Intrinsic system for viral vector transgene regulation

Abstract

A method for the regulated removal of heterologous genetic material from disseminating viral vaccine vectors is provided.

Claims

1. A method for the regulated removal of heterologous genetic material from disseminating DNA viral vaccine vectors, comprising: providing a wild-type or parental DNA viral vector which includes a wild-type or parental genomic sequence and a recombinantly engineered duplication of the said wild-type or parental genomic sequence; providing a transgene expression cassette which comprises a transgene and the necessary regulatory genetic elements for control of transgene expression; and inserting the transgene expression cassette into the DNA viral vector so that the transgene is flanked by the wild-type or parental genomic sequence and the recombinantly engineered duplication of wild-type or parental genomic sequence; whereby homologous recombination between the flanking wild-type or parental genomic sequences subsequently results in removal of the transgene and restoration of only a single copy of the wild-type or parental genomic sequence as is normally found within a wild type or parental DNA virus, thereby regenerating the wild-type or parental DNA virus genome with an absence of any non-parental virus genetic sequence; wherein the method comprises a step of selecting a length for the wild-type or parental genomic sequence to be duplicated; and in which the rate of transgene removal from the viral genome, and hence reversion of recombinant virus genome to wild-type or parental, is a function of the length of the selected duplicated wild-type or parental genomic sequence.

2. A method for the regulated removal of heterologous genetic material from DNA viral vaccine vectors, comprising: providing a wild-type or parental DNA viral vector which includes a recombinantly engineered duplication of genomic sequence of a preselected length; providing an expression cassette which comprises a transgene and the necessary regulatory genetic elements for control of transgene expression; and inserting the cassette into the DNA viral vector so that the transgene is flanked by the wild-type or parental genomic sequence and its duplicated sequence; whereby homologous recombination results in removal of the transgene to leave only a single copy of the genomic sequence as is normally found within a wild type or parental virus, thereby regenerating the wild type or parental virus genome with an absence of any non-parental virus genetic sequence; and in which the rate of transgene removal from the viral genome, and hence reversion of recombinant virus genome to wild-type or parental, is a function of the length of the duplicated sequence.

3. The method of claim 2, being an intrinsic method for regulated removal of transgene from virus vectors used for gene-replacement.

4. The method of claim 2, being a method of cancer immunotherapy requiring short-term antigen exposure, whereby a viral vector reverts back to wild-type rapidly on antigen decay.

Description

PAGE I

(1) Referring to FIG. 1 of the drawings, the exogenous transgene is inserted into the viral vector in such a manner that it is flanked by repeated viral sequences allowing directed homologous recombination between repeated sequences and concomitant removal of the inserted transgene.

(2) Recombination resulting in removal of the transgene will leave only a single copy of the flanking homology sequence thereby generating the wild type (parental) virus genome, with the absence of any non-viral genetic sequence.

(3) The rate of transgene removal from the viral genome (period required for reversion of recombinant virus genome to parental) will be a function of the specific DNA virus being used, the length of repeat sequence and the size and nature of the inserted transgene.

(4) The length of transgene expression will be definable and variable allowing an intrinsic means for fine tuning of transgene removal. Modulation of decay rate will permit transgene expression appropriate for the vaccination or gene-replacement strategy being deployed and improved safety profiles with reversion of recombinant viruses back to parental wild-type.

(5) This mechanism is applicable to all DNA virus-based viral vectors.

(6) This principle can be applied, for example, to a method for the rapid removal of heterologous sequences from genetically modified viruses.

(7) These may include viruses and viral vectors (replicating or attenuated) for use in: a. Cancer immunotherapy requiring short-term antigen exposure. Viral vectors would revert back to wild-type rapidly on antigen decay. b. Vaccination strategies for prevention of bacterial, viral, helminth or fungal infections. Viral vectors would revert back to wild-type rapidly on antigen decay. c. Alternative vaccine strategies including self-antigens such as those used for immunocontraception. Viral vectors would revert back to wild-type rapidly on antigen decay. g. Gene replacement, representing an intrinsic method for regulated removal of transgene from virus vectors used for gene-replacement.

(8) Embodiments of the present invention also provides a method for the rapid removal of heterologous genetic material from disseminating viral vaccine vectors.

