DESC:FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
(Section 10 and Rule 13)

1. TITLE OF THE INVENTION:
SURFACE MODIFICATION OF LENTIVIRAL PARTICLE AND PRODUCER CELL THEREOF

2. APPLICANT:
MICRO CRISPR Pvt. Ltd., an Indian Company, of the address Survey No: 1574, Muktanand Marg, Chala, Vapi-396191, Gujarat, India

3. The following specification particularly describes the invention and the manner in which it is to be performed:

FIELD OF INVENTION
[001] The present disclosure relates to a synthetically modified lentiviral particle produced by a producer cell. More particularly, the present disclosure relates to surface modification of a lentiviral particle by a producer cell.
BACKGROUND OF INVENTION
[002] Viral particles are one of the most preferred vectors to introduce foreign genetic material into a host cell. These viral particles, for transduction of host cells, are produced by a producer cell, for example Human Embryonic Kidney 293 (HEK293) cells.
[003] The human immunodeficiency virus (HIV) based lentiviral particle has the ability to infect both dividing and non-dividing host cells, for example, T cells. However, the transduction efficiency in quiescent (i.e., non-dividing) T cells is typically lower compared to its activated counterpart (i.e., dividing cells).
[004] The steps to infect a cell (or host cell) using the lentiviral particle involves binding the lentiviral particle to the plasma membrane of the cell, endocytosis, envelope fusion, reverse transcription to form a provirus followed by integration of the viral genome into the host genome.
[005] Conventionally, the lentiviral particles produced by conventional producer cells are pseudo-typed with vesicular stomatitis virus G (VSV-G) protein, which make use of the low-density lipoprotein receptor (LDL-R) on the host cell as their entry point. LDL-R are expressed only upon activation of the host cells, thus limiting the tropism of such VSV-G pseudo-typed lentiviral particles and thereby limiting the transduction rate/efficiency.
[006] Hence, there arises a need to develop an alternative that overcomes the drawbacks of the conventionally available lentiviral particle.

SUMMARY OF INVENTION
[007] Particular embodiments of the present disclosure are described herein below with reference to the accompanying drawings; however, it is to be understood that the disclosed embodiments are mere examples of the disclosure, which may be embodied in various forms. Well-known functions or constructions are not described in detail to avoid obscuring the present disclosure in unnecessary detail. Therefore, specific structural and functional details disclosed herein are not to be interpreted as limiting, but merely as a basis for the claims and as a representative basis for teaching one skilled in the art to variously employ the present disclosure in virtually any appropriately detailed structure.
[008] In an exemplary embodiment, the present disclosure relates to a genetic cassette including one or more promoters, and one or more genes disposed downstream of the one or more promoters. The one or more genes including at least one of one or more first genes encoding for a single chain variable fragment (scFv) each, and one or more second genes encoding for a ligand each. The scFv is at least one of anti-CD3scFv and anti-CD34scFv. The ligand is at least one of CD38, CD70, CD71, CD80, CD90, CD117, CD252, and CD275.
[009] In another exemplary embodiment, the present disclosure relates to a modified plasmid including a vector and a genetic cassette as described above ligated to the vector.
[0010] In another exemplary embodiment, the present disclosure relates to a producer cell including a modified plasmid as described above.
[0011] In yet another exemplary embodiment, the present disclosure relates to a lentiviral particle produced by a producer cell as described above. The lentiviral particle encapsulated within a portion of an outer membrane of the producer cell.
BRIEF DESCRIPTION OF DRAWINGS
[0012] The summary above, as well as the following detailed description of illustrative embodiments, is better understood when read in conjunction with the apportioned drawings. For the purpose of illustrating the present disclosure, exemplary constructions of the disclosure are shown in the drawings. However, the disclosure is not limited to specific methods and instrumentality disclosed herein. Moreover, those in the art will understand that the drawings are not to scale.
[0013] Fig. 1 depicts an ¬¬¬¬exemplary genetic cassette 100 according to an embodiment of the present disclosure.
[0014] Fig. 2 depicts an ¬¬¬¬exemplary method 200 to produce a plurality of lentiviral particles and transduce a pre-defined host cell according to an embodiment of the present disclosure.
[0015] Fig. 2a depicts an ¬¬¬¬exemplary producer cell 300 producing a plurality of lentiviral particles 400 according to an embodiment of the present disclosure.
[0016] Fig. 2b depicts an ¬¬¬¬exemplary T cell 500 being transduced by the one or more lentiviral particles 400 according to an embodiment of the present disclosure.
[0017] Fig. 2c depicts an ¬¬¬¬exemplary HSC 600 being transduced by the one or more lentiviral particles 400 according to an embodiment of the present disclosure.
