Retroviral vector for the administration and expression of replicon RNA expressing heterologous nucleic acids
Inventors
BERGLUND, Peter Lars Aksel • Archer, Jacob Freeman • Lin, Tsai-Yu
Assignees
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Publication Number
US-11135283-B2
Publication Date
2021-10-05
Expiration Date
Abstract
The present disclosure relates generally to gene delivery using a chimeric, retroviral-RNA replicon vector particle for increased expression of transgenes in a host cell. In particular, the chimeric vectors described herein can be used in any of a variety of settings including gene therapy and vaccine settings.
Core Innovation
The invention provides a chimeric retroviral-RNA replicon vector particle comprising an envelope glycoprotein, a retroviral gag polyprotein comprising matrix, capsid and nucleocapsid proteins, and a retroviral protease. The particle further includes an alphaviral replicon sequence that carries an alphavirus replication signal with at least a 5UTR and a first copy of a portion of the nsP1 coding sequence comprising at least a conserved sequence element (CSE).
The alphaviral replicon sequence also includes a retroviral packaging sequence modified to contain no stop codons, a polynucleotide sequence encoding a 2A endoprotease cleavage site, and a polynucleotide sequence encoding a full-length replicase polyprotein (REP) comprising a second copy of a portion of the nsP1 coding sequence comprising at least the CSE. The second copy is codon modified and in frame with the remainder of the sequence encoding the full-length REP. The vector particle further comprises a heterologous nucleic acid sequence of interest (SOI) that does not encode the retroviral proteins defined by the particle components.
A packaging system is also provided for producing the chimeric retroviral-RNA replicon vector particle, wherein the particle is reverse transcriptase independent. The packaging system includes a packaging cell transfected or otherwise modified to contain a first nucleic acid molecule encoding an envelope, a second nucleic acid molecule encoding gag and pol proteins (optionally encoding a nonfunctional reverse transcriptase protein), and a third nucleic acid molecule comprising the alphaviral replicon sequence with the defined 5UTR, nsP1 CSE elements, stop-codon-free retroviral packaging sequence, 2A endoprotease cleavage site, codon-modified in-frame full-length REP, and a heterologous nucleic acid sequence of interest.
Claims Coverage
The document includes two independent claims: one directed to the chimeric retroviral-RNA replicon vector particle and one directed to a packaging system for producing the particle that is reverse transcriptase independent. The independent claim set recites a set of core inventive structural features and a production architecture that supports reverse-transcriptase independence.
Chimeric retroviral-RNA replicon vector particle with alphaviral replicon and defined retroviral components
A chimeric retroviral-RNA replicon vector particle comprising an envelope glycoprotein; a retroviral gag polyprotein comprising matrix, capsid and nucleocapsid proteins; a retroviral protease; and an alphaviral replicon sequence including at least a 5UTR, a first nsP1 coding portion containing at least a conserved sequence element (CSE), a retroviral packaging sequence modified to contain no stop codons, a polynucleotide encoding a 2A endoprotease cleavage site, a polynucleotide encoding a full-length replicase polyprotein (REP) with a second nsP1 CSE-containing portion that is codon modified and in frame, and a heterologous nucleic acid sequence of interest (SOI) that does not encode the retroviral proteins.
Reverse transcriptase independent packaging system for producing the chimeric retroviral-RNA replicon vector particle
A packaging system for producing a chimeric retroviral-RNA replicon vector particle, wherein the particle is reverse transcriptase independent, comprising a packaging cell transfected or otherwise modified to contain: a first nucleic acid molecule encoding an envelope; a second nucleic acid molecule encoding gag and pol proteins optionally encoding a nonfunctional reverse transcriptase protein; and a third nucleic acid molecule comprising the alphaviral replicon sequence with at least a 5UTR, a first nsP1 coding portion containing at least a CSE, a retroviral packaging sequence modified to contain no stop codons, a polynucleotide encoding a 2A endoprotease cleavage site, a polynucleotide encoding a full-length replicase polyprotein (REP) with a codon modified in-frame second nsP1 CSE-containing portion, and a heterologous nucleic acid sequence of interest (SOI).
Overall, the claim coverage centers on a chimeric retroviral-RNA replicon vector particle that combines retroviral structural and protease components with a defined alphaviral replicon architecture carrying nsP1 CSE elements, a stop-codon-free retroviral packaging sequence, and a 2A endoprotease cleavage site, together with an SOI that excludes the retroviral proteins. The packaging system claim further specifies a reverse transcriptase independent production setup using a packaging cell containing the defined envelope, gag/pol, and alphaviral replicon nucleic acid molecules.
Stated Advantages
Higher transgene expression, as shown by increased GFP expression for chimeric alphaviral replicon vectors.
In vivo luciferase kinetics are described for chimeric alphaviral replicon vectors.
Documented Applications
Therapeutic and prophylactic vaccination using pharmaceutical compositions to induce immune responses via dendritic cell targeting, including Sindbis E2/DC-SIGN targeting, with delivery of tumor/infectious antigens and/or immunomodulators.
Treatment of cancer by administering a pharmaceutical composition including a SOI encoding one or more tumor-associated antigens, optionally in combination with an immune checkpoint inhibitor and/or cytokines.
Treatment of an infectious disease by administering a pharmaceutical composition including a SOI encoding infectious disease antigens and/or immunomodulators.
Use of vaccine-related pharmaceutical compositions including an adjuvant chemical formula (I) for vaccine use.
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