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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2005;25:2143.)
© 2005 American Heart Association, Inc.

Atherosclerosis and Lipoproteins

Complete Rescue of Lipoprotein Lipase–Deficient Mice by Somatic Gene Transfer of the Naturally Occurring LPL^S447X Beneficial Mutation

Colin J.D. Ross; Guoqing Liu; Jan Albert Kuivenhoven; Jaap Twisk; Jaap Rip; Willemijn van Dop; Katherine J.D. Ashbourne Excoffon; Suzanne M.E. Lewis; John J. Kastelein; Michael R. Hayden

From the Department of Medical Genetics (C.J.D.R., G.L., K.J.D.A.E., S.M.E.L., M.R.H.), University of British Columbia, Centre for Molecular Medicine and Therapeutics, Vancouver, Canada; the Department of Experimental Vascular Medicine (J.A.K., J.R., W.v.D., J.J.K.), University of Amsterdam, Academic Medical Center, the Netherlands; Amsterdam Molecular Therapeutics (J.T.), the Netherlands.

Correspondence to Michael R. Hayden, Centre for Molecular Medicine and Therapeutics, 950 West 28th Ave, Vancouver, BC, Canada V5Z-4H4. E-mail mrh{at}cmmt.ubc.ca

	Abstract

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The naturally occurring human lipoprotein lipase S447X variant (LPL^S447X) exemplifies a gain-of function mutation with significant benefits including decreased plasma triglycerides (TG), increased high-density lipoprotein (HDL) cholesterol, and reduced risk of coronary artery disease. The S447X variant may be associated with higher LPL catalytic activity; however, in vitro data supporting this hypothesis are contradictory. We wanted to investigate the in vivo mechanism by which the LPL^S447X variant improves the lipid profile of S447X carriers. We conducted a functional assessment of human LPL^S447X compared with LPL^WT in mice. LPL variants were compared in the absence of endogenous mouse LPL in newborn LPL^–/– mice by adenoviral-mediated gene transfer. LPL^–/– mice normally exhibit severe hypertriglyceridemia and die within 48 hours of birth. LPL^WT gene transfer prolonged the survival of mice up to 21 days. In contrast, LPL^S447X completely rescued 95% of the mice to adulthood and increased LPL catalytic activity in postheparin plasma 2.1-fold compared with LPL^WT at day 3 (P=0.003). LPL^S447X also reduced plasma TG 99% from baseline (P<0.001), 2-fold more than LPL^WT, (P<0.01) and increased plasma HDL cholesterol 2.9-fold higher than LPL^WT (P<0.01). These data provide in vivo evidence that the increased catalytic activity of LPL^S447X improves plasma TG clearance and increases the HDL cholesterol pool compared with LPL^WT.

We investigated the in vivo mechanism by which naturally occurring LPL^S447X variant improves the lipid profile of S447X carriers by comparing human LPL^S447X to LPL^WT in newborn LPL^–/– mice. LPL^WT prolonged newborn survival to 21 days. In contrast, the increased catalytic activity of LPL^S447X completely rescued LPL^–/– mice to adulthood.

Key Words: beneficial mutation • chylomicronemia • lipoprotein lipase • type 1 hyperlipoproteinemia

	Introduction

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Lipoprotein lipase (LPL) plays a central role in lipoprotein metabolism and energy homeostasis of all vertebrates. LPL hydrolyzes circulating triglycerides (TG) of very-low-density lipoprotein (VLDL) and chylomicrons at the luminal side of the endothelium to produce free fatty acids (FFA) and glycerol. LPL is synthesized mainly in adipose tissue, skeletal, and cardiac muscle. The generated FFA are used for energy production in muscle or stored in adipose tissue.¹ LPL also contributes to the high-density lipoprotein-cholesterol (HDL-C) pool during the hydrolysis of these lipoproteins.²

