Lentiviral vectors for therapeutic expression of BTK in haematopoietic cells
Inventors
ELKOUBY, Liron • Diamant, Noam • SHACHNAI-PINKAS, Liat
Assignees
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Publication Number
US-11464872-B2
Publication Date
2022-10-11
Expiration Date
Abstract
The present application, in some embodiments, is directed to a polynucleotide including: (a) a first nucleic acid molecule including a sequence of a human endogenous Bruton's tyrosine kinase (BTK) promoter; and (b) a second nucleic acid molecule including a codon optimized sequence encoding a BTK or a functional analog thereof. Further provided are an expression vector, a cell, and a composition, all of which include the polynucleotide of the invention, and a method of using same, such as for treating X-linked Agammaglobulinemia (XLA) in a subject in need thereof.
Core Innovation
The invention relates to a polynucleotide comprising a first nucleic acid molecule and a second nucleic acid molecule operably linked for BTK expression in a human subject, cell derived therefrom, or both. The first nucleic acid molecule comprises a sequence of between 799 to 1,533 nucleotides of a human endogenous Bruton's tyrosine kinase (BTK) promoter, and the second nucleic acid molecule comprises a codon optimized sequence encoding BTK or a functional analog thereof. The polynucleotide comprises the nucleic acid sequence set forth in SEQ ID NO: 23.
The described optimized BTK lentiviral gene therapy for X-linked agammaglobulinemia (XLA) uses a human endogenous BTK promoter fragment together with a codon-optimized BTK sequence operably linked for expression in human target cells. Expression vectors are described, including lentivirus-based and self-inactivating lentiviral backbones, configured to achieve physiological, cell-specific BTK expression. The description also addresses risk associated with unregulated high BTK by emphasizing the regulatory and expression characteristics of the promoter and untranslated/5′ features.
In addition to the polynucleotide and vector configurations, the invention includes hematopoietic stem cells and transplantable pharmaceutical compositions containing the polynucleotide. A transduction/selection framework based on vector copy number (VCN) is described in the context of ex vivo transduction. Documented examples report candidate screening in human CD34+ cells, with improved BTK expression for SEQ ID NO: 23 (NTX109) versus other constructs, and show that promoter length and BTK 5′UTR features modulate expression and regulation.
Claims Coverage
The document provides one independent claim and several dependent claims, focusing on a specific polynucleotide and its use in lentiviral expression, hematopoietic stem cells, and therapeutic treatment of X-linked agammaglobulinemia (XLA) with defined viability/activity outcomes.
BTK promoter-driven SEQ ID NO: 23 codon optimized BTK polynucleotide
A polynucleotide comprising a first nucleic acid molecule comprising a sequence of between 799 to 1,533 nucleotides of a human endogenous Bruton's tyrosine kinase (BTK) promoter, and a second nucleic acid molecule comprising a codon optimized sequence encoding a BTK or a functional analog thereof, wherein the first nucleic acid molecule and the second nucleic acid molecule are operably linked for BTK expression in a human subject, cell derived therefrom, or both, and wherein the polynucleotide comprises the nucleic acid sequence set forth in SEQ ID NO: 23.
Expression vector comprising the BTK promoter-driven SEQ ID NO: 23 polynucleotide
An expression vector comprising the polynucleotide.
Lentivirus-based expression vector comprising the BTK promoter-driven SEQ ID NO: 23 polynucleotide
The expression vector specified as a lentivirus-based expression vector comprising the polynucleotide.
Hematopoietic stem cell containing the BTK promoter-driven SEQ ID NO: 23 polynucleotide
A hematopoietic stem cell comprising the polynucleotide.
XLA treatment via autologous ex vivo transduced hematopoietic stem cells
A method to enhance B cell viability or activity in a human with X-linked agammaglobulinemia (XLA) by transplanting autologous hematopoietic stem cells that were transduced with the lentivirus-based expression vector.
Viability or activity includes survival, proliferation, and/or differentiation
The method wherein viability or activity comprises survival, proliferation, and/or differentiation, or any combination thereof.
Overall, the claim set centers on a SEQ ID NO: 23 polynucleotide combining a human endogenous BTK promoter fragment with a codon optimized BTK or functional analog sequence operably linked for BTK expression in human cells, and narrows to its use in lentivirus-based expression vectors, hematopoietic stem cells, and an autologous ex vivo transplant approach for enhancing B cell viability or activity in XLA.
Stated Advantages
Physiological, cell-specific BTK expression.
Modulation of expression and regulation via promoter length and BTK 5′UTR features.
Improved BTK expression for SEQ ID NO: 23 (NTX109) versus other constructs.
Restoration of B-cell differentiation/maturation in BTK-deficient Xid mice.
Lineage-appropriate (low in T cells) BTK expression in BTK-deficient Xid mice.
Partial rescue of IgM secretion after LPS stimulation in BTK-deficient Xid mice.
Documented Applications
Optimized BTK lentiviral gene therapy for X-linked agammaglobulinemia (XLA).
Ex vivo transduction and evaluation in human CD34+ cells using candidate screening for SEQ ID NO: 23 (NTX109).
In vivo potency testing in BTK-deficient Xid mice, including assessment of B-cell differentiation/maturation, lineage-appropriate BTK expression, and partial rescue of IgM secretion after LPS stimulation.
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