Split AAV Vectors Reconstitute CPS1 Gene for Ureagenesis Restoration
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Solution Overview
Problem
Current treatments for Carbamoyl phosphate synthetase 1 (CPS1) deficiency, such as dietary protein restriction, are only marginally effective and do not address the underlying metabolic disorder, leading to recurrent hyperammonemia and progressive neurological decline.
Innovation Solution
The use of split adeno-associated virus (AAV) vectors (sAAVs) that divide the CPS1 cDNA payload into two smaller overlapping segments, allowing for concatemerization via homologous recombination in hepatocytes to reconstitute the CPS1 gene and express the CPS1 protein.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If dietary protein restriction is used to treat CPS1 deficiency, then ammonia levels may be marginally controlled, but patients experience recurrent hyperammonemia and progressive neurological decline
Solution Approach 1:
The CPS1 cDNA payload is divided into two smaller overlapping segments that can be packaged into separate AAV vectors. These segments are designed to concatemerize via homologous recombination in hepatocytes to reconstitute the full functional CPS1 gene, enabling reliable ammonia control without the limitations of dietary restriction
Solution Approach 2:
AAV vectors serve as intermediaries to deliver and express functional CPS1 protein in hepatocytes. The vectors mediate the restoration of ureagenesis by providing the missing enzyme activity, thereby controlling ammonia levels and preventing neurological decline
2Quantity of substance
If full-length CPS1 cDNA is packaged into AAV vector, then sufficient gene material is provided, but the vector capacity is exceeded
Solution Approach 1:
The full-length CPS1 cDNA is segmented into two smaller overlapping polynucleotide sequences that can be individually packaged into AAV vectors. The segments are designed with overlapping regions that enable in vivo concatemerization to reconstruct the full functional gene, thus overcoming the vector capacity limitation while maintaining complete gene material
3Quantity of substance
If larger capacity vectors like lentiviruses or adenoviruses are used, then CPS1 can be accommodated, but unwanted genomic integration and immunogenicity occur
Solution Approach 1:
The CPS1 gene is divided into segments that fit within AAV vector capacity, avoiding the need for larger vectors that cause harmful effects. The segmented approach allows AAV vectors to deliver sufficient gene material while maintaining safety profiles
Solution Approach 2:
AAV vectors serve as safe intermediaries that deliver CPS1 gene segments without causing unwanted genomic integration or immunogenicity. The vectors mediate gene delivery while maintaining the safety advantages of AAV, avoiding the harmful effects associated with alternative vector systems
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach effectively controls plasma ammonia levels and promotes normal behavior in CPS1-deficient mice, providing a potential therapeutic strategy for treating CPS1 deficiency in humans.
Implementation Method 1
separate portions/segments of CPS1 were packaged into viruses individually and allowed to concatemerize in in vivo homologous recombination to reconstitute the transgenic payload and express the CPS1 protein following viral co-transduction of the same hepatocyte
Data Source
AI summary
Carbamoyl phosphate synthetase 1 (CPS1) deficiency is a metabolic disorder of the liver that results m abnormal nitrogen metabolism. To illustrate the ability of gene therapy to treat CPS1 deficiency, two adeno-associated viruses encoding portions of a codon optimized CPS1 were generated and tested in a conditional CPS1 knock out mouse model. When administered to mice having knocked out endogenous CPS1 expression, mice from this model demonstrate homologous recombination and reconstitution of the codon optimized CPS1 gene, expression of the CPS1 protein and the associated control of plasma ammonia following the administered AAVs comprising the CPS1 gene sequences. While all control mice perish, the mice in this model live and have normal behavior. As there is no effective therapy for human patients with the CPS1 disorder, this invention can address this unmet need for these patients.