RU2779300C1 - Method for enhancing targeted editing of the cxcr4 gene in human cd4 lymphocytes in order to create cell resistance to hiv-1 - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biochemistry, in particular to donor DNA for the delivery of a protective peptide from gp41 MT-C34. Methods for delivery of a protective peptide from gp41 MT-C34 are also disclosed.

EFFECT: invention makes it possible to effectively edit the CXCR4 gene in CD4 lymphocytes.

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Description

Technical field

The invention relates to the fields of molecular biology, virology and cell technology. The invention can potentially be used to treat HIV.

The work was financially supported by the Ministry of Science and Higher Education of the Russian Federation under Agreement No. 075-15-2019-1661 of October 31, 2019 and with the financial support of the Russian Foundation for Basic Research under agreement 18-29-07052.

State of the art

Previously, our laboratory developed a technology for creating T-cell resistance to HIV-1 based on genetically encoded GPI-anchored peptides from gp41, patent application 2020142800 dated 12/24/2020. The technology is a genetic construct that includes the sequence of the GPI-anchored CD52 protein, between two parts of which (the protein export signal and the GPI-anchoring signal) one of the protective peptides from the HIV-1 gp41 envelope protein is inserted, as well as methods for delivering the genetic construct to the cell membrane using the CRISPR/Cas9 genomic editing system.

The CRISPR/Cas9 genomic editing system is a relatively new technology that is being developed by a large number of research groups around the world. The technology has great potential and a wide range of possibilities for the treatment of HIV infection. Despite significant advances in this area, increasing the efficiency of therapeutic sequences introduced using the CRISPR/Cas9 system is an important task that must be solved for the effective application of the technology in the clinic.

To achieve this goal, research is underway in the field of enhancement of homologous recombination (HDR) by various modifications of donor DNA. It has been shown that elongation of the homology arms leads to an increase in HDR efficiency (Tsunekawa Y, Terhune RK, Fujita I, Shitamukai A, Suetsugu T, Matsuzaki F. Developing a de novo targeted knock-in method based on in utero electroporation into the mammalian brain. Kurihara T, Kouyama-Suzuki E, Satoga M, et al., DNA repair protein RAD51 enhances the CRISPR/Cas9-mediated knock-in efficiency. in brain neurons Biochem Biophys Res Commun 2020;524(3):621-628 doi:https://doi.org/10.1016/j.bbrc.2020.01.132) in mouse pyramidal neurons. Several studies have shown that the use of plasmid DNA as a donor can increase HDR efficiency (Sung JJ, Park C-Y, Leem JW, Cho MS, Kim D-W. Restoration of FVIII expression by targeted gene insertion in the FVIII locus in hemophilia A patient-derived iPSCs Exp Mol Med. 2019;51(4):1-9 doi:10.1038/s12276-019-0243-1; Liu J-T, Corbett JL, Heslop JA, Duncan SA. by co-targeting ATP1a1. Peer J. 2020; 8:e9060. doi:10.7717/peerj.9060). An increase in HDR efficiency was achieved by modifying donor DNA by introducing full-length DNA into it (Yao X, Wang X, Hu X, et al. Homology-mediated end joining-based targeted integration using CRISPR/Cas9. Cell Res. 2017;27(6): Yao X, Wang X, Liu J, Shi L, Huang P, Yang H. CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy. J Vis Exp. 2018;(133):56844. doi:10.3791/56844) or truncated guide RNA (gRNA) recognition sites (Nguyen DN, Roth TL, Li PJ, et al. Polymer-stabilized Cas9 nanoparticles and modified repair templates increase genome editing efficiency Nat Biotechnol 2020;38(1):44-49 doi:10.1038/s41587-019-0325-6).

Another approach to solving this problem is to modify Cas9. It has been shown that an increase in the number of nuclear localization signals (NLS) in Cas9 can improve the efficiency of construct delivery to the nucleus, which leads to an increase in the level of editing (Torres-Ruiz R, Martinez-Lage M, Martin MC, et al., Efficient Recreation of t( 11;22) EWSR1-FLI1(+) in Human Stem Cells Using CRISPR/Cas9 Stem cell reports 2017;8(5):1408-1420 doi:10.1016/j.stemcr.2017.04.014; Maggio I, Zittersteijn HA, Wang Q, et al., Integrating gene delivery and gene-editing technologies by adenoviral vector transfer of optimized CRISPR-Cas9 components, Gene Ther 2020;27(5):209-225 doi:10.1038/s41434-019- 0119-y).