See page 2018

In humans, >70 LPL gene mutations have been described that result in complete or partial loss of LPL function.¹ In contrast, the S447X mutation caused by a single nucleotide mutation causes a C-terminal 2-amino acid truncation present in 18% to 22% of the population³ that exemplifies a gain-of-function mutation. Several studies examining the relationships between LPL variants and lipid and lipoprotein levels in humans have demonstrated that the S447X mutation is associated with significant benefits including decreased plasma TG,^3–12 increased HDL-C,^3–13 reduced blood pressure,¹¹ improved vascular function,¹⁴ and a significantly reduced risk of coronary artery disease.^4–9

The mechanism by which LPL^S447X reduces plasma TG and raises HDL-C is unknown and several hypotheses have been postulated. LPL heterodimers composed of both wild-type and LPL^S447X in heterozygous carriers of the S447X mutation might be more stable than dimers composed of wild-type monomers.¹³ The S447X mutation might be in linkage disequilibrium with another linked variation in the LPL gene, promoter, or another gene responsible for the identified phenotype.³ Or the LPL^S447X variant may be directly responsible for an increase in catalytic activity which manifests with lower plasma TGs and higher HDL-C levels in S447X carriers.

In humans, initial reports suggested that there was no difference in the catalytic activity of LPL^S447X versus LPL^WT.^3,15 More recent reports have provided evidence that the S447X variant is associated with the highest quartile of LPL catalytic activity, which manifests with lower plasma TGs and higher HDL-C levels.^10,16 In vitro data supporting this hypothesis have been sought in several mutagenesis and transfection systems, but thus far the results of these studies have been contradictory; showing either increased (14% to 97%)^17,18 or decreased (–5% to –44%)^19–21 specific activity of the variant. Thus, to date, the in vitro data on LPL^S447X are equivocal with regard to the actual effect of the premature truncation on LPL function. Until now, LPL^S447X has not been studied in a well-controlled animal model system.

We conducted a functional assessment of the S447X mutation using gene transfer to LPL-deficient mice to investigate the effect of the LPL^S447X variant on the lipid profile. Homozygous disruption of the LPL gene in mice results in severe chylomicronemia and neonatal lethality within 48 hours of birth.^22,23 We show that although gene transfer of the wild-type LPL gene to newborn LPL^–/– mice is unable to rescue the mice to adulthood, administration of LPL^S447X rescues >95% of the mice to adulthood with a significantly greater reduction of plasma TG, mediated by an increased in vivo catalytic activity of LPL^S447X. These data provide in vivo evidence that the increased catalytic activity of LPL^S447X improves the clearance of plasma TG and the size of the HDL-C pool compared with wild-type LPL.

	Methods

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Recombinant Adenoviral Vectors
A recombinant adenovirus serotype 5 vector containing a human wild-type LPL cDNA (Ad-LPL^WT), or human LPL^S447X cDNA (Ad-LPL^S447X), or a reporter alkaline phosphatase (AP) cDNA (Ad-AP) under control of the cytomegalovirus (CMV) promoter/enhancer were constructed as described.²⁴ The infectivity of the batches of these vectors was determining by the number of plaque forming units (PFU).

Cell Lines
CHO Flp-In cell lines (Invitrogen) were stably cotransfected with expression vector pcDNA/FRT/LPL^WT or pcDNA/FRT/LPL^S447X (Invitrogen) and pOG44 (Invitrogen) using Polyfect transfection reagent (Westburg) according to the manufacturers protocol. Cells were cultured on regular medium supplemented with Hygromycin B (600 µg/mL) in Ultra CHO medium. Heparin was added (5 U/mL) to the culture medium to inhibit LPL reuptake for the collection of medium containing LPL^S447X or LPL^WT.

Animals
LPL^+/– mice (kindly provided by Dr Clay Semenkovich, Washington University, St. Louis, Mo) maintained on a C57BL/6J background were raised on regular rodent diet with free access to water. Breedings were set up to obtain LPL^–/– offspring using ^+/– mice. Blood samples from pups were obtained from 2 µL saphenous vein bleeds or whole body bleeds at termination. Postheparin (PH) plasma was collected from pups 30 minutes after intraperitoneal injection of 50 U heparin, and from adult mice 10 minutes after an intravenous injection of 100 U/kg heparin. Lactescent facial veins identified some LPL^–/– pups on the day of birth and were confirmed by DNA genotyping.

