Nucleic acid molecules containing spacers and methods of use thereof
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Publication Number
US-11230720-B2
Publication Date
2022-01-25
Expiration Date
Abstract
The present invention relates nucleic acid molecules and concatemers containing spacer sequences useful for the efficient packaging of viral particles so as to minimize the incorporation of contaminant nucleic acids into these vectors, as well as methods of producing such viral particles.
Core Innovation
The invention relates to nucleic acid molecules for parvoviral genome packaging that include spacer sequences SS1 and SS2 flanking inverted terminal repeats ITR1 and ITR2. The nucleic acids are arranged in a 5′-to-3′ direction in architectures such as SS1-ITR1-CS-ITR2-SS2 and SS1-ITR1-HPM-ITR2-SS2, and in repeating forms such as (SS1-ITR1-CS-ITR2-SS2)n and (SS1-ITR1-HPM-ITR2-SS2)n, where n is an integer greater than 1. The SS1 and SS2 each comprise a portion having at least 85% sequence identity to nucleic acid sequences selected from SEQ ID NOS: 3-5 and 9-11.
The nucleic acid molecules further constrain the spacer sequences so that neither SS1 nor SS2 comprises an open reading frame that encodes more than 100 amino acids. The document defines spacer sequences and specifies identity thresholds intended to reduce incorporation of contaminant nucleic acids during genome packaging. Constraints described include exclusion of prokaryotic or baculoviral transcription factor binding sites and limiting CpG-rich contaminant incorporation by constraining total CpG content thresholds within SS1 and/or SS2.
The invention also includes modular nucleic-acid architectures in which additional elements such as heterologous polynucleotide molecules, homology arms HR1 and HR2, eukaryotic promoters, and polyadenylation sites can be placed between ITR1 and ITR2. The document describes vectors and viral particle populations associated with these nucleic acids and characterized by low levels of nucleic-acid-containing particles as contaminants.
Claims Coverage
The independent claims define multiple nucleic acid architectures that use spacer sequences SS1 and SS2 with sequence-identity and open reading frame constraints, combined with specific arrangements of ITR1/ITR2 with modular payload regions (CS or HPM) and, in some variants, homology arms and repeating structures. Across the independent claims, five inventive features are presented.
Spacer-flanked ITR architecture with defined spacers
A nucleic acid molecule arranged as SS1-ITR1-CS-ITR2-SS2 in a 5′-to-3′ direction, where SS1 and SS2 each comprise a portion having at least 85% sequence identity to nucleic acid sequences of SEQ ID NOS: 3-5 and 9-11, and where neither SS1 nor SS2 comprises an open reading frame that encodes more than 100 amino acids.
Heterologous polynucleotide between ITRs with defined spacers
A nucleic acid molecule arranged as SS1-ITR1-HPM-ITR2-SS2 in a 5′-to-3′ direction, where SS1 and SS2 each comprise a portion having at least 85% sequence identity to nucleic acid sequences of SEQ ID NOS: 3-5 and 9-11, and where neither SS1 nor SS2 comprises an open reading frame that encodes more than 100 amino acids.
Homology arms between ITRs with defined spacers
A nucleic acid molecule arranged as SS1-ITR1-HR1-CS-HR2-ITR2-SS2 in a 5′-to-3′ direction, where SS1 and SS2 each comprise a portion having at least 85% sequence identity to nucleic acid sequences selected from SEQ ID NOS: 3-5 and 9-11, and where neither SS1 nor SS2 comprises an open reading frame that encodes more than 100 amino acids.
Repeating-unit spacer-flanked ITR genome cassettes
A nucleic acid molecule of formula (SS1-ITR1-CS-ITR2-SS2)n, where n is an integer greater than 1, the components are operably linked in a 5′-to-3′ direction, SS1 and SS2 together comprise a portion having at least 85% sequence identity to nucleic acid sequences selected from SEQ ID NOS: 3-5 and 9-11, and neither SS1 nor SS2 comprises an open reading frame that encodes more than 100 amino acids.
Repeating-unit spacer-flanked ITR cassettes with heterologous polynucleotide
A nucleic acid molecule of formula (SS1-ITR1-HPM-ITR2-SS2)n, where n is an integer greater than 1, the components are operably linked in a 5′-to-3′ direction, SS1 and SS2 together comprise a portion having at least 85% sequence identity to nucleic acid sequences selected from SEQ ID NOS: 3-5 and 9-11, and neither SS1 nor SS2 comprises an open reading frame that encodes more than 100 amino acids.
Across the independent claims, the core claim coverage is the use of spacer sequences SS1 and SS2 meeting an at-least-85% identity requirement to SEQ ID NOS: 3-5 and 9-11, combined with restricting open reading frame size, in defined modular arrangements between ITR1 and ITR2 using CS, HPM, and, in some constructs, HR1/HR2 and repeating units.
Stated Advantages
Reducing contaminant packaging by constraints on spacer sequences, including exclusion of contaminant nucleic acids during genome packaging.
Producing viral particle populations characterized by less than 1% contaminant nucleic-acid–containing particles.
Documented Applications
Producing a plurality of viral particles by introducing the nucleic acid into host cells in a cell culture medium and isolating viral particles from the cell culture medium [procedural detail omitted for safety].
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