Factor VIII Gene Truncation for AAV Packaging
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Solution Overview
Problem
Current treatments for hemophilia A, such as intravenous injections of plasma-derived or recombinant factor VIII proteins, are costly and require frequent administration due to the short half-life of the protein, and gene therapy approaches face challenges with inefficient expression and packaging of the factor VIII gene into AAV vectors.
Innovation Solution
A modified nucleotide sequence encoding a factor VIII protein with a shortened B domain of 90-111 base pairs, optimized for codon usage, which includes six asparagine residues for glycosylation, allowing for high expression levels and efficient packaging into AAV vectors.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If the full-length factor VIII gene is used for gene therapy, then therapeutic effect is achieved, but vector packaging capacity is exceeded and expression efficiency is low
Solution Approach 1:
The patent extracts and removes the B domain from the factor VIII gene, creating a truncated version that retains A1-A2 and A3-C1-C2 domains. This extraction reduces the gene size from 7.0 kb to 4.4 kb, enabling it to fit within AAV vector packaging capacity while maintaining therapeutic activity through the preserved domains.
Solution Approach 2:
The factor VIII gene is segmented into functional domains (A1-A2 and A3-C1-C2), with the B domain removed. This segmentation allows the remaining domains to be expressed independently while maintaining the protein's coagulation function, solving the packaging capacity issue without losing therapeutic effect.
2Device complexity
If the factor VIII gene is truncated to improve packaging, then vector capacity is improved, but protein expression efficiency decreases
Solution Approach 1:
The patent applies codon optimization to change the nucleotide sequence parameters of the truncated factor VIII gene. By optimizing codon usage to match the host cell's preferences, the expression efficiency is enhanced despite the truncation, achieving high protein production levels from the reduced gene.
Solution Approach 2:
The patent introduces specific modifications to local regions of the gene, including the addition of a poly-A signal and optimization of specific codons in the remaining domains. These localized changes improve transcription and translation efficiency without requiring restoration of the entire gene.
3Reliability
If higher vector doses are used to achieve therapeutic levels, then therapeutic effect is improved, but toxicity increases
Solution Approach 1:
The patent creates a simplified copy of the factor VIII gene without the B domain, which reduces the genetic load on the host cell. This reduced-genome approach allows therapeutic protein levels to be achieved with lower vector doses, thereby reducing toxicity while maintaining therapeutic effect.
Solution Approach 2:
By optimizing the codon sequence and adding appropriate regulatory elements like poly-A signals, the patent enhances the expression efficiency of the truncated gene. This parameter optimization ensures that lower vector doses can achieve the same therapeutic protein levels, reducing associated toxicity.
Data Source
AI summary
There is provided a nucleic acid molecule comprising a nucleotide sequence encoding for a functional factor VIII protein, wherein the portion of the nucleotide sequence encoding for the B domain of the factor VIII protein is between 90 and 111 nucleotides in length and encodes for an amino acid sequence comprising a sequence having at least 85% identity to SEQ ID NO: 4 and which comprises six asparagine residues. Also provided is a functional factor VIII protein, a vector comprising the above nucleic acid molecule, a host cell, a transgenic animal, a method of treating haemophilia, e.g. haemophilia A, and a method for the preparation of a parvoviral gene delivery vector.