (FIX), an enzyme that is essential for blood coagulation. The clotting of blood prevents blood loss and subsequent injury to tissues deprived of oxygen and nutrients. The concentration of functional FIX in patients with severe HB is less than 1% of that seen in healthy individuals. Patients with HB suffer from frequent bleeding episodes, which can be associated with crippling arthropathy (bleeding into the joints) and early death. Current treatment involves lifelong dependency on frequent intravenous injections (2-3 times per week) of FIX protein concentrate. However, this treatment is not curative and is extremely expensive.
In molecular biology, a vector is an agent that carries a piece of DNA into a cell, thereby introducing new genetic information. Viruses naturally invade host cells and use the host machinery to assist in replicating the viral DNA and ultimately propagating the virus. Researchers are exploiting this ability to develop new gene therapy approaches to treat disease. In brief, desirable genetic information is incorporated into the genetic code of a virus, and that virus is then injected into a patient. In theory, the DNA delivered by the viral vector will correct the aberration or deficiency that is causing the disease. Gene therapy offers the potential to cure HB. The single administration of a vector encoding FIX could provide continuous endogenous production of the factor. Even a small increase in the level of circulating FIX to more than 1% of normal levels can ameliorate the severe bleeding phenotype.
At present, adeno-associated virus (AAV) vector–mediated gene transfer has shown the greatest promise for long-term correction of HB in the preclinical setting. However, clinical trials using serotype 2–based AAV vectors achieved only transient improvement in the expression of FIX. These results suggested that the capsid protein coating the virus stimulated an immune reaction that destroyed the host cells containing the new genetic information. Thus, stable long-term expression of therapeutic levels of FIX requires that the vector be modified to prevent (or minimize) a T-cell–mediated immune response.
Under the direction of Andrew M. Davidoff, MD, (Surgery), Arthur W. Nienhuis, MD (Hematology), and Ulrike M. Reiss, MD (Hematology), investigators at St. Jude are testing a novel gene therapy approach to treating and ultimately curing severe HB. As reported in New England Journal of Medicine1, this Phase I/II trial differs from previous clinical trials of AAV-mediated gene transfer in three important aspects.
First, working with the St. Jude Vector Development & Production Laboratory, the researchers developed a FIX-expression cassette that is packaged as complementary dimers within a single virus particle. Self-complementary AAV vectors mediate transgene expression at substantially higher levels than single-strand AAV vectors.
Second, to avoid antibody-mediated immune responses to AAV, the vectors were pseudotyped with capsid of serotype 8 (AAV8), which is less common in humans than AAV2 is and thus less likely to stimulate an immune reaction.
Finally, AAV8 preferentially targets hepatocytes (liver cells). Thus, the vector can be injected into a peripheral vein, a simple, noninvasive approach that is safe for patients who are prone to bleeding. Once injected, the vector circulates in the blood until it reaches the liver, where it infects hepatocytes and transfers its genetic information.
Of the first six adult male participants with severe HB to participate in the trial, five received regular FIX prophylaxis 2 to 3 times per week before gene transfer. Participants were enrolled sequentially into one of three dose cohorts (low, intermediate, and high). No acute changes were noted during or after the infusion in any participant, despite the persistence of the vector in bodily fluids, including plasma, for as many as 15 days after gene transfer. None of the participants experienced clinically significant changes in serum chemistries, including liver function tests during the first 6 weeks after vector infusion, and none had neutralizing antibodies to FIX at any time point. No T-cell–mediated immune responses to the FIX transgene or putative products of translation were detected in any participant. Neither participant in the low-dose cohort had a significant T-cell response after gene transfer.
The frequency of AAV8 capsid– specific T cells was increased in both participants in the intermediate-dose cohort. In the high-dose cohort, one participant showed an increased number of circulating AAV8 capsid–specific T cells at 8 weeks after gene transfer that coincided with increased liver enzyme activity. A brief course of steroids quickly reversed the elevation in liver enzymes but did not suppress FIX activity. By 10 weeks, the capsid-specific T cells were again undetectable. The other participant showed no T-cell reactivity to AAV8 capsid for as long as 8 weeks after gene transfer, though this may have been due to low cell recovery and viability.
After gene transfer, all six participants showed an increase in FIX levels that was roughly vector dose dependent. Three of the six participants no longer require FIX concentrate prophylaxis to prevent spontaneous hemorrhage, and three have been able to extend the interval between prophylaxes. These participants had severe hemophilic arthropathy before they entered the study. Maximal protection from bleeding in this subgroup may require higher endogenous expression of FIX.
Dr. Davidoff and colleagues achieved a critical step toward the goal of eliminating the need for chronic intravenous infusions for patients with severe HB. Furthermore, this work is ground breaking in that it is the first to show sustained expression of a transferred gene in humans. A single administration of the vector into a peripheral vein consistently induced long-term expression of the FIX transgene at therapeutic levels, without acute or long-lasting toxicity in participants with severe HB. Although concerns about immune–mediated, AAV capsid–induced liver damage persist, the preliminary data suggest that this process can be controlled by a brief course of steroids, without any loss of transgene expression and with clinical improvement.
Future efforts will include accruing four more participants, following each participant for a longer period to fully define the benefits and risks of this therapy, and optimizing vector dosing. This gene therapy approach, despite its risk of mild, transient liver dysfunction, has the potential to convert the severe bleeding phenotype into a milder form of the disease or to reverse it entirely.
1Nathwani AC, Tuddenham EG, Rangarajan S, Rosales C, McIntosh J, Linch DC, Chowdary P, Riddell A, Pie AJ, Harrington C, O'Beirne J, Smith K, Pasi J, Glader B, Rustagi P, Ng CY, Kay MA, Zhou J, Spence Y, Morton CL, Allay J, Coleman J, Sleep S, Cunningham JM, Srivastava D, Basner-Tschakarjan E, Mingozzi F, High KA, Gray JT, Reiss UM, Nienhuis AW, Davidoff AM. Adenovirus-associated virus vector-mediated gene transfer in hemophilia B. N Engl J Med Dec 22;365(25):2357-65, 2011. Epub 2011 Dec 10. PubMed PMID: 22149959; PubMed Central PMCID: PMC3265081. Abstract | Full Text
Photo: Ulrike Reiss, MD, Andrew Davidoff, MD, Arthur Nienhuis, MD, and John Gray, PhD