For LPL gene transfer 1x10⁸ PFU of vector diluted in PBS was injected intramuscularly with a 33-gauge needle to 4 sites in the left and right hind limb vastus lateralis muscles and to the left and right forelimb triceps muscles of newborn LPL^–/– mice (25 µL/limb). Administration of adenovirus within 1 day of birth tolerized the mice to the vector and to the foreign LPL. The 7-month-old LPL^–/– mice were retreated by injection in 4 sites in the left and right triceps and gastrocnemius muscles with vector diluted in PBS (75 µL/limb, total of 1x10⁹ PFU). No adverse effects were observed in the treated animals. All procedures involving animals were performed in accordance with protocols from the CCAC and the UBC Animal Care Committee.

Biochemical Measurements
LPL activity was assayed as previously described²⁴ using radioactive trioleoylglycerol emulsion substrate. Human LPL (hLPL) immunoreactive protein was assayed by enzyme-linked immunosorbent assay. In brief, Maxisorp-immuno plates (Nunc International, cat. 467466) were coated with 2 µg/well of purified chicken anti-LPL IgY-P2 antibody in carbonate buffer, pH 9.6, overnight at 4°C. After washing wells with PBS/0.1% tween-20, samples were added and incubated overnight at 4°C. Bound LPL was visualized by incubation with 100 µg/well of horseradish peroxidase (Roche, cat. 1428861)-labeled 5D2 monoclonal anti-LPL antibody (a gift from Dr John Brunzell, University of Washington, Seattle, Wash) for 4 hours at room temperature in the dark. Color was developed with ODP substrate (Sigma Chemical Co, cat. P8412), and LPL protein values were determined against an LPL standard curve (Sigma, cat. L-2254).

TC (Sigma, cat. 40125P), TG (Boehringer Mannheim, cat. 450032), and HDL-C (Sigma, cat. 40125P after 20% polyethylene glycol precipitation) were determined using commercial kits following manufacturer protocols. Pooled plasma lipoprotein profiles were generated by fast protein liquid filtration chromatography (fast protein liquid [FPLC]) (Pharmacia LKB Biotechnology Inc, Piscataway, NJ).²⁴ Extremely hyperlipidemic plasma samples were centrifuged (30 000 rpm, 10 minutes) in a Beckman Airfuge (Optima TLX) and passed through a 0.2-µm filter before FPLC. Semi-quantitative hLPL mRNA levels were determined in tissue homogenates using reverse-transcription polymerase chain reaction with human-specific LPL primers standardized with an 18S RNA control.

LPL thermostability was determined by collecting media from stably-transfected CHO cell lines expressing LPL^WT or LPL^S447X, followed by measurement of LPL activity in the media after incubation for up to 360 minutes at 37°C. LPL chemical stability was determined by measuring LPL activity in media collected from stable CHO cell lines expressing LPL^WT or LPL^S447X after incubation of the media in the presence of increasing concentrations of guanidine hydrochloride (0 to 0.4 mol/L) for 5 minutes at 37°C.

Statistical Analyses
Results are given as mean±SD. Statistical significance was tested using a 2-tailed Student t test, or ANOVA with post-hoc Tukey multiple comparison test when comparing >2 groups. Pearson correlation and significance were determined with Graphpad Prism, version 3.02.

	Results

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Complete Rescue of Newborn LPL^–/– Mice Achieved With LPL^S447X
To delineate the mechanism by which S447X improves the plasma lipid profile in mice, we compared the expression of LPL^S447X and LPL^WT in newborn LPL^–/– mice using adenoviral-mediated gene transfer. Ad-LPL^WT (1x10⁸ PFU/animal) administered to 4 sites in the muscle of newborn LPL^–/– mice prolonged the average survival of newborn LPL^–/– mice to 6.7 days (Figure 1) but was unable to rescue the mice beyond 21 days. During this short life span, the pups demonstrated persistent severe hypertriglyceridemia, exemplified by lactescent facial and saphenous veins engorged with chylomicrons. In contrast, administration of Ad-LPL^S447X normalized the lactescent veins of LPL^–/– pups within 1 day and rescued 95% of the mice to adulthood (>1.5 years) (Figure 1), a rate of survival similar to that of wild-type mice. A control vector expressing alkaline phosphatase (Ad-AP; 1x10⁸ PFU/animal) did not prolong the life of LPL^–/– mice. A 5-fold lower dose of Ad-LPL^S447X prolonged the average survival of LPL^–/– pups to 15 days but was unable to rescue the mice to adulthood.