An efficient way to deliver a donor DNA construct to the nucleus is to add to it an enhancer fragment from the SV40 virus promoter, the so-called DTS (DNA nuclear transporting sequence). Fragment addition enhances plasmid nuclear import in vivo (Dean DA, Dean BS, Muller S, Smith LC. Sequence requirements for plasmid nuclear import. Exp Cell Res. 1999;253(2):713-722. doi:10.1006/excr. 1999.4716). This is explained by the fact that DTS SV40 has landing sites for several transcription factors that are expressed and work in almost all cell types. The NFkB landing site sequence (Mesika A, Grigoreva I, Zohar M, Reich Z. A Regulated, NFκB-Assisted Import of Plasmid DNA into Mammalian Cell Nuclei. Mol Ther. 2001;3( 5):653-657 doi:https://doi.org/10.1006/mthe.2001.0312).

The use of the DTS site in the donor construct is mentioned only in one work devoted to the production of knockin in rat zygotes (Remy S, Chenouard V, Tesson L, et al. Generation of gene-edited rats by delivery of CRISPR/Cas9 protein and donor DNA into intact zygotes using electroporation Sci Rep. 2017;7(1):16554.doi:10.1038/s41598-017-16328-y). However, the authors failed to achieve an increase in the knockin level, which may be due to the very large (4 kb) size of the donor construct.

The described methods have been used to develop effective treatments for various genetic diseases, but have not been used to create HIV-resistant cells.

The patented method is a significant improvement of the previously created system, which makes it possible to introduce into the genome of T-cells a construct that makes them resistant to HIV with greater efficiency.

The technical problem - improving the technology for creating T-cell resistance to HIV-1 based on genetically encoded GPI-anchored peptides from gp41 - is solved by creating an effective technology for targeted editing of the CXCR4 gene in CD4 lymphocytes. The technical result is an increase in the efficiency of introducing a protective structure and an increase in the resistance of cells to HIV.

The improvements made have made it possible to achieve a significant increase in the effectiveness of the introduction of a protective structure, which is a key factor for the potential use of technology for HIV therapy. At the same time, the advantages of the original technology, namely, the cost-effectiveness of donor DNA production and the compatibility of the technology with various genome editing systems based on CRISPR/Cas9 and others, which makes it easy to adapt it to the latest developments in this area, were retained.

Disclosure of the essence of the invention

The present invention represents a significant improvement of our previously developed technology for creating T-cell resistance to HIV-1 based on genetically encoded GPI-anchored peptides from gp41, in which the protective peptide from gp41 is delivered to the surface of the plasma membrane of the target cell by introducing its encoding nucleotide sequence into the genome using CRISPR/Cas9 as part of a genetic construct, which is the CD52 protein, between the two parts of which (the protein export signal and the GPI anchoring signal) the target peptide is inserted.

An increase in the efficiency of introduction of the target construct was achieved by the following modifications of the donor DNA: 1. Donor DNA is introduced into the cell in the context of plasmid DNA. 2. Arms of homology increased from approximately 100 to 500 nucleotides. 3. Surrounding the sequence of the target construct, gRNA and DTS sites increase the efficiency of donor DNA delivery to the target site in the genome.

To implement the invention, two components are introduced into cells: a plasmid encoding Cas9 nuclease and gRNA, in the sequence of which there are several NLS sites (pX330-Cas9-BFP-gRNAX4ex2) and a plasmid encoding donor DNA. Guide RNA directs Cas9 to a specific location in the genome. The donor DNA contains the target genetic construct flanked by homology arms, at the ends of which are the Cas9 landing site (X4ex2 protospacer) and the DTS transcription factor binding site, which increase the efficiency of donor DNA delivery to the editing site, and serves as a template for homologous recombination.

In an alternative embodiment of the invention, the plasmid encoding the Cas9 nuclease and gRNA is replaced by a ribonucleoprotein complex (RNP) comprising recombinant Cas9 protein complexed with gRNA obtained by in vitro transcription.

The present invention provides methods for efficiently delivering a target genetic construct to a cell membrane.

