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JP-7855208-B2 - Non-embedded virus delivery systems and related methods

JP7855208B2JP 7855208 B2JP7855208 B2JP 7855208B2JP-7855208-B2

Inventors

  • パウザ， チャールズ デイビッド
  • ラフーゼン， テイラー

Assignees

  • アメリカン ジーン テクノロジーズ インターナショナル インコーポレイテッド

Dates

Publication Date
20260508
Application Date
20170607
Priority Date
20160608

Claims (14)

  1. A combination comprising a first virus delivery system, a second virus delivery system, a third virus delivery system, and a fourth virus delivery system for use in the course of treatment, Each virus delivery system is a non-integrated virus delivery system that includes a virus carrier containing a defective integrase gene, The aforementioned course of action is included in the course of action in the first quadrant, the course of action in the second quadrant, the course of action in the third quadrant, or the course of action in the fourth quadrant. If the aforementioned treatment sequence is included in the treatment sequence of the first quadrant, the first virus delivery system is selected, and the first virus delivery system includes a heterologous human papillomavirus (HPV) viral episomal DNA replication origin containing a first 5' cleaved human papillomavirus (HPV) LCR fragment, and does not include an initiator protein. If the aforementioned treatment sequence is included in the treatment sequence of the second quadrant, the second virus delivery system is selected, and the second virus delivery system includes a heterologous human papillomavirus (HPV) viral episomal DNA replication origin comprising at least one initiator protein and a second 5' cleaved HPV LCR fragment. If the aforementioned treatment sequence is included in the treatment sequence of the third quadrant, the third viral delivery system is selected, and the third viral delivery system includes a heterologous human papillomavirus (HPV) viral episomal DNA replication origin comprising at least one initiator protein and a third 5' cleaved HPV LCR fragment. If the aforementioned treatment sequence is included in the treatment sequence of the fourth quadrant, the fourth viral delivery system is selected, and the fourth viral delivery system includes a heterologous human papillomavirus (HPV) viral episomal DNA replication origin containing a fourth 5'-cleaved HPV LCR fragment, and does not include an initiator protein. The HPV LCR is HPV16 LCR, and is a nucleic acid containing a sequence encoding one YYI binding site, three AP1 binding sites, four E2 binding sites, and one E1 binding site. The first 5'-cleaved HPV LCR fragment and the second 5'-cleaved HPV LCR fragment are nucleic acid fragments of HPV LCR having a truncation in their 5' region and including an E1 binding site and an E2 binding site, wherein the E1 binding site and the E2 binding site are as follows: a) One E1 binding site and two E2 binding sites, wherein two of the four E2 binding sites are removed from the HPV LCR; or, b) One E1 binding site and three E2 binding sites, wherein one of the four E2 binding sites is removed from the HPV LCR; It consists of, The third 5'-cleaved HPV LCR fragment and the fourth 5'-cleaved HPV LCR fragment are nucleic acid fragments of HPV LCR having truncations in their 5' region, and are nucleic acids comprising three AP1 binding sites, YY1 binding sites, and E1 binding sites and E2 binding sites, wherein the E1 binding sites and E2 binding sites consist of one E1 binding site and four E2 binding sites. The at least one initiator protein is specific to the first 5'-cleaved HPV LCR fragment, the second 5'-cleaved HPV LCR fragment, the third 5'-cleaved HPV LCR fragment, or the fourth 5'-cleaved HPV LCR fragment, The aforementioned at least one initiator protein comprises at least one of E1, E1-C, E2, or E1-T2A-E2, The treatment process in the first quadrant includes at least one of gene editing or safety testing . The treatment process in the second quadrant includes at least one of the following: cell reprogramming, expression of long-acting growth factors, or checkpoint suppression. The treatment course in the third quadrant includes at least one of the following: passive immunity, immune stimulation , or expression of transcription/differentiation factors. The course of treatment in the fourth quadrant includes at least one of placebo control or use for dose escalation . A combination of items.
  2. The combination according to claim 1, wherein at least one of the first 5'-cleaved HPV LCR fragment and the second 5'-cleaved HPV LCR fragment lacks an AP1 transcription factor binding site or any portion thereof.
  3. The combination according to claim 1, wherein at least one of the first 5'-cleaved HPV LCR fragment and the second 5'-cleaved HPV LCR fragment contains two or fewer AP1 transcription factor binding sites or portions thereof.
  4. The combination according to claim 1, wherein at least one of the third 5'-cleaved HPV LCR fragment and the fourth 5'-cleaved HPV LCR fragment contains at least three AP1 transcription factor binding sites.
  5. The combination according to claim 1, wherein at least one of the first 5'-cut HPV LCR fragment and the second 5'-cut HPV LCR fragment has a length of less than about 200 base pairs.
  6. The combination according to claim 1, wherein at least one of the first 5'-cut HPV LCR fragment and the second 5'-cut HPV LCR fragment comprises a length of about 200 base pairs to about 300 base pairs.
  7. The combination according to claim 1, wherein at least one of the first 5'-cut HPV LCR fragment and the second 5'-cut HPV LCR fragment comprises a length of about 300 base pairs to about 550 base pairs.
  8. The combination according to claim 1, wherein at least one of the third 5'-cut HPV LCR fragment and the fourth 5'-cut HPV LCR fragment comprises a length of about 550 base pairs to about 750 base pairs.
  9. The combination according to any one of claims 1 to 4, wherein at least one of the first 5'-cut HPV LCR fragment and the second 5'-cut HPV LCR fragment has at least 90% sequence identity with any one of sequence numbers 3, 4, and 5 .
  10. The combination according to any one of claims 1 to 4, wherein at least one of the third 5'-cut HPV LCR fragment or the fourth 5'-cut HPV LCR fragment has at least 90% sequence identity with either one of sequence numbers 1 and 2 .
  11. The combination according to any one of claims 1 to 10 , characterized in that the cells are brought into contact with the first virus delivery system, the second virus delivery system, the third virus delivery system, or the fourth virus delivery system.
  12. The combination according to claim 11, wherein the first virus delivery system and the second virus delivery system further comprise the first gene cargo, and the third virus delivery system and the fourth virus delivery system further comprise the second gene cargo.
  13. The combination according to claim 12 , wherein contact between the cells and the first virus delivery system results in more than approximately 0.02 basal episomal copies of the first gene cargo per cell.
  14. The combination according to claim 12 , wherein contact between the cells and the fourth virus delivery system results in a basal episomal copy of the second gene cargo of less than approximately 0.02 per cell.

