WO2023286088A1 - Methods and compositions for viral vector transduction - Google Patents
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Abstract
The present invention discloses methods and compositions for5enhancing the transduction efficiency of a viral vector. Themethod comprises processing the target cell for inactivatingepidermal growth factor receptor-1, producing one or morelentiviral vectors from a mammalian cell to obtain lentiviralparticles, and introducing the lentiviral particles into the10incubated target cell. The inactivation of the epidermal growthfactor receptor-1 in the target cell is achieved by eitherincubating the target cells with an EGFR1 inhibitor or subjectingthe target cells for knockout of a gene expressing EGFR1, thusenhancing the lentiviral transduction. The EGFR1 inhibitor is15selected from a group comprising 1-tert-Butyl-3-[6-(2,6-dichlorophenyl)-2-[[4-(diethylamino)butyl]amino]pyrido[2,3-dpyrimidin-7-yl]urea, 6-[4-[(4-Ethylpiperazin-1-yl)methyl]phenyl]-N-[(1R)-1-phenylethyl]-7H-pyrrolo[2,3-d]pyrimidin-4-amine, and2-Bromo-4-[(6,7-dimethoxyquinazolin-4-yl)amino]phenol. Thus,20the invention provides compositions and methods that are lesscytotoxic, exhibit less cell-cycle inhibitory activity upon longerincubation of the inhibitory compound in the target cells.
Description
METHODS AND COMPOSITIONS FOR VIRAL VECTOR
TRANSDUCTION
FIELD OF INVENTION
The invention generally relates to the field of molecular biology and particularly to gene delivery vehicles. The invention discloses methods and compositions for improving the transduction efficiency of viral vectors.
BACKGROUND
Viral vectors are the most sought-after tools for delivering genetic material into cells. The commonly used vectors include retrovirus, lentivirus, adenovirus, adeno-associated virus, and herpes simplex virus. A variety of viral vectors are known for their application in gene therapy and the development of vaccines. Therefore, to enhance the viral transduction in the target cells several methods and compositions are evaluated.
The existing methods and compositions target particular host signaling pathways, affecting cell growth, cell-cycle progression and have cytotoxic effect. For example, the mechanistic Target of Rapamycin (mTOR) pathway has a crucial role in cell-cycle progression and cell growth. The inhibition of these pathways using their respective inhibitors such as rapamycin and other mTOR inhibitors is not tolerable by host cells for prolonged time and suppresses cell-cycle progression and cell division. Therefore, targeting essential genes or pathways may be a limiting factor in the existing technology. Therefore,
necessitating methods and compositions that are less cytotoxic, and the cell cycle progression and cell division being unaffected in the target cells.
BRIEF DESCRIPTION OF DRAWINGS FIG. 1 shows a graphical representation of the comparison of control sample and the effect of the different EGFR1 inhibitors on the target cells during transduction, according to an embodiment of the invention.
FIG. 2 shows an image of control and PD161570 treated A549 cells that are transduced and expressing the fluorescent protein, according to an embodiment of the invention.
FIG. 3 shows a graphical representation of the comparison of control (a) transduced population either untreated (b), or PD161570-treated (c) THP-1 cells, E6.1 T-cells and U937 cells expressing the fluorescent protein after lentiviral transduction, according to an embodiment of the invention.
FIG 4 shows the graphical representation of the comparison of control (a) and the transduced population either untreated (b), or PD161570-treated (c) CD4+ cells and CD14+ cells expressing the fluorescent protein after lentiviral transduction, according to an embodiment of the invention.
FIG. 5 shows a graphical representation of the effect of the different concentrations of PD161570 on the target cells, according to an embodiment of the invention.
FIG 6 shows an image of transduced, control TZM-GFP cells and target TZM-GFP cells treated with PD161570 expressing fluorescent protein, according to an embodiment of the invention.