The target genetic construct encoded in the donor DNA is the sequence of the GPI-anchored protein CD52, between the export signal and the membrane anchoring signal of which is the sequence encoding the protective peptide from gp41, MT-C34. SEQ ID NO: 1 is a protein export signal. SEQ ID NO:2 serves as a signal for membrane anchoring of the construct. The sequence encoding the protective peptide from gp41 MT-C34 - SEQ ID NO: 3. This design delivers the peptide to the cell surface in a specific zone - the lipid raft, where the CD4 receptor and co-receptors CCR5 and CXCR4 are localized, recognized by HIV and used by it to enter the cell (Fig. 1). The peptide exposed on the cell surface interacts with the N-terminal gp41 heptad repeat of the viral fusion protein and prevents the formation of a six-stranded bundle, thus protecting the cell from HIV penetration.

Donor DNA is represented by the following sequence: X4ex2 protospacer (SEQ ID NO: 4), 5' homology arm (SEQ ID NO: 5), P2A sequence, which allows the ribosome to skip the formation of a covalent bond between glycine and proline, in other words, allows to obtain from one RNA separate translation of CXCR4 peptide and protein (SEQ ID NO: 6), protein export signal (SEQ ID NO: 1), sequence encoding protective peptide from gp41 MT-C34 (SEQ ID NO: 3), signal for membrane anchoring (SEQ ID NO: 2), polyadenylation sequence (SEQ ID NO: 7), 3'-arm homology (SEQ ID NO: 8), DTS sequence (SEQ ID NO: 9) as shown in Figure 2 A. Donor DNA is introduced into cell in the context of the pJet commercial cloning vector (Thermo scientific USA).

The most efficient gRNA directing Cas9 to the second exon, CXCR4 (SEQ ID NO: 10).

Brief description of the drawings

Figure 1. Scheme of the location of the protective peptide on the surface of the cell membrane.

Figure 2. Schemes of sequences used as donor DNA to introduce target mutations: A - patented technology; B - original technology.

Figure 3. Comparison of the efficiency of introducing knockin by the original and modified methods into the CEM/R5 cell line: A - Comparison of the efficiency of introducing the knockin construct into the genome of CEM-R5 cells using the original donor DNA construct (X4) and using the modified donor DNA (sg-X4 -DTS). B - Increase in the efficiency of introducing knockin when using the plasmid pX330-Cas9-BFP-gRNAX4ex2 compared to wild-type Cas9. * - p <0.05 ** - p <0.01 by Student's t-test.

Figure 4. Raji to CD4+ MT-C34 intracellular infection test A - Luciferase activity of construct knockin cells (MT-C34+), control cells (MT-C34-) and the Raji cell line. B - data on the content of the viral Gag protein in the medium three days after infection with HIV-1 CD4+ lymphocytes without (MT-C34-) and with (MT-C34+) knockin constructs. Data obtained by ELISA for p24.

Implementation of the invention

Example 1. Obtaining a cell line carrying the target construct, followed by evaluation of the effectiveness of the method

Based on the CEM-CCRF (CEM) CD4+ lymphoblast cell line, a subline of CEM/R5 cells was obtained that are susceptible to infection with HIV-1 of different tropism due to the expression of CD4, CCR5, and CXCR4 molecules, one of which was introduced by lentiviral transduction of a CCR5-expressing construct. .

Based on this subline, by introducing a knockin, cell lines carrying the target constructs with the peptide were obtained. For this, electroporation was used using a Neon Thermo Fisher device in the mode recommended by the manufacturer.

The mixture for electroporation of 1 million cells included the following components:

- 4 μg pX330-Cas9-BFP-gRNAX4ex2 - plasmid encoding wild-type Cas9, BFP fluorescent protein and gRNA;

- 6 μg donor template in the context of the commercial vector pJet (Thermo scientific USA) described above (Fig. 2A);

- commercial buffer R up to 110 µl.

The cell line CEM/R5/MT-C34-new with the peptide in the context of the GPI-anchored CD52 protein was obtained by the described method. Sorting of cells with the target construct was carried out by immunofluorescent staining. Antibodies to the MT-C34 peptide obtained in our laboratory were used as primary antibodies; antibodies conjugated with the Alexa546 fluorescent label were used as secondary antibodies. Isolation of cells with the target construct was carried out by FACS - sorting to achieve a culture purity of at least 98%.

Using RNP, the CEM/R5/MT-C34-RNP-new cell line was obtained by electroporation of CEM/R5 cells. The mixture for electroporation of 1 million cells included:

- RNP complex;

- donor template in the context of the commercial pJet vector described above (FIG. 2A);

- commercial buffer R up to 110 µl;

The mode recommended by Neon Thermo Fisher was used.