[0018] Figs. 3 – 6 depict experimental observations associated with the lentiviral particles 400 according to one or more embodiments of the present disclosure.
DETAILED DESCRIPTION OF THE ACCOMPANYING DRAWINGS
[0019] Prior to describing the invention in detail, definitions of certain words or phrases used throughout this patent document will be defined: the terms "include" and "comprise", as well as derivatives thereof, mean inclusion without limitation; the term "or" is inclusive, meaning and/or; the phrases "coupled with" and "associated therewith", as well as derivatives thereof, may mean to include, be included within, interconnect with, contain, be contained within, connect to or with, coupled to or with, be communicable with, cooperate with, interleave, juxtapose, be proximate to, be bound to or with, have a property of, or the like. Definitions of certain words and phrases are provided throughout this patent document, and those of ordinary skill in the art will understand that such definitions apply in many, if not most, instances to prior as well as future uses of such defined words and phrases.
[0020] Reference throughout this specification to “one embodiment,” “an embodiment,” or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, appearances of the phrases “in one embodiment,” “in an embodiment,” and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment, but mean “one or more but not all embodiments” unless expressly specified otherwise. The terms “including,” “comprising,” “having,” and variations thereof mean “including but not limited to” unless expressly specified otherwise. An enumerated listing of items does not imply that any or all of the items are mutually exclusive and/or mutually inclusive, unless expressly specified otherwise. The terms “a,” “an,” and “the” also refer to “one or more” unless expressly specified otherwise.
[0021] Although the operations of exemplary embodiments of the disclosed method may be described in a particular, sequential order for convenient presentation, it should be understood that the disclosed embodiments can encompass an order of operations other than the particular, sequential order disclosed. For example, operations described sequentially may in some cases be rearranged or performed concurrently. Further, descriptions and disclosures provided in association with one particular embodiment are not limited to that embodiment, and may be applied to any embodiment disclosed herein. Moreover, for the sake of simplicity, the attached figures may not show the various ways in which the disclosed system, method, and apparatus can be used in combination with other systems, methods, and apparatuses.
[0022] Furthermore, the described features, advantages, and characteristics of the embodiments may be combined in any suitable manner. One skilled in the relevant art will recognize that the embodiments may be practiced without one or more of the specific features or advantages of a particular embodiment. In other instances, additional features and advantages may be recognized in certain embodiments that may not be present in all embodiments. These features and advantages of the embodiments will become more fully apparent from the following description and apportioned claims, or may be learned by the practice of embodiments as set forth hereinafter.
[0023] The present disclosure discloses a synthetic producer cell (or producer cell) configured to produce a plurality of lentiviral particles (or particles). The particles are provided with one or more surface modifications to enable the particles to transduce a pre-defined host cell. As an example, the host cell may be at least one of a T cell and a hematopoietic stem cell (HSC).
[0024] The producer cell, as an example, is genetically modified Human Embryonic Kidney 293T (HEK293T) cells.
[0025] Although the present disclosure is described with the example of HEK293T cells, other cells having the said genetic modifications are also within the scope of the teachings of the present disclosure.
[0026] The particles of the present disclosure display surface modification that provides high transduction efficiency of the host cells even if the host cells are in their quiescent stage (i.e., non-dividing stage). In other words, the particles of the present disclosure can transduce host cells prior to their activation thereby reducing ex vivo manipulation time of the host cells.
[0027] The surface modification of the particles may include at least one of a surface mounted single chain variable fragment (or scFv) and surface mounted ligands (or ligands). The scFv may have binding affinity towards one or more surface antigens of the host cell. And, the ligands may have binding affinity towards one or more surface receptors of the host cell.
[0028] The scFv retains binding specificity and display improved tissue penetration, blood clearance, receptor targeted delivery thus, significantly enhancing the transduction efficiency of the host cells. Further, the ligands specific to the receptors of the host cell further enhances the transduction efficiency in both dividing (activated) and non-dividing (quiescent) host cells.
[0029] Now referring to the figures, Fig. 1 depicts an exemplary genetic cassette 100 provided within a producer cell (not shown). The genetic cassette 100 is a polynucleotide molecule having a pre-defined structure including, but not limited to, single stranded deoxynucleic acid (ssDNA), double stranded deoxynucleic acid (dsDNA), ribonucleic acid (RNA), etc. The genetic cassette 100 may either be integrated to the genome of the producer cell or may be disposed as a free-floating plasmid within the producer cell. In an exemplary embodiment, the genetic cassette 100 has a dsDNA structure disposed within the producer cells as free-floating plasmids. In another exemplary embodiment, the genetic cassette 100 has a dsDNA structure that is integrated to the genome of the producer cell. In an exemplary embodiment, the producer cell is genetically modified HEK293T cell.