View larger version (12K): [in this window] [in a new window]   Figure 1. LPL–/– neonates treated with Ad-LPLS447X (1x108 PFU/animal, n=21) survive to adulthood, whereas newborn LPL–/– mice treated with Ad-LPLWT survive a maximum of 21 days (n=13), and untreated LPL–/– mice survive 2 days.

Enhanced TG Reduction in Newborn LPL^–/– Mice Treated With LPL^S447X
LPL^S447X was more effective than LPL^WT in lowering plasma TG in LPL^–/– neonates (n=10/group). By day 3, the plasma TG of Ad-LPL^S447X–treated newborn LPL^–/– mice were reduced 99.0% compared with Ad-LPL^WT–treated pups (131±128 versus 12 749±2183 mg/dL, P<0.001), and reduced 99.6% compared with untreated LPL^–/– mice (33 100±6074 mg/dL; P<0.001) (Figure 2A). Between days 3 and 7, Ad-LPL^WT reduced the plasma TG in newborn LPL^–/– mice by 41% to 61%, compared with a 98% to 99% reduction with Ad-LPL^S447X, resulting in an average 2-fold lower plasma TG in LPL^–/– mice expressing LPL^S447X compared with mice expressing LPL^WT (P<0.001).

View larger version (20K): [in this window] [in a new window]   Figure 2. In newborn LPL–/– mice (n=10/group), Ad-LPLS447X reduced plasma TG 99.6% vs untreated controls, and 99% vs LPLWT within 3 days (A). PH plasma LPLS447X activity was 23% of normal vs 11% for LPLWT (B), whereas LPLS447X and LPLWT protein levels were similar and significantly increased vs controls (C). Plasma TG correlated inversely with PH plasma LPL activity (D).

Increased LPL Activity in Newborn LPL^–/– Mice Treated With LPL^S447X
The PH plasma LPL activity in of newborn LPL^–/– pups 3 days after administration of Ad-LPL^S447X was 2.1-fold higher compared with LPL^WT (23±8% versus 11±5% of normal; P=0.003) (Figure 2B). For reference, the LPL activity of wild-type pups was 1007±151 mU/mL. The levels of hLPL protein in PH plasma were similar in mice treated with either LPL^S447X or LPL^WT (3160±1306 versus 2736±1577 ng/mL, not significant) and hLPL protein was undetectable in untreated mice (Figure 2C). At day 3, the specific activity of LPL^S447X in PH plasma of LPL^–/– mice was 1.6-fold higher compared with LPL^WT (79.6±28.3 versus 49.6±13.8 mU/µg; P=0.023). The lower plasma TG in mice expressing LPL^S447X correlated with increased LPL catalytic activity (r=–0.83, r²=0.69; P<0.0001) (Figure 2D).

Adult Rescued LPL^–/– Mice Display Hypertriglyceridemia and Low HDL
As the number of cells in the mouse expanded as the mice grew, because the vector was not integrated, the number of cells carrying the episomal vector DNA became diluted over time, resulting in an overall apparent loss of expression, which was actually caused by a decrease in the number of vector containing cells compared with the total number of cells in the animal. The decreased levels of LPL^S447X in adult LPL^–/– mice were unable to meet the normal TG catabolism requirements as demonstrated by the gradual increase of plasma TG levels with age (Figure 3A). As summarized in the Table, rescued adult LPL^–/– mice displayed low plasma LPL activity levels (4% normal), extreme hypertriglyceridemia (TG increased 200-fold versus wild-type), 4.4-fold increased plasma FFA, 8.4-fold increased plasma TC, and a 10-fold reduction in plasma HDL-C (9.3% normal). The lipoprotein profile of LPL^–/– mice was significantly different from wild-type mice, as demonstrated by FPLC analysis showing a massive CM/VLDL peak and a reduced HDL peak (Figure 3B). The rescued mice survived into adulthood with no premature mortality.

