Controlled expression of viral proteins
AssigneeVoyager Therapeutics, Inc.
InventorsJeffrey Morley SLACK, Christopher Cain Lam NGUYEN, Peter SLADE, Ryan Joseph NISTLER, Amanda Udumma IBE-ENWO
Life of this patent
Estimating the term…
Abstract
The present disclosure describes methods and systems for use in the production of adeno-associated virus (AAV) particles, including recombinant adeno-associated virus (rAAV) particles. In certain embodiments, the production process and system use Spodoptera frugiperda insect cells (such as Sf9 or Sf21) as viral production cells (VPCs).
Claims71
An AAV expression construct comprising:
- a VP-coding region comprising a nucleotide sequence encoding at least one, two, or three VP proteins, chosen from a VP1 protein, a VP2 protein, a VP3 protein, or a combination thereof;
- at least a portion of a baculovirus genome, e.g., a variant baculovirus genome, and
- a modified Kozak sequence comprising the nucleotide sequence of SEQ ID NO:
- 252, or a nucleotide sequence comprising no more than one, two, or three different nucleotides relative to SEQ ID NO:
- 252.
An AAV expression construct comprising:
- (i) a Rep-coding region, comprising a nucleotide sequence encoding a Rep protein chosen from Rep52, Rep40, Rep68, Rep78 protein, or a combination thereof, e.g., a Rep52 protein and/or a Rep78 protein;
- and
- (ii) a VP-coding region comprising a nucleotide sequence encoding at least one, two, or three VP proteins, chosen from a VP1 protein, a VP2 protein, a VP3 protein, or a combination thereof;
- wherein the AAV expression construct comprises at least a portion of a baculovirus genome, e.g., a variant baculovirus genome, comprising a disruption of at least two non-essential genes (e.g., auxiliary and/or per os infectivity factor genes), wherein the at least two non-essential genes are independently chosen from egt, p74 (PIF0), p26, SOD, ChiA, v-cath, p10, polyhedrin, ctx, odv-e56, PIF1, PIF2, PIF3, PIF4, PIF5, Tn7, AcORF-91, AcORF-108, AcORF-52, v-ubi, or p94;
- and
- wherein the Rep-coding region is operably linked to a first promoter, e.g., a baculovirus early promoter or a baculovirus early-late promoter (e.g., a gp64 promoter), and a second promoter, e.g., a baculovirus later or a baculovirus very late promoter (e.g., a polh promoter), optionally, wherein:
- (a) the first promoter results in transcription of the Rep-coding region prior to transcription of the VP-coding region;
- (b) the Rep-coding region is present downstream of a homologous repeat region hr5;
- and/or
- (c) the VP-coding region is present in the SOD locus.
An AAV payload expression construct comprising a payload coding region comprising a nucleotide sequence encoding a payload wherein the AAV expression construct comprises at least a portion of a baculovirus genome, e.g., a variant baculovirus genome, comprising a disruption of at least two non-essential genes (e.g., auxiliary and/or per os infectivity factor genes), wherein the at least two non-essential genes are independently chosen from egt, p74 (PIF0), p26, SOD, ChiA, v-cath, p10, polyhedrin, ctx, odv-e56, PIF1, PIF2, PIF3, PIF4, PIF5, Tn7, AcORF-91, AcORF-108, AcORF-52, v-ubi, or p94.
Dependencies are inferred from the claim text.