To compare the effectiveness of introducing knockin by the modified and modified methods, electroporation of CEM/R5 cells was performed. The mixture for electroporation of 1 million cells included the following components:

- 3 µg pcDNA3.3-hCas9 (Addgene #41815) - plasmid encoding wild-type Cas9;

- 1 μg vector pKS gRNA BB expressing gRNA, which included gRNA SEQ ID NO: 4;

- 1 μg donor template obtained by PCR amplification, and representing the sequence described in figure 2B;

- commercial buffer R up to 110 µl

The mode recommended by Neon Thermo Fisher was used.

On the third day after electroporation, staining was carried out as described above. It has been established that the modified method is much more efficient than the modified one. The results are obtained by conducting three independent experiments and are presented in figures 3A and B.

Example 2. Introduction of the target construct into human CD4 lymphocytes with subsequent evaluation of the efficiency of the construct

Primary PBMCs were isolated from donated blood. To do this, under sterile conditions, donor blood was mixed with PBS in a ratio of 1:1, after which it was layered on a ficoll solution (PanEco). The resulting two-phase mixture was centrifuged for 40 minutes at a centrifugal acceleration of 400 g with minimal acceleration and deceleration. As a result, the erythrocyte mass settled to the bottom of the tube, and the target cells formed a layer on the ficoll surface. PBMC were selected and washed with PBS. The resulting cell mass was incubated for 10 minutes in erythrocyte lysis buffer (Nh4Cl 155mM, KHCO3 10mM, EDTA 0.1 mM, pH=7.3) and washed in PBS.

Isolation of CD4 lymphocytes was performed using the Dynabeads Untouched Kit (Invitrogen, USA) following the manufacturer's instructions. After isolation, the cells were activated with CD2/CD3/CD28 magnetic beads (Miltenyi Biotec, Germany) according to the manufacturer's instructions and incubated with the addition of 100 U/ml of recombinant human interleukin-2 (Rontoleykin, Biotech, Russia). On day 3 after activation, cells were electroporated using a Neon Thermo Fisher instrument at 1,600 V, 10 ms x 3.

The mixture for electroporation of 2 million cells included the following components:

- 4 µg pX330-Cas9-BFP-gRNAX4ex2 - plasmid encoding wild-type Cas9 protein, BFP fluorescent protein and gRNA;

- 6 μg donor template in the context of the commercial pJet vector described above (Fig. 2)

- commercial buffer R up to 110 µl

Using the described method, a population of CD+ lymphocytes was obtained with a knockin of the target construct (CD4+MT-C34) with the MT-C34 peptide in the context of CD52. Sorting of cells with the target construct was carried out by immunofluorescent staining. Antibodies to the MT-C34 peptide obtained in our laboratory were used as primary antibodies; antibodies conjugated with the Alexa546 fluorescent label were used as secondary antibodies. Isolation of cells with the target construct was carried out by FACS - sorting.

To study the effectiveness of the genetic construct introduced into the cells, infectious tests for intercellular transmission were carried out with the HIV virus pseudotyped with the Env protein of the ZM135 strain.

To do this, Raji cells were electroporated using a Neon Thermo Fisher instrument using the manufacturer's recommended regimen. The electroporation mixture included plasmids pUCHR-inLuc-mR, pCMV-d8.2R and envelop ZM135. After 6 hours, the electroporated cells were mixed with test CD4+MT-C34 cells in a ratio of 3:1. Conducted infection tests showed that the construct effectively protects primary cells from HIV-1 penetration. The results are presented in figure 4.

The invention can potentially be used for HIV therapy.

Cited Literature

1. Tsunekawa Y, Terhune RK, Fujita I, Shitamukai A, Suetsugu T, Matsuzaki F. Developing a de novo targeted knock-in method based on in utero electroporation into the mammalian brain. Development. 2016;143(17):3216-3222. doi:10.1242/dev.136325

2. Kurihara T, Kouyama-Suzuki E, Satoga M, et al., DNA repair protein RAD51 enhances the CRISPR/Cas9-mediated knock-in efficiency in brain neurons. Biochem Biophys Res Commun. 2020;524(3):621-628. doi:https://doi.org/10.1016/j.bbrc.2020.01.132

