RU2769567C1 - Technology for creating t-cell resistance to hiv-1 based on genetically encoded gpi-anchored peptides from gp41 - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology, in particular, to methods for editing the CXCR4 cell gene in order to prevent infection of human CD4 lymphoblasts with HIV-1 using the CRISPR/Cas editing system. The methods involve electroporation of CD4 lymphoblast cells with an electroporation mixture consisting of an effective amount of guide RNA, Cas9 endonuclease, and donor DNA, as well as knockin or directed insertion of a nucleotide sequence encoding a protective peptide of the gp41 group under the endogenous promoter of the CXCR4 gene.

EFFECT: invention is effective in treatment of HIV-1.

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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

Selection of gp41-dependent fusion as a target for antiviral therapy.

In the treatment of human immunodeficiency virus (HIV), peptide fusion inhibitors are widely used. They prevent the last stage of HIV penetration into the target cell by blocking the formation of a six-helix bundle, which is involved in the fusion of the viral and cell membranes.

Enfuvirtide (Lalezari et al., 2003), a short peptide from the C-terminal heptad repeat of gp41 (C-peptide), was the first approved drug in this group. This drug has a number of disadvantages, including the emergence of resistant strains of HIV and the development of side effects in patients.

After his approval, this area of science began to develop actively, a large number of studies appeared. However, none of the published soluble peptides (US2005089840A1, WO2016171980A1) can completely protect cells from infection for life.

As a result, systems for delivering the peptide to the membrane of the target cell began to appear.

A method for exposing a peptide fusion inhibitor on a membrane in the context of the HIV chemokine co-receptor CXCR4 is described (Lesli et al., 2016), in which the fusion of the peptide with the receptor occurs through a linkage of two amino acids, leucine and lysine, and a lentiviral vector is used as the inhibitor delivery method. Clinical trials are underway.

A method for delivering the 2P23 peptide to the membrane surface in the context of a GPI-anchored protein is also described (Xiaoran et al., 2019), however, the described construct is larger, and only a lentiviral vector is proposed as a delivery method. Anchoring the 2P23 fusion inhibitor to the cell membrane can completely block infection by HIV-1 and enfuvirtide-resistant mutants. Membrane-bound 2P23 effectively blocks HIV infection and renders CD4+ T cells resistant to virus infection.

In 2019, our laboratory developed a system for delivering epitope tags to the membrane, which allows for the rapid isolation of cells with CRISPR/Cas9-induced targeted genetic modification by immunofluorescence and cell sorting. It was found that the shortest and most efficient construct is the GPI-anchored CD52 protein, where the Flag or HA epitope tag sequence was inserted between the export signal and the GPI-anchoring signal (Zotova et al., 2019). We modified the exposed sequence of the epitope tag in the delivery system by replacing it with the sequence of one of the C-peptides inhibiting the fusion of HIV with the cell membrane, and thus made the system not only capable of selecting edited cells, but also ensured the resistance of the modified cells to HIV infection. various tropes.

To achieve the highest efficiency, we have developed a technology that makes it possible to expose two peptides on the membrane simultaneously and to select cells by two protective peptides.

The developed technology makes it possible to obtain cell populations immune to HIV infection by introducing minimal off-target changes in the native cell genome through the use of the ribonucleoprotein (RNP) complex of Cas9 nuclease with guide (guide) RNA. Due to the small size of the embedded genetic construct, the proposed technology has a number of advantages. First, the process of producing donor DNA is economical. Second, delivery of the construct to cells and its integration by the mechanism of homologous recombination is more efficient. The compatibility of the technology with different genome editing systems based on CRISPR/Cas9 and others makes it easy to adapt it to the latest developments in this area.

Disclosure of the essence of the invention

The present invention is a technology for HIV gene therapy, in which a protective peptide from gp41 is delivered to the surface of the plasma membrane of a target cell by introducing a nucleotide sequence encoding it into the genome using CRISPR/Cas9 as part of a genetic construct, which is a CD52 protein, between the two parts of which (protein export signal and GPI anchoring signal) the target peptide is inserted, either by fusion of the target peptide to the N-terminus of CXCR4.

Alternatively, delivery of a genetic construct representing the CD52 protein, between the two parts of which (the protein export signal and the anchoring signal) the target peptide is inserted, is carried out using a lentiviral vector.

