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Published on: March 12, 2018
Correction in female PKU mice by repeated administration of mPAH cDNA using phiBT1 integration system
1Department of Gene and Cell Medicine, Mount Sinai School of Medicine, New York, New York 10029, USA.
Insights
Repeated gene therapy using the phiBT1 system corrected phenylketonuria (PKU) in female mice by cumulatively increasing transgene expression in diverse liver cells. This approach offers a promising strategy for metabolic disorder gene therapy.
Area of Science:
- Genetics
- Metabolic Disorders
- Gene Therapy
Background:
- Phenylketonuria (PKU) is a genetic metabolic disorder caused by phenylalanine hydroxylase (PAH) deficiency, leading to severe intellectual disability.
- Previous studies achieved partial gene correction in male PKU mice but not females using genome-targeted delivery of murine PAH (mPAH) cDNA via the phiBT1 system.
Purpose of the Study:
- To investigate the efficacy of sequential transgene administration via the phiBT1 integration system for treating phenylketonuria (PKU) in female mice.
- To evaluate the potential for cumulative transgene expression and correction of metabolic phenotypes through repeated gene therapy applications.
Main Methods:
- Sequential administration of distinct fluorescent protein (GFP and RFP) expressing cassettes to demonstrate non-overlapping hepatocyte targeting.
- Weekly administration of mPAH cDNA via the phiBT1 integration system to female PKU mice over 10 weeks.
- Assessment of hyperphenylalaninemic and hypopigmentation phenotypes, along with liver pathology, following treatment.
Main Results:
- Sequential administration of GFP and RFP cassettes resulted in distinct, non-overlapping populations of fluorescent hepatocytes in vivo.
- Complete correction of hyperphenylalaninemia and hypopigmentation phenotypes was achieved in female PKU mice after 10 weekly mPAH cDNA administrations.
- No significant liver pathology was observed even after 10 consecutive administrations of the phiBT1 integration system, indicating safety.
Conclusions:
- Repeated transgene administration via the phiBT1 system enables genome-targeted integration in a diverse hepatocyte population.
- Cumulative transgene expression can overcome limitations of low integration frequencies, supporting its use in gene therapy for metabolic disorders.
- This enhanced gene therapy approach shows promise for effectively treating phenylketonuria and potentially other metabolic diseases.
Abstract:
Phenylketonuria (PKU) is a metabolic disorder secondary to a hepatic deficiency of phenylalanine hydroxylase (PAH) that predisposes affected children to develop severe and irreversible mental retardation. We have previously reported the complete and permanent correction of the hyperphenylalaninemic and hypopigmentation phenotypes in male, but not female, PKU mice after genome-targeted delivery of murine PAH (mPAH) complementary DNA (cDNA) in a phiBT1 bacteriophage integration system. Here we show that sequential administration of green fluorescent protein (GFP)- and red fluorescent protein (RFP)-expressing cassettes in the phiBT1 integration system led to distinct and non-overlapping populations of green and red fluorescent hepatocytes in vivo. The hyperphenylalaninemic and hypopigmentation phenotypes of female PKU mice were completely corrected after 10 weekly administrations of mPAH cDNA. Importantly, there was no apparent liver pathology in mice even after 10 consecutive administrations of the phiBT1 integration system. The results indicate that repeated administration of transgenes in the phiBT1 integration system can lead to their genome-targeted integration in a diverse population of hepatocytes and result in the elevation of transgene expression levels in a cumulative manner, which can be utilized to overcome insufficient transgene expression owing to low genome integration frequencies in a gene therapy paradigm for metabolic disorders.