3. Sung JJ, Park C-Y, Leem JW, Cho MS, Kim D-W. Restoration of FVIII expression by targeted gene insertion in the FVIII locus in hemophilia A patient-derived iPSCs. Exp Mol Med. 2019;51(4):1-9. doi:10.1038/s12276-019-0243-1

4. Liu J-T, Corbett JL, Heslop JA, Duncan SA. Enhanced genome editing in human iPSCs with CRISPR-CAS9 by co-targeting ATP1a1. PeerJ. 2020;8:e9060. doi:10.7717/peerj.9060

5. Yao X, Wang X, Hu X, et al. Homology-mediated end joining-based targeted integration using CRISPR/Cas9. Cell Res. 2017;27(6):801-814. doi:10.1038/cr.2017.76

6. Yao X, Wang X, Liu J, Shi L, Huang P, Yang H. CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy. J Vis Exp. 2018;(133):56844. doi:10.3791/56844

7. Nguyen DN, Roth TL, Li PJ, et al. Polymer-stabilized Cas9 nanoparticles and modified repair templates increase genome editing efficiency. Nat Biotechnol. 2020;38(1):44-49. doi:10.1038/s41587-019-0325-6

8. Torres-Ruiz R, Martinez-Lage M, Martin MC, et al., Efficient Recreation of t(11;22) EWSR1-FLI1(+) in Human Stem Cells Using CRISPR/Cas9. Stem cell reports. 2017;8(5):1408-1420. doi:10.1016/j.stemcr.2017.04.014

9. Maggio I, Zittersteijn HA, Wang Q, et al., Integrating gene delivery and gene-editing technologies by adenoviral vector transfer of optimized CRISPR-Cas9 components. Gene Ther. 2020;27(5):209-225. doi:10.1038/s41434-019-0119-y

10. Dean DA, Dean BS, Muller S, Smith LC. Sequence requirements for plasmid nuclear import. Exp Cell Res. 1999;253(2):713-722. doi:10.1006/excr.1999.4716

11. Mesika A, Grigoreva I, Zohar M, Reich Z. A Regulated, NFκB-Assisted Import of Plasmid DNA into Mammalian Cell Nuclei. Mol Ther. 2001;3(5):653-657. doi:https://doi.org/10.1006/mthe.2001.0312

12. Remy S, Chenouard V, Tesson L, et al., Generation of gene-edited rats by delivery of CRISPR/Cas9 protein and donor DNA into intact zygotes using electroporation. sci rep. 2017;7(1):16554. doi:10.1038/s41598-017-16328-y/.

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SEQUENCE LIST

<110> Federal State Budgetary Institution of Science Institute of Gene Biology

Russian Academy of Sciences (Institute of Gene Biology Russian Academy of Sciences)

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human in order to create cell resistance to HIV-1

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<---

Claims (12)

1. Donor DNA for delivery of the protective peptide from gp41 MT-C34 by the sequence of SEQ ID NO: 3 to the surface of the plasma membrane, while the donor DNA has the following sequence: • protospacer X4ex2 SEQ ID NO: 4, • 5'-arm homology SEQ ID NO: 5, • the P2A sequence, which makes it possible to obtain a separate translation of the peptide and protein CXCR4 SEQ ID NO: 6 from one RNA, • protein export signal SEQ ID NO: 1, • the sequence encoding the protective peptide from gp41 MT-C34 SEQ ID NO: 3, • signal for membrane anchoring SEQ ID NO: 2, • polyadenylation sequence SEQ ID NO: 7, • 3'-arm homology SEQ ID NO: 8, • transcription factor binding site DTS SEQ ID NO: 9. 2. A method for delivering a protective peptide from gp41 MT-C34 with the sequence SEQ ID NO: 3 to the surface of the plasma membrane of the target cell by introducing into the cell an effective amount of: 1) the first plasmid pX330-Cas9-BFP-gRNAX4ex2 encoding Cas9 nuclease and gRNA sequence SEQ ID NO: 10, in the sequence of which there are several NLS sites; and 2) a second plasmid encoding the donor DNA of claim 1. 3. A method for delivering a protective peptide from gp41 MT-C34 SEQ ID NO: 3 to the surface of the plasma membrane of a target cell by introducing into the cell an effective amount of: 1) a ribonucleoprotein complex comprising a recombinant Cas9 protein in complex with an gRNA sequence of SEQ ID NO: 10 ; and 2) a plasmid encoding the donor DNA of claim 1.

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