FIG 7 shows an image of transduced wild-type and EGFR1 knockout TZM-GFP cells expressing fluorescent protein, according to an embodiment of the invention.
FIG 8 shows a graphical representation providing a comparison between the TZM-GFP wild-type cell and TZM-GFP EGFR1 knockout cell, according to an embodiment of the invention.
FIG. 9 shows a graphical representation providing a comparison of the EGFR1 inhibitor and rapamycin to assess their effects on lentiviral vector transduction, according to an embodiment of the invention.
SUMMARY OF THE INVENTION
The present invention overcomes the drawback of the prior arts by providing methods and compositions for viral transduction without affecting the cell cycle progression and cell division.
An aspect of the invention provides methods for enhancing the transduction efficiency of a viral vector. The method comprises, processing the target cell with at least one inhibitor compound for epidermal growth factor receptor-1 (EGFR1), producing one or more lentiviral vectors from a mammalian cell to obtain lentiviral particles, and introducing the lentiviral particles into the incubated target cell. The inhibition of the epidermal growth
factor receptor-1 in the target cell enhances the lentiviral transduction.
Another aspect of the invention provides composition of the EGFR1 inhibitors. The EGFR1 inhibitor is selected from a group comprising 1-tert-Butyl-3-[6-(2,6-dichlorophenyl)-2-[[4-
(diethylamino)butyl]amino]pyrido[2,3-d]pyrimidin-7-yl]urea also referred as PD161570, 6-[4-[(4-Ethylpiperazin-1- yl)methyl]phenyl]-N-[(1 R)-1 -phenylethyl]-7H-pyrrolo[2,3- d]pyrimidin-4-amine also referred as AEE 788, or 2-Bromo-4- [(6,7-dimethoxyquinazolin-4-yl)amino]phenol also referred as
WFII-P154 or a derivative thereof.
Another aspect of the invention provides a method for transduction by inactivating the EGFR1 gene. The method comprises processing the target cell for inactivating an epidermal growth factor receptor-1 (EGFR1) by subjecting the target cell for knockout of the EGFR1 from the target cell using a CRISPR/Cas9 system, producing one or more lentiviral vectors from a mammalian cell to obtain lentiviral particles, and introducing the lentiviral particles into the processed target cell. The inactivation of epidermal growth factor receptor-1 in the target cell enhances the lentiviral transduction.
DETAILED DESCRIPTION OF THE INVENTION
The definitions, terms, and terminology adopted in the disclosure have their usual meaning and interpretations, unless otherwise specified. Various embodiments of the invention
provide methods and compositions for improving viral transduction.
The invention provides methods and compositions for enhancing the transduction efficiency of a viral vector. The method comprises transducing a target cell with one or more vectors in the presence of compounds that inhibit the epidermal growth factor receptor-1 (EGFR1) signaling pathway. The method employs the use of an inhibitor compound that targets EGFR1. The viral vector disclosed herein is a retroviral vector such as a lentiviral vector and includes the HIV-1 based vectors.
According to an embodiment of the invention, the invention discloses a method for lentiviral transduction in the target cell. The method comprises processing the target cell for inactivating an epidermal growth factor receptor-1 by incubating the target cells with an EGFR1 inhibitor, producing one or more lentiviral vectors from a mammalian cell to obtain lentiviral particles, and introducing the lentiviral particles into the incubated target cell. The inhibition of the epidermal growth factor receptor-1 in the target cell enhances the lentiviral transduction. The production of lentiviral particles requires a plurality of lentiviral vectors. At least one transfer vector, at least one packaging vector, and at least one envelope vector are essential for vector production and transduction.
