Synthetic FVIII B Domain Substitute for Hemophilia A Gene Editing
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Solution Overview
Problem
Current treatments for hemophilia A, such as virus-based gene therapy, are inadequate due to episomal maintenance of the therapeutic coding sequence, which is not effectively replicated in dividing cells, particularly in children, and protein replacement therapy is inconvenient, costly, and prone to breakthrough bleeding events.
Innovation Solution
Gene editing methods using a DNA endonuclease, guide RNA, and a donor template with a synthetic Factor VIII coding sequence that includes a B domain substitute with reduced N-linked glycosylation sites to enhance expression and integration, allowing for improved production and secretion of functional FVIII protein.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If AAV-based gene therapy is used to deliver FVIII coding sequence, then therapeutic protein expression is achieved, but the episomal coding sequence is not effectively replicated in dividing cells leading to loss of therapeutic effect
Solution Approach 1:
The patent extracts the problematic B domain from the FVIII coding sequence, creating a truncated version that can be efficiently packaged in AAV vectors while maintaining therapeutic function. This extracted sequence is then integrated into the host genome rather than maintained episomally, resolving the replication issue in dividing cells.
Solution Approach 2:
The patent performs preliminary genome integration of the FVIII coding sequence before cell division occurs. By establishing genomic integration in advance, the therapeutic sequence is ensured to be replicated along with host DNA in subsequent cell divisions, preventing loss of therapeutic effect.
2Productivity
If the B domain is removed from FVIII coding sequence to enable AAV packaging, then vector packaging efficiency is improved, but protein secretion is impaired due to loss of N-linked glycosylation sites
Solution Approach 1:
The patent applies local quality modification by selectively adding back specific N-linked glycosylation sites (asparagine residues at positions 196, 212, and 558) in the B domain substitute region, while keeping the rest of the B domain removed. This localized addition of glycosylation capability restores secretion efficiency without compromising packaging efficiency.
Solution Approach 2:
The patent creates a composite FVIII construct that combines elements of the full-length FVIII (A1-A2-C1-C2 domains and selected B domain residues) with a truncated version. The B domain substitute contains strategically placed glycosylation sites from the original B domain, creating a hybrid structure that achieves both packaging and secretion requirements.
3Quantity of substance
If protein replacement therapy is used to treat hemophilia A, then FVIII levels are maintained, but frequent intravenous administration is required which is inconvenient and costly
Solution Approach 1:
The patent enables the patient's own cells to produce FVIII protein through genomic integration of the FVIII coding sequence. Once integrated, the cells continuously produce and secrete functional FVIII without requiring external administration, making the treatment system self-sustaining and eliminating the need for frequent infusions.
4Duration of action of stationary object
If gene editing is used to integrate FVIII coding sequence into host genome, then permanent expression is achieved, but immune responses may occur against the therapeutic protein
Solution Approach 1:
The patent modifies the FVIII protein structure by removing the B domain and adding specific glycosylation sites, creating a variant with altered immunogenicity properties. These parameter changes in the protein structure (composition and post-translational modifications) may reduce immune recognition while maintaining function, allowing for durable expression.
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
The approach provides long-lasting and effective expression of functional Factor VIII protein, reducing the risk of immune responses and improving treatment outcomes for both children and adults with hemophilia A by enhancing integration and secretion of the protein.
Implementation Method 1
a DNA endonuclease or a nucleic acid encoding the DNA endonuclease
Implementation Method 2
a guide RNA (gRNA) having a spacer sequence complementary to a host cell locus
Implementation Method 3
a donor template having a nucleic acid sequence encoding a synthetic FVIII protein
Implementation Method 4
Removal of the B domain, which contains up to 18 N-linked glycosylation sites, results in impaired secretion of FVIII protein
Data Source
AI summary
Provided herein, in some embodiments, are materials and methods for treating hemophilia A in a subject ex vivo or in vivo. Also provided herein, in some embodiments, are materials and methods for knocking in a coding sequence encoding a synthetic FVIII having a B domain substitute into a genome.