View larger version (28K): [in this window] [in a new window]   Figure 3. Time course analysis of plasma TG in LPL–/– mice treated with Ad-LPLS447X (), Ad-LPLWT (•), or untreated mice () (A). A transient TG reduction was observed at approximately day 21, corresponded to weaning of the animals. (B) The FPLC profile of 4-month-old mice shows an increased CM/VLDL peak and a reduced HDL peak.

View this table: [in this window] [in a new window]   Plasma Lipids and Lipase Activity of Adult Male LPL–/– Mice (10 to 14 Months Old) Rescued at Birth With Ad-LPLS447X and Wild-Type Male Controls

Adult Rescued LPL^–/– Mice Are Fertile but Lactation-Deficient
The fertility of male and female LPL^–/– mice was normal. Litters of LPL^–/– offspring were generated from homozygous–homozygous breeding; however, the offspring of LPL^–/– mothers, including newly treated LPL^–/– pups or untreated LPL^+/– pups, did not survive >3 days. This impaired survival was caused by a defect in maternal lactation. Milk could not be collected from the LPL^–/– mothers and newborn mice were consistently absent of milk in their stomach, but when transferred to a foster mother the rescued LPL^–/– pups survived normally.

Retreatment of Adult LPL^–/– Mice With LPL^S447X Reduces Plasma TG and Increases HDL
To compare the S447X variant and wild-type LPL in adult mice in an environment without endogenous murine LPL, we used the unique resource of rescued adult LPL^–/– mice (n=5/group) retreated with a second dose of either Ad-LPL^S447X, Ad-LPL^WT, or Ad-AP (intramuscularly, 1x10⁹ PFU/animal) at 7 months of age. In this experiment, retreatment with Ad-LPL^WT resulted in a 44% reduction in plasma TG levels within 2 days (from 6279±1733 to 3508±987 mg/dL; P<0.01) and a modest 40% increase (not significant) in plasma HDL-C (Figure 4a and 4b). Retreatment with Ad-LPL^S447X further reduced plasma TG 2.2-fold more than LPL^WT (P<0.01); plasma TG were reduced 96% to near-normal levels (from 6419±1141 to 254±262 mg/dL; P<0.001) (Figure 4A). HDL-C was concomitantly increased 4.5-fold with Ad-LPL^S447X (from 6.8±1.5 to 31.6±12.9 mg/dL; P<0.001) (Figure 4B), resulting in a 2.9-fold higher plasma HDL-C levels compared with Ad-LPL^WT by day 7 (P<0.01).

View larger version (45K): [in this window] [in a new window]   Figure 4. Seven-month-old LPL–/– mice (n=5/group) were readministered Ad-LPLS447X (), Ad-LPLWT (•), or control vector Ad-AP (). LPLS447X significantly lowered plasma TG (A), increased HDL-C (B), and increased PH plasma LPL activity compared with LPLWT (C). LPLS447X had similar PH plasma hLPL protein levels compared with LPLWT (D). Plasma TG correlated inversely with PH plasma LPL activity (E). LPLS447X visibly reduced plasma lipemia compared with LPLWT (F) and the plasma FPLC profile after Ad-LPLS447X shows a reduced CM/VLDL peak and an increased HDL peak compared with Ad-LPLWT (G).