To implement the invention, three components are introduced into cells: Cas9 nuclease, guide RNA (gRNA) and donor DNA. Guide RNA directs Cas9 to a specific location in the genome. The donor DNA contains the target genetic construct and homology arms surrounding the insertion site and serves as a template for homologous recombination. In one of the embodiments of the invention, targeted editing can be performed by transfection with Cas9 and gRNA coding plasmids (for example, pcDNA3.3-hCas9 (Addgene #41815)), in other embodiments of the invention - using the RNP complex, including the recombinant Cas9 protein in complex with gRNA obtained by in vitro transcription.

In one embodiment of the invention, the target genetic construct encoded in the donor DNA is based on the sequence of the GPI-anchored CD52 protein. SEQ ID No. 1 or 2 is a protein export signal. SEQ ID No. 3 serves as a signal for membrane anchoring of the construct. Between SEQ ID No. 1/2 and No. 3 is a sequence encoding one of the protective peptides from gp41 (SEQ ID No. 4-9). 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 penetrate the cell. 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 entry (Fig. 1A).

In another embodiment of the invention, a peptide from gp41 fused to the N-terminus of the CXCR4 co-receptor acts in the same way to prevent the formation of a six-stranded bundle (Fig. 1B).

C34, MT-C34, HP23L, 2p23, nHP23L, and n2p23 were the most effective as protective peptides in constructs (SEQ ID nos. 4-9, respectively).

The present invention also provides methods for the efficient delivery of targeted genetic constructs to the cell membrane:

- insertion into the genome by means of CRISPR/Cas9 under an endogenous promoter, for example, the CXCR4 gene, for which gRNA (SEQ ID No. 10) is selected, the donor DNA includes 5' and 3' homology arms of SEQ ID No. 11 and 12, respectively, between which the target CD52-peptide sequence is enclosed, followed by the P2A sequence (SEQ ID No. 13), which allows the ribosome to skip the formation of a covalent bond between glycine and proline, in other words, allows one to obtain separate translation of the peptide and CXCR4 protein from one RNA (Fig. 2A);

- fusion of the peptide to the N-terminus of the chemokine receptor CXCR4 (Fig. 1B) using the CRISPR/Cas9 genomic editing system. In this case, guide RNA (SEQ ID No. 10) is used to target Cas9, donor DNA in this case includes the 5' and 3' homology arms of SEQ ID No. 11 and 12, respectively, between which one of the target peptides is located (SEQ ID No. 5 , 6-9, Fig. 2B). In this case, immediately after translation of the peptide, translation of CXCR4 begins, which leads to the fusion of the peptide with its N-terminus.

Since it is possible to make modifications to two alleles of the gene, it is possible to simultaneously express two peptides from gp41 on the cell surface, including combining peptides fused with the coreceptor and anchored on the membrane, two fused with the coreceptor, or two anchored peptides in the cell. To do this, when constructs are introduced into a cell during electroporation, two donor DNAs are used simultaneously, shown in Fig. 2, in a ratio of 1:1.

As co-receptor fusion peptides, MT-C34, HP23L, 2p23, nHP23L, and n2p23 can be used, but are not limited to (SEQ ID Nos. 5, 6-9, respectively).

As a membrane anchor, MT-C34 (SEQ ID No. 5) can be used, but is not limited to.

The combination of the anchored MT-C34 peptide and 2p23 or HP23L fused to CXCR4 showed high efficiency.

For an alternative delivery of the genetic construct, which is the CD52 protein, between the two parts of which the target peptide C34, MT-C34, HP23L, 2p23, nHP23L and n2p23 is inserted (SEQ ID no. 4-9, respectively), a lentiviral vector is used (Fig. 3A ). To facilitate sorting of cells carrying the target construct, the sequence of the fluorescent protein mClover, separated from the target construct by the P2A sequence, was introduced into the vector. This system allows translation of mClover and a protection construct from a single messenger RNA. Due to this, the transduced cells fluoresce and can be sorted.

Brief description of the drawings

Figure 1. Scheme of the location of protective peptides on the surface of the cell membrane: A - in the context of the GPI-anchored CD52 protein, B - fused to the N-terminus of the CXCR4 protein.

Figure 2. Schemes of sequences used as donor DNA to introduce target mutations: A - a protective peptide from gp41 in the context of the GPI-anchored CD52 protein is inserted before CXCR4 through P2A, B - the peptide is fused to the N-terminus of CXCR4.

Figure 3. Scheme of a lentiviral vector used to introduce a protective peptide from gp41 in the context of the GPI-anchored CD52 protein with the ability to sort cells carrying a target mutation in the green fluorescent protein mClover (A), as well as a vector that is used as a template for PCR- donor DNA amplification (B).