[0030] As shown in Fig. 1, the genetic cassette 100 extends between a 5’ end and a 3’ end. The genetic cassette 100 may optionally include a complementary strand (not shown) extending between a 3’ end and a 5’ end as is known for dsDNA structures. The genetic cassette 100 may be flanked with a long terminal repeat (LTR) region. The genetic cassette 100 further includes one or more promoters 103, one or more genes, etc. The one or more genes disposed downstream of the one or more promoters 103.
[0031] In an exemplary embodiment, the genetic cassette 100 is flanked by a 5’ LTR region 101 disposed at the 5’ end and encoded by SEQ ID No. 1. And, the genetic cassette 100 is flanked by a 3’ LTR region 101a disposed at the 3’ end and encoded by SEQ ID No. 2. The LTR regions 101, and 101a helps the genetic cassette 100 to integrate into the genome of the producer cell.
[0032] The promoter 103 is disposed towards the 5’ end, adjacent to the 5’ LTR region 101. The promoter 103 helps in expression of the one or more genes present on the genetic cassette 100. The promoter 103 may be at least one of cytomegalovirus (CMV) promoter encoded by SEQ ID No. 3, human phosphoglycerate kinase 1 (hPGK) promoter encoded by SEQ ID No. 4, etc. In an exemplary embodiment, the genetic cassette 100 includes one CMV promoter. The CMV promoter is a strong viral promoter that drives high levels of gene expression in mammalian cells.
[0033] In the depicted embodiment, the one or more genes of the genetic cassette 100 includes one or more first genes 110 and one or more second genes 120. The first genes 110 encode for a single chain variable fragment (scFv) each. The first gene 110 is disposed downstream of the promoter 103. The scFv encoded by the first gene 110 may be at least one of anti-CD3scFv (the first gene 110 being encoded by at least one of SEQ ID No. 5-7) and anti-CD34scFv (the first gene 110 being encoded by SEQ ID No. 8). The anti-CD3scFv has a binding affinity towards CD3 ligand(s) specific to T cell (an exemplary host cell) surfaces. The anti-CD34scFv has a binding affinity towards CD34 ligand(s) specific to hematopoietic stem cells (HSCs, an exemplary host cell) surfaces.
[0034] The second genes 120 encodes for a ligand each. The second gene 120 is disposed downstream of the promoter 103. The second gene 120 may either be disposed upstream or downstream of the first gene 110. In an exemplary embodiment, as shown in Fig. 1, the second gene 120 is disposed downstream of the first gene 110. The ligand may be at least one of CD38 (the second gene 120 being encoded by SEQ ID No. 9), CD70 (the second gene 120 being encoded by SEQ ID No. 10), CD71 (the second gene 120 being encoded by SEQ ID No. 11), CD80 (the second gene 120 being encoded by SEQ ID No. 12), CD90 (the second gene 120 being encoded by SEQ ID No. 13), CD117 (the second gene 120 being encoded by SEQ ID No. 14), CD252 (the second gene 120 being encoded by SEQ ID No. 15), CD275 (the second gene 120 being encoded by SEQ ID No. 16). The ligand CD70, CD80, CD252 and CD275 have a binding affinity towards cell surface receptors of T cells (an exemplary host cell). The ligand CD38, CD71, CD90 and CD117 have a binding affinity towards cell surface receptors of HSCs (an exemplary host cell).
[0035] Additionally or optionally, the one or more genes of the genetic cassette 100 further include at least one third gene 130 encoding for at least one of envelop component (VSV-G) (the third gene 130 being encoded by SEQ ID No. 17), first packaging plasmid component (Gag-pol) (the third gene 130 being at least partially encoded by SEQ ID No. 18) and second packaging plasmid component (Rev) (the third gene 130 being encoded by SEQ ID No. 19). The envelop component (VSV-G) acts as a viral envelope protein that facilitate host cell tropism and increase stability of the lentiviral particles. The first packaging plasmid component has two sub-components, namely Gag and Pol. The Gag sub-component encodes for structural proteins (like matrix, capsid, nucleocapsid, etc.) of the lentiviral particles. The Pol sub-component encodes for essential enzymes like reverse transcriptase, integrase, and protease. The second packaging plasmid component ensures and regulates efficient nuclear export of messenger RNAs (mRNAs).
[0036] The third gene 130 is disposed downstream of the promoter 103. The third gene 130 being disposed either upstream of the first gene 110, the second gene 120, or downstream of the second gene 120. In an exemplary embodiment, as shown in Fig. 1, the third gene 130 is disposed downstream of the second gene 120.