(9) These may include, but are not limited to, viruses and viral vectors for use in animal populations where spread of viral vector rather than direct inoculation is advantageous, but where a non-permanent presence of the recombinant virus within the environment is desirable. Non-exclusive examples are given below: a. Captive animal populations requiring short-term vaccination/dissemination (as above, bacterial-, viral-, fungal-, helminth- or self-antigens) followed by reversion of viral vector to parental virus. b. Companion animals requiring short-term vaccination/dissemination (as above, bacterial-, viral-, fungal-, helminth- or self-antigens) followed by reversion of viral vector to parental virus. c. Livestock animals requiring short-term vaccination/dissemination (as above, bacterial-, viral-, fungal- helminth- or self-antigens) followed by reversion of viral vector to parental. e. Wild animals requiring short-term vaccination/dissemination (as above, bacterial-, viral-, fungal- or helminth-self-antigens) followed by reversion of viral vector to parental.

(10) Embodiments of the present invention also provides a method for the delayed removal of heterologous genetic material from disseminating viral vaccine vectors.

(11) This would include but is not limited to examples given above.

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(12) Technical Progress/Highlights: TA2 WPIB

(13) Intrinsic Attenuation

(14) The results provided in the FIGURES demonstrate that homologous recombination can be used to remove expressed transgenes as described in FIG. 1.

(15) LacZ cassette (transgene) remains within a proportion of virions for at least 4 passages with a repeat length of 250 bp and is not lost from control virions with 0 bp repeat length consistent with hypothesis.

(16) Rates of loss are influenced by flanking homology length, which is quantified in subsequent experiments (see below).

(17) TABLE-US-00001 TABLE I Reconstitution of iMCMV B-Gal BAC Constructs in Permissive Murine Fibroblasts Cytopathic Effect (CPE) Recombinant iMCMV BAC clones (Day 12 post- (reconstituted in M210B4 mouse fibroblasts) transfection) MCMV pArk14 +++ [Parental -Gal negative] iMCMV(250) +++ [MCMVM157 LacZ/Kan.sup.R flanked by 250 nt repeats] (DH10B clone 1) iMCMV(25) +++ [MCMVM157 LacZ/Kan.sup.R flanked by 25 nt repeats] (DH10B clone 1) iMCMV(0) + [MCMV M157 LacZ/Kan.sup.R flanked by 0 nt repeats] Table 1. Growth of iMCMV following reconstitution in permissive M210.B4 mouse fibroblasts. The iMCMV BAC clones were constructed by E/T recombination. The different iMCMV BACs contain the -Gal + Kan.sup.R cassette flanked by indicated region of homology inserted within the non-essential M157 MCMV ORF. BACs were selected on the basis of Kan-resistance and characterized by PCR. Complete genome sequence analysis confirmed genome integrity, and functional analysis in bacteria showed -Gal (LacZ) gene metabolized X-gal (resulting in blue colonies) demonstrating stable expression of a functional -Gal. MCMV pArk14 does not contain the LacZ gene. Table 1 shows results from reconstitution of iMCMV BACs in MCMV permissive murine M210.B4 fibroblasts. Key: +++ = 50% CPE; + = <25% (2-3 plaques).

PAGE 3 & 4

(18) Pages 3 and 4 of the drawings relate to the loss of a LacZ/KN cassette in a virus at P1 and P2.

(19) LacZ is present in 0 nt, 25 nt and 250 nt MCMV Bgal strains at P1 and P2.

(20) Red highlighted bands (at right on page 4) show that at P1 there is already excision of LacZ/Kn between 25 nt and 250 nt flank sequences.

(21) Sequencing confirmed that the highlighted bands corresponds to excised LacZ/Kn.

(22) Loss of the LacZ/Kn is not detected in Ont flank samples.

(23) M PCR with primers m156intFOR and m 158REV:

(24) vARK 14 (WT): 1326 bp

(25) 25 nt flank with insert: 6884 bp

(26) 250 nt flank with insert: 7109 bp

(27) lacZ/Kn excised between repeat sequences: 702 bp

(28) L PCR with primers LacZintFOR and LacZintREV: 3024 bp

PAGES 5 & 6

(29) Pages 5 and 6 of the drawings relate to loss of LacZ/KN cassette in a virus at P3 and P4.