Figure 4. Examples of immunofluorescent staining of cell lines carrying a peptide that inhibits HIV entry: A - cell lines obtained by lentiviral transduction based on HEK293T/CD4/R5 with peptides C34 and MT-C34, HP23L B - cell lines obtained by lentiviral transduction based on Raji/CD4/R5 with peptides C34 and MT-C34. B - cell lines derived from CEM/R5 by CRISPR/Cas9-mediated biallelic knockin of peptides 2P23 fused to CXCR4 and MT-C34 expressed in lipid rafts separately from CXCR4.

Figure 5. Graphical representation of the results of infection tests: A - Infection with free viral particles on HEK293T/CD4/R5/peptide cells, B - Intercellular infection test from Raji to Raji/CD4/R5/peptide, C - Intercellular infection test from Raji to CEM/R5/peptide.

Implementation of the invention

Example 1 Generation of Cell Lines Carrying Target Constructs with CRISPR/Cas9 and Efficacy Study

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

The electroporation mixture included the following components:

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

- pKS gRNA BB vector expressing guide RNA, which includes gRNA SEQ ID No. 11;

- donor template obtained by PCR amplification, and representing the sequence described above (Fig. 2).

Obtaining a PCR donor:

PCR donors were obtained by the method described earlier by D.V. Mazurov. (Mazurov, 2020). Lentiviral vectors (Fig. 3B) carrying the target peptide serve as templates for PCR. As primers, sequences of SEQ ID nos. 14-23 containing homology arms were used (Table 1).

Table 1

Design Plasmid containing the target sequence Primers: Forward/Reverse MT34-CD52 5'-CXCR4start-in: SEQ ID NO: 14
3'-CXCR4start-in: SEQ ID NO: 15 pUCHR-CD-MT34-52-mClover 2p23-CD52 5'-CXCR4start-in: SEQ ID NO: 14
3'-CXCR4start-in: SEQ ID NO: 15 pUCHR-CD-2p23-52-mClover HP23L-CD52 5'-CXCR4start-in: SEQ ID NO: 14
3'-CXCR4start-in: SEQ ID NO: 15 pUCHR-CD-HP23L-52-mClover MT34-fusion 5'-MT34-X4-in: SEQ ID No. 16
3'-MT34-X4-in: SEQ ID No. 17 pUCHR-CD-MT34-52-mClover 2p23-fusion 5'-2P23-X4-in: SEQ ID No. 18
3'-2P23-X4-in: SEQ ID No. 19 pUCHR-CD-2p23-52-mClover HP23L-fusion 5'-HP23L-X4-in: SEQ ID No. 20
3'-HP23L-X4-in: SEQ ID No. 21 pUCHR-CD-HP23L-52-mClover n2p23-fusion 5'-n2P23-X4-in: SEQ ID NO:22
3'-n2P23-X4-in:
use 3'-2P23-X4-in (SEQ ID No. 19) pUCHR-CD-n2p23-52-mClover nHP23L-fusion 5'-nHP23L-X4-in: SEQ ID NO:23
3'-nHP23L-X4-in:
use 3'-HP23L-X4-in (SEQ ID No. 21) pUCHR-CD-nHP23L-52-mClover

Amplification was carried out in the following mode:

Using the described method, cell lines were obtained with a peptide in the context of CD52: CEM/R5/MT34-CD52 fused with CXCR4: CEM/R5/MT34-fusion, CEM/R5/2p23-fusion, CEM/R5/HP23L-fusion, CEM/ R5/n2p23-fusion, CEM/R5/nHP23L-fusion and biallelic knockin cell lines: CEM/R5/MT34-CD52/2p23-fusion, CEM/R5/MT34-fusion/2p23-fusion, CEM/R5/MT34- CD52/HP23L-fusion, CEM/R5/MT34-fusion/HP23L-fusion (FIG. 4B). Sorting of cells with the target construct was carried out by immunofluorescent staining. Antibodies to the corresponding peptides obtained in our laboratory were used as primary antibodies; commercial antibodies conjugated with a 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/MT34-CD52/2p23-fusion-RNP cell line was obtained by electroporation of CEM/R5 cells. The electroporation mixture included:

- RNP-gRNA protein

- PCR donor including the MT34 peptide corresponding to the scheme of FIG. 2A

- PCR donor including the 2p23 peptide according to the scheme of FIG. 2B

The mode recommended by Neon Thermo Fisher was used.

To study the effectiveness of the genetic constructs introduced into the cell, infectious tests for intercellular transmission with HIV viruses with different tropisms were carried out.