Ad-LPL^WT increased PH plasma LPL activity from a residual background level (adenovirus-derived) of 4.9±1.3% to 10.4±2.0% (P<0.05) (Figure 4c), and LPL^WT protein levels were increased from 358±143 to 2712±1102 ng/mL by day 7 (Figure 4D). Ad-LPL^S447X increased PH plasma LPL activity 2.2-fold higher than LPL^WT to 23.2±10.0% of normal (P<0.01) (Figure 4C), whereas PH plasma hLPL protein levels were in the same range as LPL^WT (2537±218 ng/mL) (Figure 4D). At day 7, the PH plasma specific activity of LPL^S447X was 1.7-fold higher compared with LPL^WT (39.9±9.9 versus 23.5±12.6 mU/µg; P=0.036). The lower plasma TG in mice expressing LPL^S447X correlated with increased PH plasma LPL catalytic activity (r=–0.75, r²=0.57; P<0.0001) (Figure 4E).

The visible plasma hyperlipidemia was resolved within 2 days with Ad-LPL^S447X (Figure 4F) and the lipoprotein profile was significantly improved with 9-fold lower TG in CM/VLDL fractions (area under the curve [AUC]) and 5-fold higher cholesterol in HDL fractions, as demonstrated by FPLC (Figure 4G). Treatment with Ad-LPL^WT did not alter the visible plasma hyperlipidemia, and the lipoprotein profile had 1.7-fold lower TG (AUC) in chylomicron (CM)/VLDL fractions and 1.6-fold higher cholesterol in HDL fractions.

The injected gastrocnemius muscle of treated LPL^–/– mice expressing LPL^S447X or LPL^WT showed similar levels of LPL activity (12.4±3.6 versus 18.7±13.0 mU LPL activity/µg protein, not significant) and LPL protein (61.5±18.3 versus 57.8±33.9 ng LPL protein/µg protein, not significant) at day 10, indicating similar transduction efficiencies of both adenoviral constructs. In the gastrocnemius muscle of control mice, LPL activity (3.6±2.2 mU/µg protein) and LPL protein (15.1±2.0 ng/µg) remained at background levels.

Similar mRNA Levels, Clearance, and Stability of LPL^WT and LPL^S447X
The levels of hLPL mRNA in the injected gastrocnemius muscles were compared by semiquantitation of human specific LPL mRNA in LPL^–/– mice treated with either Ad-LPL^WT or Ad-LPL^S447X after 1, 3, and 60 days (Figure 5A). There were no significant differences in human LPL mRNA levels in the LPL^–/– mice expressing either LPL^S447X or LPL^WT at each time point. The in vivo clearance rates of LPL^S447X and LPL^WT were also similar (t=6 minutes each) after intravenous injection of 3.5 µg of each protein into the circulation of wild-type mice (Figure 5B). The in vitro thermo and chemical stabilities of LPL^S447X and LPL^WT were similar, as determined by the comparison of baseline LPL activity after exposure of LPL^S447X or LPL^WT to increasing concentrations of guanidine hydrochloride (Figure 5C), or 37°C over time (Figure 5D).

View larger version (32K): [in this window] [in a new window]   Figure 5. LPLS447X and LPLWT show similar relative mRNA levels in Ad-LPLS447X or Ad-LPLWT–treated LPL–/– mice (A), and similar clearance rates of protein from plasma in mice injected intravenously with 3.5 µg of LPLS447X (B). LPLS447X and LPLWT show similar in vitro chemical (C) and thermo (D) stability.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Adenoviral gene transfer of the LPL^S447X variant cDNA to LPL-deficient mice showed a 2.1- to 2.2-fold increased LPL catalytic activity in PH plasma, compared with the transfer of wild-type LPL. This resulted in a 2- to 2.2-fold greater reduction in plasma TG and a 2.9-fold greater increase in plasma HDL-C levels. LPL^S447X gene transfer completely rescued newborn LPL^–/– mice to adulthood, which would have otherwise died within 2 days, and this survival was attributable to the increased catalytic activity of LPL^S447X. In contrast, gene transfer of the wild-type gene prolonged the survival of treated mice to a maximum of 21 days. These data provide in vivo evidence that the LPL^S447X variant has increased catalytic activity underlying increased TG clearance and increased HDL-C compared with wild-type LPL.

