Liver targeting adeno-associated viral vectors
Inventors
Colosi, Peter Cameron • Ramirez, Silvia
Assignees
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Publication Number
US-11821008-B2
Publication Date
2023-11-21
Expiration Date
Abstract
The invention relates to novel adeno-associated virus (AAV) capsid proteins, AAV particles comprising a novel capsid protein, polynucleotides encoding these capsid proteins and AAV vectors expressing these capsid proteins. The invention also relates to methods of making the herein described AAV vectors expressing the novel capsid proteins of the invention and associated therapeutic uses of thereof.
Core Innovation
The patent describes recombinant adeno-associated virus (rAAV) systems that include a capsid protein comprising VP1, VP2, and VP3 regions derived from specified SEQ ID NOS, together with variable regions and constant regions in between. In particular embodiments, the capsid protein is at least 98% identical to VP1, VP2, or VP3 regions of selected SEQ ID NOS and comprises a GH loop identical to the GH loop of any one of the specified SEQ ID NOS, with the GH loop comprising variable regions IV to VIII or variable regions IV to VII.
The rAAV systems further include a transgene comprising a heterologous gene operably linked to a regulatory sequence controlling expression of the heterologous gene in a host cell. The documented constructs include AAV particles/vectors and encoded polynucleotides with defined SEQ ID NOS and mapped variable regions, and the nucleic acid sequence is operably linked to heterologous regulatory elements for expression of the capsid protein and/or the encoded transgene in a host cell.
The patent addresses delivering heterologous transgenes in a physiologically compatible manner and emphasizes liver-targeted delivery and liver cell specificity. It asserts improved performance including resistance to IVIG/neutralization and improved transduction, with emphasis on particular capsids including capsid Bba-49.
Claims Coverage
The document includes multiple independent claims covering rAAV capsid proteins and transgenes, AAV-encoding vectors, and methods of producing rAAV, with capsid sequence definitions centered on VP1/VP2/VP3 identity and capsid loop and variable region constraints. Across the independent claims, the coverage focuses on four inventive features.
Recombinant AAV with VP regions and transgene expression control
An rAAV comprising a capsid protein with an amino acid sequence at least 98% identical to VP1, VP2, or VP3 regions of specified SEQ ID NOS, wherein the capsid protein comprises variable regions IV to VII of any one of the specified SEQ ID NOS and constant regions in between, together with a transgene comprising a heterologous gene operably linked to a regulatory sequence controlling expression of the heterologous gene in a host cell.
Recombinant AAV with GH loop constraint and VP regions
An rAAV comprising a capsid protein with an amino acid sequence at least 98% identical to VP1, VP2, or VP3 regions of specified SEQ ID NOS, wherein the capsid protein comprises a GH loop identical to the GH loop of any one of the specified SEQ ID NOS, the GH loop comprising variable regions IV to VIII, together with a transgene comprising a heterologous gene operably linked to a regulatory sequence controlling expression of the heterologous gene in a host cell.
Vector nucleic acid encoding GH loop capsid protein for capsid expression
A vector comprising a nucleic acid sequence encoding an AAV capsid protein with an amino acid sequence at least 98% identical to VP1, VP2, or VP3 regions of specified SEQ ID NOS, wherein the capsid protein comprises a GH loop identical to the GH loop of any one of the specified SEQ ID NOS, the GH loop comprising variable regions IV to VIII, and wherein the nucleic acid sequence is operably linked to a heterologous regulatory element that controls expression of the capsid protein in a host cell.
Producing rAAV by culturing cells expressing rep and defined capsid protein
A method of producing a recombinant adeno-associated virus comprising culturing in a cell culture a cell comprising an AAV vector comprising AAV inverted terminal repeat sequences flanking a transgene with a heterologous gene operably linked to a regulatory sequence controlling expression in a host cell, wherein the cell expresses AAV rep and a capsid protein defined by VP1, VP2, or VP3 regions meeting identity requirements to specified SEQ ID NOS and comprising either a GH loop identical to the GH loop of any one of the specified SEQ ID NOS with variable regions IV to VIII, or variable regions IV to VII and constant regions in between; and collecting the rAAV from the supernatant of the cell culture.
Overall, the independent claim set defines rAAVs and AAV vectors by capsid protein sequence relationships to specified SEQ ID NOS for VP1/VP2/VP3 regions, optionally including a GH loop identical to a reference GH loop and specified variable region ranges, and combines these capsids with transgenes containing heterologous genes under control of regulatory sequences. The independent method claims add producing rAAV by culturing cells expressing AAV rep and the defined capsid protein followed by collecting rAAV from the culture supernatant.
Stated Advantages
Liver cell specificity.
Resistance to IVIG/neutralization.
Improved transduction performance, including with capsid Bba-49.
Documented Applications
Therapeutic/medicinal use for delivering heterologous transgenes, including liver-targeted delivery.
Treatment for disorders or diseases including hemophilia A, hemophilia B, Wilson's disease, hereditary angioedema (HAE), alpha 1 antitrypsin deficiency, or galactosemia.
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