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 one of the envelops: NL4-3, JRFL or ZM135. After 6 hours, the electroporated cells were mixed with the tested CEM/R5/peptide cells in a ratio of 2:1. All results were obtained in three independent experiments. Conducted infection tests showed that the technology effectively protects cells from HIV penetration (Fig. 5B).

Due to the compatibility of the technology with different systems based on CRISPR/Cas9, the technology is easily adaptable in accordance with the latest developments in this area.

Example 2 Introducing Target Construct into a Cell Using a Lentiviral Vector and Efficiency Study

To implement an alternative option for introducing a construct into a cell, based on the HEK293T and Raji cell lines, lines were obtained that are susceptible to HIV infection of different tropisms - HEK293T/CD4/R5 and Raji/CD4/R5, expressing CD4, CCR5 and CXCR4, where CD4 and CCR5 were exogenous and stably integrated with a lentiviral vector. Then, on their basis, using a lentiviral vector, cell lines carrying one of the GPI-anchored peptides on the membrane were obtained: HEK293T/CD4/R5/С34, HEK293T/CD4/R5/MT-C34, HEK293T/CD4/R5/MT- C34-R, HEK293T/CD4/R5/HP23L, HEK293T/CD4/R5/2p23 (Fig. 4A); Raji/CD4/R5/C34, Raji/CD4/R5/MT-C34, Raji/CD4/R5/HP23L, Raji/CD4/R5/2p23, Raji/CD4/R5nHP23L, and Raji/CD4/R5n2p23 (Fig. 4B) .

To do this, lentiviral pseudoparticles were obtained by lipofective transfection of HEK293T with three plasmids: pUCHR-mClover-P2A-CDpeptide52 containing the sequence of the target peptide in the context of CD52 (Fig. 3A), pCMVΔ8.2R - HIV packaging plasmid (Addgene # 12263) and pCMV- VSV-G (Addgene # 8454) expressing the G coat protein of vesicular stomatitis virus. After transfection, the cells were incubated for 48 hours, after which the supernatant was collected and added to the HEK293T/CD4/R5 and Raji/CD4/R5 cell lines at a dose required to obtain no more than 30% of cells with the target construct. Cells with the target construct were sorted by mClover until a culture purity of at least 98% was achieved.

To assess the degree of protection of cells from HIV, the obtained cells were analyzed in a single-cycle pseudovirus test. Infection tests were performed on HEK293T/CD4/R5/peptide cells with isolated viral particles. For this, pseudoviral particles coated with one of three HIV-1 envelope proteins were produced: CXCR4 - tropic NL4-3 and CCR5 - tropic JRFL and ZM-135. HEK293T cells were transfected using the Lipofectamine 2000 reagent (ThermoFisher) according to the manufacturer's instructions with three plasmids: pUCHR-inLuc-mR, carrying the luciferase gene and necessary for detecting virus entry into the cell; packaging pCMV-d8.2R, and one of the envelope proteins NL4-3, JRFL, or ZM135. After 48 hours, the viral supernatant was collected and filtered. An infection test was performed by adding the viral supernatant to the tested HEK293T/CD4/R5/peptide cell line. The HEK293T/CD4/R5 cell line was used as a control. After 48 hours, the cells were harvested, lysed and the luciferase activity was measured. The results are shown in Figure 5A. The test showed that most of the GPI-anchored gp41 peptides effectively protect the cell from HIV-1 entry.

For the HIV-1 intercellular transmission test, Raji cells were electroporated with pUCHR-inLuc-mR, pCMV-d8.2R plasmids and one of the Env envelope proteins from HIV-1 strains NL4-3, JRFL, or ZM135. Transfected cells were incubated for 6 hours and mixed with Raji/CD4/R5/peptide target cells at a ratio of 2:1 (Fig. 5B). After 72 hours of co-cultivation, the level of infection was measured by luciferase activity on lysed cells. The test results showed that the invented constructs effectively protect cells from HIV penetration not only when infected with free viral particles, but also block intercellular HIV-1 infection transmitted by direct contact of the virus producer cell with the target cell. All results shown were obtained in at least three independent experiments, which confirms the described effects and their statistical significance.

The invention can potentially be used on human CD4+ cells for HIV therapy.

Cited Literature

1. Mazurov D.V. Selection of edited cells by the SORTS method. Chapter 22 in the monograph "Methods for editing genes and genomes", Novosibirsk, Publishing House of the Siberian Branch of the Russian Academy of Sciences, 2020.

