doi: 10.1161/01.ATV.0000203502.01793.8d
© 2006 American Heart Association, Inc.
Letters to the Editor |
Gene Therapy With Lipoprotein Lipase Variant S447X
Blackburn Cardiovascular Genetics Laboratory, Robarts Research Institute, London, Ontario, Canada
To the Editor:
Ross et al recently reported a dazzling series of in vivo experiments1 showing reversal of abnormal biochemical phenotypes in Lpl–/– mice through adenoviral-mediated gene transfer of the so-called "gain-of-function" S447X prematurely truncated human variant of lipoprotein lipase (LPL or LIPD). Furthermore, all readouts in lipase-deficient mice treated with this human variant were at least as good as (and usually better than) those in mice treated with wild-type human LPL,1 providing some of the best evidence to date for a "gain-of-function" associated with this human variant, albeit in mice. These experiments offer hope for LPL-deficient patients, whose life quality and duration are compromised by their dyslipidemia. Indeed, somatic gene transfer experiments using adeno-associated virus (AAV) to deliver S447X (AAV1-LPLS447X) into the muscle of some patients with elevated plasma triglycerides (TGs) and low plasma LPL activity are underway.2,3
As this groundbreaking work proceeds, 2 points are worth remembering. First, the LPL S447X variant was initially discovered in patients with severe hypertriglyceridemia through genomic DNA screening experiments that were designed to detect loss-of-function mutations in LPL.4 One of these was my patient: a heterozygous woman of European ancestry 45 years of age with fasting chylomicronemia and recurring pancreatitis, whose plasma TGs ranged between 25 and 80 mmol/L despite good compliance with a low-fat diet and fibrate therapy. Her APOC2 was normal and her plasma postheparin LPL activity was &50% of normal, similar to some patients in whom AAV1-LPLS447X therapy is being contemplated.2,3 She had no other mutations in LPL and or in any other gene so far studied. In fact, S447X was originally implicated as being causative for her dyslipidemia, before its presence in many completely healthy individuals implied that it must be a normal variant.4 Another patient, a 30-year-old female, had plasma triglycerides ranging between 30 and 100 mmol/L and recurring pancreatitis, with depressed postheparin LPL activity and normal APOC2; she was a compound heterozygote for the LDL loss-of-function mutation G188E on one allele and S447X on the other allele. Thus, suggestions that somatic transfer of AAV1-LPLS447X may help patients with LPL deficiency and perhaps those with other molecular forms of hypertriglyceridemia5 must be considered with caution. Certainly, germline presence of S447X did not protect some patients against severe hypertriglyceridemia.4 Patients with pure homozygous molecularly proven LPL deficiency (ie, analogues of the Lpl–/– mouse) rather than those with other forms of hypertriglyceridemia would be expected to benefit the most.
The second point is that without parallel-blinded somatic transfer using wild-type LPL, any benefit observed with somatic transfer of AAV1-LPLS447X will not resolve the issue, which, for some of us, is still contentious of whether S447X really has a biologically or clinically meaningful "beneficial" or "gain-of-function" advantage over wild-type LPL in humans. Persistent attribution of a "gain-of-function" to this nonsense variant during human somatic gene transfer experiments in the absence of appropriate controls with wild-type LPL might be unintentionally misleading, especially if it is ever shown that similar clinical outcomes could have been achieved using the wild-type LPL gene.
Acknowledgments
This work was supported by the Jacob J. Wolfe distinguished medical research chair, the Edith Schulich Vinet Canada research chair (Tier I) in human genetics, a career investigator award from the Heart and Stroke Foundation of Ontario, and operating grants from the Canadian Institutes for Health Research, the Heart and Stroke Foundation of Ontario, the Ontario Research and Development Challenge Fund (project 0507), and by Genome Canada.
References
1. Ross CJ, Liu G, Kuivenhoven JA, Twisk J, Rip J, van Dop W, Excoffon KJ, Lewis SM, Kastelein JJ, Hayden MR. Complete rescue of lipoprotein lipase-deficient mice by somatic gene transfer of the naturally occurring LPLS447X beneficial mutation. Arterioscler Thromb Vasc Biol. 2005; 25: 2143–2150.
