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Retroviral-mediated gene transfer and expression of human lipoprotein lipase in somatic cells
M E Lewis1, I J Forsythe, J D Marth
1Department of Medical Genetics, University of British Columbia, Vancouver, Canada.
Insights
Gene therapy using retroviral vectors successfully transferred and expressed human lipoprotein lipase (LPL) in various cell types. Primary human fibroblasts showed significant LPL activity, indicating potential for treating LPL deficiency.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Lipoprotein lipase (LPL) deficiency causes severe health issues, including pancreatitis and failure to thrive.
- Heterozygous carriers may face increased risk of coronary artery disease.
- Gene therapy offers a potential strategy to correct LPL deficiency.
Purpose of the Study:
- To develop a gene therapy approach for LPL deficiency.
- To assess the efficacy of retroviral vectors for LPL gene transfer and expression.
Main Methods:
- Human LPL cDNA was inserted into myeloproliferative sarcoma virus (MPSV)-based retroviral vectors.
- Gene transfer and expression were evaluated in various cell types, including fibroblasts and hematopoietic cell lines.
- Bioactive LPL levels were quantified in transduced cells.
Main Results:
- Stable gene transfer and enhanced human LPL expression were achieved in multiple somatic cell types.
- Primary human fibroblasts demonstrated substantial increases in LPL immunoreactive mass (24-fold) and activity (50-fold).
- LPL expression levels varied significantly across different cell lines.
Conclusions:
- Human fibroblasts can effectively mature and secrete bioactive LPL in vitro.
- Fibroblasts show promise as a cellular vehicle for LPL gene delivery in treating LPL deficiency.
- Further research is needed to optimize LPL expression levels for therapeutic applications.
Abstract:
Lipoprotein lipase (LPL) is an enzyme responsible for the hydrolysis of triglyceride-rich circulating lipoproteins. Humans with complete defects in LPL activity present from infancy with failure to thrive, eruptive xanthomas, pancreatitis, and lactescent plasma. In addition, heterozygous carriers for this disorder may be at increased risk for the development of coronary artery disease. In view of a potential strategy for correcting complete or partial LPL deficiency, a 1.56-kb human LPL cDNA was inserted into a series of recombinant myeloproliferative sarcoma virus (MPSV)-based retroviral vectors under transcriptional control of the constitutive MPSV long terminal repeat (LTR). Stable gene transfer and enhanced expression of human LPL was observed at both the RNA and protein level in a variety of somatic cell types in vitro. Genetically modified cell populations included mouse NIH-3T3 fibroblasts and C2C12 myoblasts, primary human fibroblasts, and established human hematopoietic cell lines of erythroid (K562), myelocytic (HL60), and monocytic (U937,THP-1) type. The achieved levels of bioactive human LPL were found to vary widely between the different transduced cell lines, which may be critical to an approach to gene therapy. Transduced primary human fibroblasts yielded maximal elevation of LPL immunoreactive mass and activity of at least 24- and 50-fold, respectively, above constitutively expressed levels for this cell type. Human fibroblasts, therefore, appear to accommodate in vitro the complex processes readily leading to the maturation and secretion of bioactive human LPL and may serve as an effective cellular vehicle for LPL gene delivery and expression in human LPL deficiency.