Description

This application is a follow-up to U.S. Provisional Patent Application No. 62/347,552, filed on June 8, 2016, titled "NON-INTEGRATING VIRAL DELIVERY SYSTEM AND METHODS OF USE THEREOF," U.S. Provisional Patent Application No. 62/431,760, filed on December 8, 2016, titled "NON-INTEGRATING VIRAL DELIVERY SYSTEM AND METHODS RELATED THERETO," and "NON-INTEGRATING VIRAL DELIVERY SYSTEM AND METHODS RELATED" This application claims priority to PCT/US16/66185, filed December 12, 2016, entitled "THERETO," the disclosures of which are incorporated herein by reference. This disclosure generally relates to viral vectors and systems for gene delivery, and to the field of use for other therapeutic, diagnostic, or investigative purposes. More specifically, embodiments of this disclosure relate to non-embedded viral vectors and systems for gene delivery, and to the field of use for other therapeutic, diagnostic, or investigative purposes. Viral vectors have been used to transduce genes and other therapeutic nucleic acid constructs into target cells due to their specific viral envelope-host cell receptor interactions and viral mechanisms for gene expression. As a result, viral vectors are used as a medium for gene transfer into many different cell types, including but not limited to isolated tissue samples, in-situ tissue targets, and cultured cell lines. The ability to introduce and express exogenous genes into cells is useful for studying gene expression, elucidating cell lineages and pathways, and providing potential for therapeutic interventions such as gene therapy and various types of immunotherapy. Several viral systems, including lentiviruses, murine retroviruses, adenoviruses, and adeno-associated viruses, have been proposed as promising therapeutic gene transfer vectors. However, numerous hurdles have prevented their widespread use as approved therapeutic agents. These hurdles include, but are not limited to, expression stability and control, genome packaging capabilities, and construct-dependent vector stability. Furthermore, the in vivo application of viral vectors can be limited by the host's immune response to viral structural proteins and/or transduced gene products, which can lead to adverse anti-vector immunological effects. Researchers have attempted to find stable expression systems as a way to overcome some of these hurdles. One approach involves utilizing gene regulatory molecules, including recombinant polypeptides or small RNAs, in such expression systems. These systems employ chromosomal integration of the transduced retroviral genome, or at least a portion thereof, into the host cell's genome. A key limitation of these approaches is that the sites of gene integration are generally random, and the number and proportion of genes integrated at any given site are often unpredictable. Therefore, vectors that rely on chromosomal integration result in the persistent maintenance of recombinant genes that can extend beyond the treatment interval, while plasmids or other non-replicating DNA are not well controlled and may collapse before the desired treatment interval is completed. Another approach is the use of transient expression systems. Under transient expression systems, the expression of the target gene is based on a non-integrated plasmid, and therefore, expression is typically lost as the cell subsequently divides, or the plasmid vector is destroyed by endogenous nucleases. Consequently, transient gene expression systems typically fail to achieve sufficient expression over time and typically require repeated treatments, which is generally understood to be an undesirable characteristic. Figure 1 shows an exemplary vector-in-vector (VIV) embodiment. Figure 1(A) shows a linear vector, and Figure 1(B) shows a ring-shaped vector. Figure 2 shows an exemplary vector-in-vector (VIV) embodiment containing the E1 initiator protein (also referred to herein as vector 1). Figure 3 shows the results of transduction in 293T cells from three separate experiments using vector 1. Figure 4 shows an exemplary vector-in-vector (VIV) embodiment (also referred to herein as vector 19) containing both E1 and E2 initiator proteins. Figure 5 shows exemplary vector-in-vector (VIV) embodiments expressing (A) mCherry and (B) VEGF, respectively. Figure 6 shows the expression of mCherry-positive cells for various described constructs when E1 and E2 are provided by plasmid (A) or lentivirus (B), respectively. Figure 7 shows an exemplary vector-in-vector (VIV) embodiment used in combination with the embodiments described in detail herein. Figure 8 shows the expression levels of VEGF for various constructs containing fragment 1 of the HPV16 long control region (LCR). Figure 9 shows an exemplary vector-in-vector (VIV) embodiment used in combination with the embodiments described in detail herein. Figure 10 is a diagram illustrating an example of the episomal morphology of a non-embedded lentiviral vector. Figure 11 shows the genomic rel