These findings rule out several alternative hypotheses for the mechanism by which LPL^S447X increases LPL activity and improves plasma lipids. First, the postulate that LPL heterodimers composed of both wild-type and LPL^S447X in heterozygous carriers of the S447X mutation might be more stable than dimers composed of wild-type monomers¹³ was not supported because the increased catalytic activity of LPL^S447X was observed in LPL deficient animals in the absence of wild-type LPL, treated only with either LPL^S447X or LPL^WT. Because this does not rule out the possibility that LPL^S447X dimers are more stable than LPL^WT dimers, we assessed the thermo and chemical stabilities of LPL^S447X and LPL^WT homodimers and found no differences between LPL^S447X and LPL^WT stability. Second, the possibility that the S447X mutation is in linkage disequilibrium with another linked change in the LPL gene or promoter that could be associated with the identified phenotype³ was not supported because by using LPL gene transfer, we delivered the identical LPL gene to the animals, except for the presence or absence of the S447X mutation.

In 820 healthy participants of the REGRESS study, LPL catalytic activity was significantly increased 11% in S447X carriers compared with noncarriers.¹⁰ As expected, the relative increase in LPL catalytic activity in human LPL^S447X carriers was less than that observed in mice expressing LPL^S447X because human S447X heterozygous carriers express both LPL^WT and LPL^S447X genes. In addition, the LPL^WT or LPL^S447X genes expressed in mice were constitutively expressed by the CMV promoter so that any differences between LPL^S447X and LPL^WT would be amplified compared with the regulated expression of LPL from endogenous promoter of human subjects.

The relative changes in plasma TG in humans and mice that expressed LPL^S447X were proportional with the increased LPL^S447X catalytic activity. In mice, the 2-fold greater reduction in plasma TG was directly proportional with the 2-fold increase in LPL^S447X catalytic activity. Similarly, the more modest 11% increase in LPL^S447X catalytic activity in human S447X carriers¹⁰ was proportional with the 8% to 21% reduction in plasma TG observed in S447X carriers.^3–12

The reasons for the increased catalytic activity caused by the S447X mutation are not clear. In vitro assessment of LPL^S447X and LPL^WT reveal that the thermal and chemical stabilities, and in vivo mRNA levels, and half-lives of these 2 proteins are similar. Therefore, altered stability, half-life, and increased transcriptional activity cannot account for the enhanced catalytic activity.

In treated LPL^–/– mice, the specific activity of LPL^S447X was significantly increased 60% to 70%. This finding is in agreement with previously reported in vitro findings showing 14% to 97% increased LPL^S447X specific activity,^17,18 but not with other reports showing a decreased LPL^S447X specific activity.^19–21 Notably, in the reports of decreased LPL^S447X-specific activity, the catalytic activity of LPL^S447X was either the same²¹ or increased 20% to 25% compared with wild-type,^19,20 but the specific activity was decreased because the LPL^S447X variant showed up to 2-fold higher levels of LPL protein when measured using a chicken polycloncal/5D2 enzyme-linked immunosorbent assay.^19,20 We have found previously that different antibodies can result in different measures of LPL protein levels, and this affects the measure of specific activity. The findings of this study clearly demonstrate, however, that the LPL^S447X variant increases the catalytic activity of LPL^S447X, and that the specific activity of LPL^S447X was increased, based on the protein levels measured by the IgY-P2/5D2 enzyme-linked immunosorbent assay.

The relatively inefficient rescue of newborn LPL^–/– mice using Ad-LPL^WT was reported by Strauss et al, who rescued 3% of newborn LPL^–/– mice to adulthood using a 50-fold higher dose of Ad-LPL^WT compared with the dose used in this study.²⁵ In contrast, LPL^S447X efficiently rescued 95% of LPL^–/– mice, which has led to the establishment of an LPL^–/– mouse colony available for further investigation. In this colony, the essential role of LPL in lactation became immediately apparent. LPL^–/– mothers could not produce milk and their offspring did not survive unless transferred to a foster mother within hours of birth. The breast milk of LPL deficient humans and cats is markedly reduced in total fat (–75%) as preformed fatty acids do not enter the lactating breast from plasma or adipose tissue.^26,27