The envelop vector is selected from a group comprising PMD2.G, pCAG-VSVG, and pFIEF-VSVG vectors. The transfer vector is selected from a group comprising PScalps, pLKO.1
puro, pLK0.1-TRC series plasmids, Tet-pLKO-puro, pWPI, pLenti CRISPR-E, pLenti CRISPR-V1/V2, EF.CMV.RFP, pLenti CMV Puro DEST, or lentiviral vector for carrying a gene/ shRNA/ CRISPR, encoding a fluorescent protein, reporter protein, therapeutic gene, or any transgene of interest. In one example the transfer vector PScalps is used for carrying a ZsGreen (GFP) for expression of a fluorescent protein. In another example the transfer vector PScalps is used to carry luciferase as transgene. The packaging vector is selected from a group comprising psPAX2, pCMV delta R8.2, pMDLg/pRRE, psPAX2- D64V, and psPAX2-D116N.
The invention provides an effective means to enhance the transduction of retroviral vectors in various human cell lines. The methods enhance the transduction efficiency of recombinant retroviral vectors in the EGFR1 positive target cells. The target cells include primary cells comprising CD4+ cell and CD14+ cell, a primary T cell and a primary monocyte respectively, a monocyte, epithelial cell comprising HeLa cell or TZM-GFP cell, A549 cell, THP-1 monocyte, Jurkat E6.1 T-cell, THP-1 derived macrophage, as well as U937 and SFISY5Y cells. The primary cells suitable for the invention are isolated from the biological sample such as the peripheral blood of healthy donors.
The invention further discloses a composition of the EGFR1 inhibitors. The EGFR1 inhibitor is selected from group comprising 1 -tert-Butyl-3-[6-(2,6-dichlorophenyl)-2-[[4-
(diethylamino)butyl]amino]pyrido[2,3-d]pyrimidin-7-yl]urea also
referred as PD 161570, 6-[4-[(4-Ethylpiperazin-1- yl)methyl]phenyl]-N-[(1 R)-1 -phenylethyl]-7H-pyrrolo[2,3- d]pyrimidin-4-amine also referred as AEE 788, and 2-Bromo-4- [(6,7-dimethoxyquinazolin-4-yl)amino]phenol also referred as WHI-P154 or a derivative thereof.
FIG. 1 shows a graphical representation of the comparison of control and the effect of the different EGFR1 inhibitors on the target cells during transduction, according to an embodiment of the invention. The target cells are treated with different EGFR1 inhibitor such as WHI-P154, AEE 788 and PD161570 and the effect of the inhibitor is compared to the control sample. The cells treated with the EGFR1 inhibitor exhibited significant increase in transduction of viral vector compared to the control.
In one example, the target cell for viral transduction is a primary CD4+ cell. The method comprising the steps of incubating the primary CD4+ cell with the EGFR1 inhibitor such as the PD161570 for a duration of 24 hours, producing lentiviral particle from a 10 cm2 cell culture plate by incubating a transfer vector such as pScalps containing a transgene ZsGreen at a concentration of 8 pg, an envelope vector such as pMD2.G at a concentration of 2 pg, packaging vector such as psPAX2 at a concentration of 6 pg introduced into the mammalian cell such as HEK293T cell cultured in Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with Fetal Bovine Serum (FBS) at a concentration in the range between 2% to 10% for producing the lentiviral particle. In some embodiments of the
invention, the lentiviral particles are produced from a 35 mm2 plate by incubating the transfer vector pScalps ZsGreen at a concentration of 4 pg, the envelope vector pMD2.G at a concentration of 0.5 pg, the packaging vector such as psPAX2 at a concentration of 3 pg. The cells are incubated in a 5%-10% C02 incubator at a temperature of about 37°C for a duration between 48h and 72h. The lentiviral particles are collected and filtered through a 0.22pm syringe filter. Finally introducing the lentiviral particles into the EGFR1 incubated primary CD4+ cell, the cells are incubated in a 5%-10% C02 incubator at a temperature of about 37°C for a duration of 48h to 72h. A control sample devoid of the EGFR1 inhibitor is parallely transduced. The cells incubated with EGFR1 inhibitor have a higher yield of the green cells depicting the increase in the lentiviral transduction.