2. Nierman MC, Rip J, Twisk J, Meulenberg JJ, Kastelein JJ, Stroes ES, Kuivenhoven JA. Gene therapy for genetic lipoprotein lipase deficiency: from promise to practice. Neth J Med. 2005; 63: 14–19.[Medline] [Order article via Infotrieve]
3. Rip J, Nierman MC, Sierts JA, Petersen W, Den Oever KV, Raalte DV, Ross CJ, Hayden MR, Bakker AC, Dijkhuizen P, Hermens WT, Twisk J, Stroes E, Kastelein JJ, Kuivenhoven JA, Meulenberg JM. Gene therapy for lipoprotein lipase deficiency: working toward clinical application. Hum Gene Ther. 2005. [Epub ahead of print].
4. Hata A, Robertson M, Emi M, Lalouel JM. Direct detection and automated sequencing of individual alleles after electrophoretic strand separation: identification of a common nonsense mutation in exon 9 of the human lipoprotein lipase gene. Nucleic Acids Res. 1990; 18: 5407–5411.
5. Rader DJ. Gain-of-function mutations and therapeutic implications: lipoprotein lipase S447X to the rescue. Arterioscler Thromb Vasc Biol. 2005; 25: 2018–2019.
In Response:
Department of Medical Genetics, University of British Columbia, Centre for Molecular Medicine and Therapeutics, Vancouver, British Columbia, Canada
Amsterdam Molecular Therapeutics, Amsterdam, The Netherlands
Department of Experimental Vascular Medicine, University of Amsterdam, Academic Medical Center, Amsterdam, The Netherlands
Department of Medical Genetics, University of British Columbia, Centre for Molecular Medicine and Therapeutics, Vancouver, British Columbia, Canada
We would like to thank Dr Hegele for his comments on our publication describing the preclinical results of gene therapy in a mouse model of lipoprotein lipase (LPL) deficiency.1 The potential for using LPL gene therapy to treat this disease has been investigated extensively,2–6 most recently using an adeno-associated viral vector (AAV) to express the LPL variant S447X in skeletal muscle of LPL-deficient mice.6 Since then, preclinical toxicity and biodistribution studies have led to regulatory approval for the clinical testing of this gene therapy vector in LPL-deficient patients.7
Dr Hegele raises 2 points. The first is that LPLS447X gene therapy may not be effective for all forms of hypertriglyceridemia. The second is related to the use of the S447X variant instead of the wild-type gene.
We agree that LPL gene therapy may not overcome all causes of hypertriglyceridemia, and this is not our aim. We are hopeful that LPL gene therapy will reduce plasma triglycerides in patients with hypertriglyceridemia caused by reduced LPL enzyme activity. For this reason, the clinical trial of AAV1-LPLS447X requires that all patients must have proven genetic LPL deficiency confirmed by DNA sequence analysis of the LPL gene.7
Referring to the report from Hata et al, Dr Hegele points out that "germ line presence of S447X did not protect at least three patients with hypertriglyceridemia." However, there are conditions for which the presence of the S447X variant would not be expected to overcome hypertriglyceridemia, such as ApoCII deficiency or inhibitory anti-LPL autoantibodies in some patients with autoimmune disease,8 and these patients would not be considered candidates for LPL gene therapy. Moreover, S447X carriers with additional LPL gene mutations would also be expected to exhibit hypertriglyceridemia. For example, Faustinella et al described patients with hypertriglyceridemia caused by homozygous Asp156Gly mutations in the LPL gene, who were also carriers of the S447X variant.9 Therefore, the beneficial effects of the S447X variant are not absolute, and other causes of hypertriglyceridemia should be taken into account.
Dr Hegele also questions the use of the S447X variant, and not wild-type LPL, as a therapeutic approach. The S447X variant was first discovered by Hata et al in an effort to identify mutations in hypertriglyceridemic patients. The S447X variant was present in 33% of normal controls (n=86), including 2 homozygous S447X carriers, and in only 9% of hypertriglyceridemic cases (P=0.037),10 and this provided the first evidence that the S447X variant may actually be a beneficial mutation. This finding has been replicated 27 times10–36 demonstrating that LPLS447X is a beneficial variant associated with an improved lipid profile or a reduced risk of cardiovascular disease (Table).
