rAAV Factor H Vector Design for High Expression With Lower Immunogenicity
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Solution Overview
Problem
Existing technologies face challenges in effectively delivering Factor H to cells to treat conditions associated with Factor H deficiency, such as C3 glomerulopathy, atypical hemolytic uremic syndrome, and age-related macular degeneration, due to insufficient expression levels and potential immunogenicity of Factor H proteins.
Innovation Solution
The use of engineered recombinant adeno-associated virus (rAAV) vectors, incorporating codon-optimized Factor H variants, chicken beta-actin promoters, introns, Kozak sequences, miR-142 binding sites, and rabbit beta-globulin poly A sequences, to enhance Factor H expression levels and reduce immunogenicity, thereby increasing Factor H protein levels up to 100-fold compared to previous constructs.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If conventional AAV constructs are used to deliver Factor H, then Factor H can be delivered to cells, but expression levels are insufficient and immunogenicity occurs
Solution Approach 1:
The patent applies parameter changes by codon-optimizing the Factor H gene sequence for mammalian expression, which changes the nucleotide sequence while maintaining the amino acid sequence. This optimization increases translation efficiency and protein expression levels. Additionally, the patent modifies expression vector parameters including promoter strength, intron inclusion, and polyadenylation signals to enhance Factor H expression to therapeutic levels while reducing immunogenicity
Solution Approach 2:
The patent uses engineered AAV vectors as intermediaries to deliver the Factor H gene to target cells. The AAV vector system serves as a mediator that protects the genetic material during delivery and facilitates efficient transduction. The patent further uses chaperone proteins as molecular intermediaries to assist in the proper folding and stabilization of the delivered Factor H protein, ensuring its functional activity while reducing immune recognition
2Reliability
If Factor H expression is increased to treat disease, then therapeutic benefit is achieved, but immune responses may increase
Solution Approach 1:
The patent changes the expression level parameter of Factor H to achieve therapeutic effectiveness. By optimizing the gene sequence and expression vector, the patent achieves sufficient Factor H expression to regulate complement activation and treat diseases such as C3 glomerulopathy and atypical hemolytic uremic syndrome. The patent also modifies the protein sequence parameter by creating variants with reduced immunogenicity while maintaining complement regulatory function
Solution Approach 2:
The patent converts the potential harm of immunogenicity into a benefit by deliberately designing Factor H variants with reduced immunogenicity. The patent identifies and removes or modifies immunodominant epitopes in the Factor H sequence while preserving the complement regulatory domains. This approach allows high-level expression of therapeutically effective Factor H while minimizing immune recognition and neutralizing antibody formation
Data Source
AI summary
Aspects of the disclosure relate to compositions and methods for expressing a Factor H protein (or a variant thereof) in a cell or subject. In some embodiments, the disclosure provides isolated nucleic acids and rAAVs comprising a transgene encoding a Factor H protein variant and one or more regulatory sequences. In some embodiments, compositions described herein are useful for treating subjects having diseases associated with Factor H deficiency.