LPL^S447X gene transfer is an effective means to lower plasma TG and could be used to treat LPL deficiency. Human LPL deficiency is characterized by profound hypertriglyceridemia and potentially lethal pancreatitis,¹ causing significant morbidity and mortality in Canada, where a founder effect has resulted in the highest worldwide frequency of LPL deficiency, affecting up to 1 in 4000 people in Eastern Quebec.²⁸ LPL deficiency is refractory to lipid-lowering drugs, and enzyme replacement therapy is ineffective because of the short half-life of exogenous LPL in plasma.²⁴ LPL gene therapy has been proposed to reduce the life-threatening risk of pancreatitis. The increased catalytic activity of LPL^S447X would improve efficacy, translating into lower vector doses and reduced vector-derived toxicity. Clinical application of LPL gene therapy requires long-term LPL expression and the adeno-associated virus (AAV) is an attractive vector for clinical consideration and it has been used in several human gene therapy studies.²⁹ Recently, we have demonstrated long-term expression of LPL^S447X from a single intramuscularly administration of AAV-LPL^S447X (8x10¹² genome copies/kg), resulting in a highly significant 97% TG reduction to near-normal levels for >12 months in LPL^–/– mice.³⁰ We have recently been able to confirm these promising findings in LPL-deficient cats (unpublished, 2005).

The S447X mutation is an example of a naturally occurring, beneficial, gain-of-function mutation. Most beneficial mutations are loss-of-function mutations, such as the 32-bp deletion in the CCR-5 receptor conferring resistance to HIV infection,³¹ and plasminogen activator inhibitor (PAI)-1 mutations that protect against coronary artery disease.³² However, there are very few examples of beneficial gain-of-function mutations. Two examples of beneficial gain-of-function mutations are malarial resistance in hemoglobin-S heterozygotes and protection from postpartum hemorrhage in factor V-Leiden carriers. However, these beneficial mutations also carry significant disadvantages, including homozygous lethality with hemoglobin-S; and a propensity for venous thrombosis in factor V-Leiden carriers. Thus, S447X mutation is an example of a naturally occurring, beneficial, gain-of-function mutation without deleterious side effects.

It should be noted that LPL has a dual function in atherogenesis through its anti-atherogenic clearance of plasma lipoproteins, whereas LPL protein in the arterial wall is pro-atherogenic through its ability to promote the retention of apolipoprotein B-rich lipoproteins.³³ In addition, macrophage LPL in the arterial wall could also be pro-atherogenic through its catalytic function as well. Nevertheless, the reduced risk of cardiovascular disease in LPL^S447X carriers demonstrates the net beneficial effect of these 2 opposing actions for LPL^S447X.

The naturally occurring LPL^S447X mutation represents a class of beneficial mutation in humans with enhanced function over the wild-type gene. The net effect of the increased LPL^S447X catalytic ability is demonstrated here by the complete rescue of newborn LPL^–/– mice using LPL^S447X gene transfer.

	Acknowledgments

 
C.J.D.R. was supported by the BCRI Mining for Miracles fellowship, a Michael Smith Foundation for Health Research Award, and a CIHR fellowship. J.A.K. is a Dr Dekker Research fellow of the Netherlands Heart Foundation (1998T011). J.J.P.K. is an Established Investigator of the Netherlands Heart Foundation (2000T039). This study was further supported by grants from the CGON, CIHR, and the BC and Yukon Heart and Stroke Foundation. M.R.H. is a holder of a Canadian Research Chair in Human Genetics. The authors thank NCE Genetics, Xenon Genetics, and Amsterdam Molecular Therapeutics for support, Fudan Miao for expert technical assistance, Dr John D. Brunzell (University of Washington, Seattle) for generously providing us with the 5D2 monoclonal antibody, and Drs Clay Semenkovich and Trey Coleman for LPL heterozygous mice.

	Footnotes

 
C.J.D.R. and G.L. contributed equally to this work.

Received April 26, 2005; accepted June 22, 2005.

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