(30) LacZ is present in 0 nt, 25 nt and 250 nt MCMV Bgal strains at P3 and P4.

(31) Red highlighted bands (right on page 6) corresponds to excision of LacZ/Kn between 25 nt and 250 nt flank sequences.

(32) Rate of gene loss is higher in virus with 250 versus 25 bp repeats.

(33) Loss of the LacZ/Kn is not detected in Ont flank samples.

(34) Take home message: LacZ cassette is retained for at least 4 passages in both 25 and 250 bp repeat viruses. Homologous recombination is occurring and leads to loss of LacZ in a proportion of virus. Rate of loss is determined by length of repeat sequence.

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(35) Intrinsic decay: A simple tool for robust biological control of a transmissible vaccine (WP2 TAIB)

(36) A) Principle of repeat sequence based intrinsic decay. The rate of homologous recombination is dictated by length of the repeat sequence (and capacity for viral encapsidation, a function of total genome size) in the virus, longer repeats should lead to more rapid loss of the transgene (or LacZ).

(37) B. Design of iMCMV strains showing plasmid and mechanism of homologous recombination leading to reversion to wild type (WT) sequence containing only a single repeat sequence.

(38) Homologous recombination between the repeat sequences leads to loss of intervening sequence and one repeat.

(39) C. Staining for retention of LacZ (viral plagues retaining LacZ stain blue following the addition of betagalactosidase). Shown is passage 4 virus titrations for iMCMV-250 and iMCMV-0.

(40) D. Quantification of transgene (LacZ) loss over serial passage. iMCMV-250 variant has complete transgene loss by passage 13, whilst iMCMV-25 retains the transgene for multiple passages. iMCMV-25 eventually loses expression at a rate slightly faster than virus without repeat sequences (iMCMV-0). We could reasonably expect more rapid transgene loss in vivo due to greater selective pressure. We will be testing new iMCMV strains with, 0, 25, 50, 75 and 100 bp repeats in vivo whilst also assessing immunogenicity, and loss of immunogenicity, to the nominal antigen NP from LCMV. We will also be assessing the role that competition between WT and iMCMV strains play in the role of transgene loss. In this instance competition may select for WT leading to loss of iMCMV strains via competition, i.e. independent of on-going recombination.

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(41) Transmissible vaccination: A tool to prevent the host species jump of zoonotic viruses from animals into humans (WPI)

(42) A) Principle of transmissible vaccination. Most highly pathogenic viruses in humans come from wildlife. Wildlife vaccination can prevent the jump into humans, BUT inaccessibility and harsh conditions prevent use of conventional (directly administered) vaccines except on rare occasions (e.g. rabies). Transmissible vaccination solves the problem of providing high vaccine coverage in inaccessible wildlife populations living in harsh environments.

(43) B) Transmissible LASV Vaccine. A transmissible vaccine against Lassa virus (LASV) for use in the wild rat (Mastomys natalensis; Mnat) reservoir provides a Proof-of-Concept with direct Real-World impact. Mastomys cytomegalovirus (MasCMV) will be used as the transmissible vaccine. TIMELINE. June, 2019: Based on ability to amplify of a 179 bp section of the 230,000 bp genome, PCR had shown the presence of MasCMV in Mnat tissues. September, 2019: Availability of tissues from an independently funded project at RML combined with development of a highly permissive Mnat epithelial cell line enabled isolation of 3 distinct species of MasCMV. This sensitive co-culturing assay showed MasCMV infection to be high in the wild Mnat population (at least 40%) and coinfection was common, indicating that prior immunity to MasCMV (even for very closely related viruses) did not prevent re-infection: these are two characteristics important for use as a transmissible vaccine. January, 2020: Real-time full-length genome sequencing has provided the most extensive characterization of any CMV in its native wild animal population. Key to the development of MasCMV as a transmissible vaccine, genome characterization has enabled movement to the second stage of the project where the MasCMVs will be cloned as infectious bacterial artificial chromosomes such that they can be genetically manipulated to construct a transmissible vaccine for LASV.

(44) Although illustrative embodiments of the invention have been disclosed in detail herein, with reference to the accompanying drawings, it is understood that the invention is not limited to the precise embodiments shown and that various changes and modifications can be effected therein by one skilled in the art without departing from the scope of the invention as defined by the appended claims and their equivalents.