2. Lalezari J.P., DeJesus E., Northfelt D.W. et al. A controlled phase II trial assessing three doses of enfuvirtide (T-20) in combination with abacavir, amprenavir, ritonavir and efavirenz in nonnucleoside reverse transcriptase inhibitor-naive HIV-infected adults. antivirus. Ther. 2003; 8:279-287.

3. Leslie GJ, Wang J, Richardson MW, Haggarty BS, Hua KL, Duong J, Secreto AJ, Jordon AP, Romano J, Kumar KE, DeClercq JJ, Gregory PD, June CH, Root MJ, Riley JL, Holmes MC, Hoxie J.A. Potent and Broad Inhibition of HIV-1 by a Peptide from the gp41 Heptad Repeat-2 Domain Conjugated to the CXCR4 Amino Terminus. PLoS Pathog. 2016 Nov 17;12(11): e1005983. doi:10.1371/journal.ppat.1005983

4. Xiaoran Tang, Hongliang Jin, Yue Chen, Li Li, Yuanmei Zhu, Huihui Chong, Yuxian He. A Membrane-Anchored Short-Peptide Fusion Inhibitor Fully Protects Target Cells from Infections of Human Immunodeficiency Virus Type 1 (HIV-1), HIV-2, and Simian Immunodeficiency Virus, Journal of Virology Oct 2019, 93 (22) e01177-19; DOI: 10.1128/JVI.01177-19

5. Zotova, A., Pichugin, A., Atemasova, A. et al. Isolation of gene-edited cells via knock-in of short glycophosphatidylinositol-anchored epitope tags. Sci Rep 9, 3132 (2019). https://doi.org/10.1038/s41598-019-40219-z

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

gene of the Russian Academy of Sciences (Institute of Gene Biology Russian Academy of

Sciences)

<120> Technology for creating T-cell resistance to HIV-1 based on

genetically encoded GPI-anchored peptides from gp41

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Claims (22)

1. A method for editing a cellular CXCR4 gene to prevent infection of human CD4 lymphoblasts with HIV-1 using the CRISPR/Cas editing system, including the following steps: a) electroporation of lymphoblast CD4 cells with an electroporation mixture consisting of an effective amount of guide RNA with the sequence SEQ ID 10, Cas9 endonuclease and donor DNA carrying a nucleotide sequence encoding a protective peptide of the gp41 group and inserted between the leader sequence of the CD52 protein and the GPI-anchoring signal of the CD52 protein ; while the donor DNA has the following formula: - 5' homology arm with SEQ ID 11; - CD52 leader sequence by SEQ ID 1 or SEQ ID 2; - protective peptide group gp41 MT-C34 sequence SEQ ID 5; - signal GPI-anchoring sequence CD52 SEQ ID 3; - the P2A sequence, which allows one to obtain translation of two protein products from one RNA, the sequence of SEQ ID 13; - homology arm 3' with SEQ ID 12; b) knockin or targeted insertion of a nucleotide sequence encoding a protective peptide of the gp41 group under the endogenous promoter of the CXCR4 gene; c) exposure of the protective peptide of the gp41 group on the cell surface in lipid rafts. 2. A method for editing a cellular CXCR4 gene to prevent infection of human CD4 lymphoblasts with HIV-1 using the CRISPR/Cas editing system, including the following steps: a) electroporation of lymphoblast CD4 cells with an electroporation mixture consisting of an effective amount of guide RNA with the sequence SEQ ID 10, Cas9 endonuclease and donor DNA carrying a nucleotide sequence encoding a protective peptide of the gp41 group, while the donor DNA has the formula: arm homology 5' sequence SEQ ID 11; a protective peptide of the gp41 group selected from C34, MT-C34, HP23L, 2p23, nHP23L, n2p23 with SEQ ID nos. 4, 5, 6, 7, 8 and 9, respectively; arm homology 3' sequence SEQ ID 12; b) knockin or targeted insertion of a nucleotide sequence encoding a protective peptide of the gp41 group under the endogenous promoter of the CXCR4 gene; c) fusion of the protective peptide of the gp41 group with the N-terminus of endogenous CXCR4. 3. A method for editing the cellular CXCR4 gene in order to prevent infection of human CD4 lymphoblasts with HIV-1 using the CRISPR/Cas editing system, including editing the cellular CXCR4 gene by the method according to claim 1 or 2, characterized in that two donor DNAs are used simultaneously in electroporation in a 1:1 ratio, resulting in a combination of the anchored peptide and the CXCR4-fused peptide.

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