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The parallel testing of AAV-mediated delivery of LPLWT and LPLS447X in humans, although certainly ideal from a scientific standpoint, is unfortunately not a realistic option at this time because of costs of development and regulatory requirements. The naturally occurring LPLS447X was selected as a transgene because it shows beneficial improvements in lipid profiles in human S447X carriers, as demonstrated in the Table. Our article also demonstrates that expression of the S447X variant is a more potent triglyceride-lowering strategy than a similar one using wild-type LPL.1 Finally, a single administration of an AAV vector expressing LPLS447X shows long-term complete correction of the hypertriglyceridemia in LPL-deficient mice,6 and preclinical toxicity and biodistribution studies have led to regulatory approval for clinical testing in LPL-deficient patients.7 Together, these provide a strong rationale for choosing the S447X variant for gene therapy in LPL-deficient patients.
References
1. Ross CJ, Liu G, Kuivenhoven JA, Twisk J, Rip J, van Dop W, Excoffon KJ, Lewis SM, Kastelein JJ, Hayden MR. Complete rescue of lipoprotein lipase-deficient mice by somatic gene transfer of the naturally occurring LPLS447X beneficial mutation. Arterioscler Thromb Vasc Biol. 2005; 25: 2143–2150.
2. Excoffon KJ, Liu G, Miao L, Wilson JE, McManus BM, Semenkovich CF, Coleman T, Benoit P, Duverger N, Branellec D, Denefle P, Hayden MR, Lewis ME. Correction of hypertriglyceridemia and impaired fat tolerance in lipoprotein lipase-deficient mice by adenovirus-mediated expression of human lipoprotein lipase. Arterioscler Thromb Vasc Biol. 1997; 17: 2532–2539.
3. Liu G, Excoffon KJ, Benoit P, Ginzinger DG, Miao L, Ehrenborg E, Duverger N, Denefle PP, Hayden MR, Lewis ME. Efficient adenovirus-mediated ectopic gene expression of human lipoprotein lipase in human hepatic (HepG2) cells. Hum Gene Ther. 1997; 8: 205–214.[Medline] [Order article via Infotrieve]
4. Liu G, Excoffon KJ, Wilson JE, McManus BM, Miao L, Benoit P, Duverger N, Branellec D, Denefle P, Hayden MR, Lewis ME. Enhanced lipolysis in normal mice expressing liver-derived human lipoprotein lipase after adenoviral gene transfer. Clin Invest Med. 1998; 21: 172–185.[Medline] [Order article via Infotrieve]
5. Liu G, Ashbourne EK, Wilson JE, McManus BM, Rogers QR, Miao L, Kastelein JJ, Lewis ME, Hayden MR. Phenotypic correction of feline lipoprotein lipase deficiency by adenoviral gene transfer. Hum Gene Ther. 2000; 11: 21–32.[CrossRef][Medline] [Order article via Infotrieve]
6. Ross CJD, Twisk J, Meulenberg JM, Liu GQ, van den Oever K, Moraal E, Hermens WT, Rip J, Kastelein JJP, Kuivenhoven JA, Hayden MR. Long-term correction of murine lipoprotein lipase deficiency with AAV1-mediated gene transfer of the naturally occurring LPLS447X beneficial mutation. Hum Gene Ther. 2004; 15: 906–919.[Medline] [Order article via Infotrieve]
7. Rip J, Nierman MC, Sierts JA, Petersen W, Den Oever KV, Raalte DV, Ross CJ, Hayden MR, Bakker AC, Dijkhuizen P, Hermens WT, Twisk J, Stroes E, Kastelein JJ, Kuivenhoven JA, Meulenberg JM. Gene therapy for lipoprotein lipase deficiency: working toward clinical application. Hum Gene Ther. 2005; 16: 1276–1286.[CrossRef][Medline] [Order article via Infotrieve]
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10. Hata A, Robertson M, Emi M, Lalouel JM. Direct detection and automated sequencing of individual alleles after electrophoretic strand separation: identification of a common nonsense mutation in exon 9 of the human lipoprotein lipase gene. Nucleic Acids Res. 1990; 18: 5407–5411.
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