FIG. 2 shows an image of control and PD161570 treated A549 cells that are transduced and expressing the fluorescent protein, according to an embodiment of the invention. The A549 cells treated with PD161570 expressed fluorescent protein thus emphasizing the increase in transduction due to the inhibition of EGFR1 .
In some embodiments of the invention, the mammalian cell may optionally comprise HEK293FT or HEK293 cells for producing lentiviral particle. According to an embodiment of the invention, the method optionally includes pre-stimulating the target cells such as the
primary cells with a recombinant cytokine, an antibody, antigen lectins, or an antibody-coated magnetic bead before initiating transduction for expansion in cell culture. In one example the CD4+ cell is pre-stimulated with cytokines to enable cell division. The target cells are pre-stimulated from a group comprising cytokines such as lnterleukin-2 in the range between 30U/ml- 100U/ml, antibodies such as anti cluster of differentiation 3 (CD3) or cluster of differentiation 28 (CD28), antigenic lectins such as phytohemagglutinin at a concentration of about 5pg/ml, polygonatum multiflorum agglutinin at a concentration in the range between 10ng/ml-30ng/ml, and antibody-coated magnetic bead such as anti-CD3 or anti-CD28 coated magnetic beads in a 1 :1 ratio with cells or a combination thereof.
In one example the target cells such as THP-1 cells, E6.1 T- cells and U937 cells are transduced with ZsGreen along with the EGFR1 inhibitor PD161570, and control sample devoid of EGFR1 inhibitor for establishing a significant increase in the transduction of the viral vector. FIG. 3 shows a graphical representation of the comparison of control (a) transduced population either untreated (b), or PD161570-treated (c) THP-1 cells, E6.1 T-cells and U937 cells expressing the fluorescent protein after lentiviral transduction, according to an embodiment of the invention. The panel (a) exhibits the results of control sample devoid of pre-stimulation process and devoid of treatment with the EGFR1 inhibitor in THP-1 cells, E6.1 T-cells and U937 cells. The panel (b) exhibits the transduction results of
untreated THP-1 cells, E6.1 T-cells and U937 cells devoid of pre-stimulation process, the panel (c) exhibits the transduction results of the pre-stimulated THP-1 cells, E6.1 T-cells and U937 cells transduced with the EGFR1 inhibitor PD161570.
FIG 4 shows the graphical representation of the comparison of control (a) and the transduced population either untreated (b), or PD161570-treated (c) CD4+ cells and CD14+ cells expressing the fluorescent protein after lentiviral transduction, according to an embodiment of the invention. The panel (a) exhibits the result of control sample devoid of pre-stimulation process and devoid of treatment with the EGFR1 inhibitor in both CD4+ and CD14+ cells. The panel (b) exhibits the result of untreated CD4+ and CD14+ cells devoid of pre-stimulation process transduced with PD161570, the panel (c) exhibits the result of the pre-stimulated CD4+ and CD14+ cells transduced in the presence of PD161570. The increase in the transduction efficiency is observed in the both pre-stimulated CD4+ and CD14+ cells.
FIG. 5 shows a graphical representation of the effect of the different concentrations of PD161570 on the target cells, according to an embodiment of the invention. The effect of the PD161570 in the target cell is analyzed at a concentration in the range of 0.1 mM, 0.2 mM, 1 mM, 5 mM, 10 mM along with a control sample is assessed using Alamar-Blue cell viability fluorescence-based assay. The results depict that the treatment of the target cells with EGFR1 inhibitor does not reduce proliferation at the given working range of concentrations.
EGFR1 inhibitor also showed no effect on cell viability of adherent as well as suspension cells in the working range as shown in FIG 5. Since the EGFR1 inhibitor exhibits no cytotoxicity in the working range of up to 10mM, therefore reflects that the inhibitor compound can be present during the entire transduction process without causing any adverse effect on the target cells or the EGFR1 inhibitor can be added at a specific interval.
In one aspect of the invention, the method includes the viral vector transduction into the primary cells at the multiplicity of infection in the range between 0.1 to10.
According to another embodiment of the invention, the method comprises processing the target cell for inactivating an epidermal growth factor receptor-1 (EGFR1) by subjecting the target cell for knockout of the EGFR1 using the CRISPR/Cas9 system for removing a gene expressing EGFR1 , producing one or more lentiviral vectors from a mammalian cell to obtain lentiviral particles, and introducing the lentiviral particles into the processed target cell. The inactivation of the EGFR1 in the target cell enhances the lentiviral transduction.
The knockout of EGFR1 in the target cells does result in any effect on the cell proliferation of the target cells upon transduction and grows normally as the wild cell type. Thus, proving that knockout of EGFR1 or inhibition of EGFR1 does not affect the cell proliferation or cell cycle in the knockout cells or in the target cells incubated with EGFR1 inhibitor respectively. FIG
6 shows an image of transduced, control TZM-GFP cells and target TZM-GFP cells treated with PD161570 expressing fluorescent protein, according to an embodiment of the invention. FIG 7 shows an image of transduced wild-type and EGFR1 knockout TZM-GFP cells expressing fluorescent protein, according to an embodiment of the invention. The inhibition of the EGFR1 either by using a EGFR1 inhibitor or by knocking the gene expressing the EGFR1 both results in increasing the transduction ratio and is evident in both cases.
FIG 8 shows a graphical representation providing a comparison between the TZM-GFP wild-type cell and TZM-GFP EGFR1 knockout cell, according to an embodiment of the invention. The transduction efficiency in knockout cells is found similar to the transduction efficiency in target cells treated with EGFR1 inhibitor.
The inactivation of the EGFR1 either by using a EGFR1 inhibitor or by removing the gene expressing the EGFR1 both results in increasing the transduction ratio and is evident in both cases.
FIG. 9 shows a graphical representation providing a comparison of the EGFR1 inhibitor and rapamycin to assess their effects on lentiviral vector transduction, according to an embodiment of the invention. The mTOR inhibitor such as the rapamycin is known to enhance viral transduction. A comparative study with respect to the transduction efficiency in the target cells incubated with the EGFR1 inhibitor and rapamycin along with a control sample is analyzed. The target cells are incubated with rapamycin at a
concentration of 20mM in comparison with EGFR1 inhibitors such as PD165170 at a concentration of 5 mM. The study revealed that in comparison with the control sample the lentiviral vector transduction in the target cells was ~2.6 fold high and PD165170 exhibited an increase of ~5 fold as depicted in FIG. 2. Although rapamycin enhances lentiviral transduction, due to the cytotoxic nature the compound is removed 6h after incubation. However, the EGFR1 inhibitor works at a concentration of nanomolar and micromolar ranges and the prolonged incubation time with the target cells does not reduce the proliferation of the cells as shown in FIG. 5.
ADVANTAGES:
The present invention provides methods with improved lentiviral transduction. The method prevents damage to the target cell with less cell cytotoxicity and reduced cell-cycle inhibitory activity upon prolonged exposure of the inhibitor compound in the target cell. Further, the inhibition of the EGFR1 is not toxic, and does not result in limiting an essential signalling pathway in the target cells unlike the composition known in the art.
INDUSTRIAL APPLICABILITY:
The invention provides methods with improved lentiviral transduction to enhance the expression of a gene of interest without apparent toxicity to the target cells. A therapeutic agent can be expressed by the method of the invention and the desired gene or protein can be over expressed. The therapeutic agent can be a therapeutic gene for curing a genetic deficiency
by lentiviral gene therapy methods, modified CAR-T gene for CAR-T cell therapy, a short hairpin RNA for gene silencing, a CRISPR/Cas9 formulation to eliminate the functionality of the desired gene, a CRISPR/Cas9 formulation to augment gene function of the desired gene, or a deliver gene such as CRISPR components to label, silence, activate, repress, genetic loci for biomedical research application and therapeutic application.
Claims
1. A method for lentiviral transduction in a target cell, the method comprising: processing the target cell for inactivating an epidermal growth factor receptor-1 (EGFR1); producing at least one lentiviral vector from a mammalian cell to obtain lentiviral particles; and introducing the lentiviral particles into the processed target cell, wherein the inactivation of the epidermal growth factor receptor-1 in the target cell enhances the lentiviral transduction.
2. The method as claimed in claim 1, wherein inactivating the EGFR1 is achieved by incubating with at least one EGFR1 inhibitor or subjecting the target cell for knockout of the
EGFR1 from the target cell.
3. The method as claimed in claim 1 , wherein the EGFR1 inhibitor is selected from a group comprising 1 -tert-Butyl-3- [6-(2,6-dichlorophenyl)-2-[[4-(diethylamino) butyljamino] pyrido[2,3-d pyrimidin-7-yl]urea, 6-[4-[(4-Ethylpiperazin-1- yl)methyl]phenyl]-N-[(1 R)-1 -phenylethyl]-7H-pyrrolo[2,3- d]pyrimidin-4-amine, and 2-Bromo-4-[(6,7- dimethoxyquinazolin-4-yl)amino]phenol.
4. The method as claimed in claim 1 , wherein the knockout of EGFR1 is achieved by CRISPR/Cas9 for removing a gene expressing EGFR1.
5. The method as claimed in claim 1, wherein the target cell includes CD4+ cell, CD14+ cell, a monocyte, FleLa cell,
TZM-GFP cell, A549 cell, THP-1 monocyte, Jurkat E6.1 T- cells, THP-1 derived macrophage, U937 cells.
6. The method as claimed in claim 1 , wherein the method further comprises pre-stimulating the target cells with compounds selected from a group comprising recombinant cytokines, an antibody, antigen lectins, and an antibody- coated magnetic bead or a combination thereof.
7. The method as claimed in claim 1, wherein the lentiviral vector includes at least one envelope vector, at least one transfer vector, and at least one packaging vector.
8. The method as claimed in claim 1, wherein the envelop vector is selected from a group comprising PMD2.G, pCAG-VSVG, and pHEF-VSVG.
9. The method as claimed in claim 1 , wherein the transfer vector is selected from a group comprising PScalps
ZsGreen, pLKO.1 puro, pLK0.1-TRC series cloning plasmids, Tet-pLKO-puro, pWPI, pLenti CRISPR-E, pLenti CRISPR-V1/V2, EF.CMV.RFP, pLenti CMV Puro DEST, or
lentiviral vector for carrying a gene/ shRNA/ CRISPR encoding a fluorescent protein, or gene of interest.
10. The method as claimed in claim 1, wherein the packaging vector is selected from a group comprising psPAX2, pCMV delta R8.2, pMDLg/pRRE, psPAX2-D64V, and psPAX2-
D116N.
11. The method as claimed in claim 1 , wherein the mammalian cell is selected from a group comprising, HEK 293T, HEK293FT, HEK293 cell.
12. The method as claimed in claim 1, wherein the lentiviral vectors are incubated in a 5%-10% C02 incubator for a duration of 48-72 hours at a temperature of 37°C.
13. The method as claimed in claim 1, wherein the lentiviral particles introduced into the processed target cells are maintained in a 5-10% C02 incubator for a duration of 48-
72 hours at a temperature of 37°C.
14. The method as claimed in claim 1 , wherein the EGFR1 inhibitor is present during the entire process of transduction or at a specific interval at a concentration in the range between 0.1 mM and 10 mM.
15. The method as claimed in claim 1 , wherein the target cells are transduced with a multiplicity of infection in the range between 0.1-10.
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