[0037] In an exemplary embodiment, the genetic cassette 100 is directed toward transduction of T cells and includes the first gene 110 that encodes for anti-CD3scFv. In another exemplary embodiment, the genetic cassette 100 is directed toward transduction of T cells and includes the first gene 110 that encodes for anti-CD3scFv and the second gene 120 that encodes for CD252. In another exemplary embodiment, the genetic cassette 100 is directed toward transduction of T cells and includes the first gene 110 that encodes for anti-CD3scFv and the second gene 120 that encodes for CD275. In another exemplary embodiment, the genetic cassette 100 is directed toward transduction of T cells and includes the first gene 110 that encodes for anti-CD3scFv and the second gene 120 that encodes for CD80. In another exemplary embodiment, the genetic cassette 100 is directed toward transduction of T cells and includes the first gene 110 that encodes for anti-CD3scFv and the second gene 120 that encodes for CD70. In another exemplary embodiment, the genetic cassette 100 is directed toward transduction of T cells and includes the first gene 110 that encodes for anti-CD3scFv, the second gene 120 that encodes for CD252 and CD275. In another exemplary embodiment, the genetic cassette 100 is directed toward transduction of T cells and includes the first gene 110 that encodes for anti-CD3scFv, the second gene 120 that encodes for CD252 and CD80. In another exemplary embodiment, the genetic cassette 100 is directed toward transduction of T cells and includes the first gene 110 that encodes for anti-CD3scFv, the second gene 120 that encodes for CD252 and CD70. In another exemplary embodiment, the genetic cassette 100 is directed toward transduction of T cells and includes the first gene 110 that encodes for anti-CD3scFv, the second gene 120 that encodes for CD275 and CD80. In another exemplary embodiment, the genetic cassette 100 is directed toward transduction of T cells and includes the first gene 110 that encodes for anti-CD3scFv, the second gene 120 that encodes for CD275 and CD70. In another exemplary embodiment, the genetic cassette 100 is directed toward transduction of T cells and includes the first gene 110 that encodes for anti-CD3scFv, the second gene 120 that encodes for CD80 and CD70. In another exemplary embodiment, the genetic cassette 100 is directed toward transduction of T cells and includes the first gene 110 that encodes for anti-CD3scFv, the second gene 120 that encodes for CD252, CD275 and CD80. In another exemplary embodiment, the genetic cassette 100 is directed toward transduction of T cells and includes the first gene 110 that encodes for anti-CD3scFv, the second gene 120 that encodes for CD252, CD275 and CD70. In another exemplary embodiment, the genetic cassette 100 is directed toward transduction of T cells and includes the first gene 110 that encodes for anti-CD3scFv, the second gene 120 that encodes for CD252, CD80 and CD70. In another exemplary embodiment, the genetic cassette 100 is directed toward transduction of T cells and includes the first gene 110 that encodes for anti-CD3scFv, the second gene 120 that encodes for CD275, CD80 and CD70. In another exemplary embodiment, the genetic cassette 100 is directed toward transduction of T cells and includes the third gene 130 that encodes for VSV-G, Gag-pol and Rev. In another exemplary embodiment, the genetic cassette 100 is directed toward transduction of T cells and includes the first gene 110 that encodes for anti-CD3scFv, the third gene 130 that encodes for VSV-G, Gag-pol and Rev. In another exemplary embodiment, the genetic cassette 100 is directed toward transduction of T cells and includes the first gene 110 that encodes for anti-CD3scFv, the second gene 120 that encodes for CD252, the third gene 130 that encodes for VSV-G, Gag-pol and Rev. In another exemplary embodiment, the genetic cassette 100 is directed toward transduction of T cells and includes the first gene 110 that encodes for anti-CD3scFv, the second gene 120 that encodes for CD275, the third gene 130 that encodes for VSV-G, Gag-pol and Rev. In another exemplary embodiment, the genetic cassette 100 is directed toward transduction of T cells and includes a first gene 110 that encodes for anti-CD3scFv, the second gene 120 that encodes for CD80, the third gene 130 that encodes for VSV-G, Gag-pol and Rev. In another exemplary embodiment, the genetic cassette 100 is directed toward transduction of T cells and includes the first gene 110 that encodes for anti-CD3scFv, the second gene 120 that encodes for CD70, the third gene 130 that encodes for VSV-G, Gag-pol and Rev.
[0038] In an exemplary embodiment, the genetic cassette 100 is directed toward transduction of HSCs and includes the first gene 110 that encodes for anti-CD34scFv. In another exemplary embodiment, the genetic cassette 100 is directed toward transduction of HSCs and includes the first gene 110 that encodes for anti-CD34scFv and the second gene 120 that encodes for CD90. In another exemplary embodiment, the genetic cassette 100 is directed toward transduction of HSCs and includes the first gene 110 that encodes for anti-CD34scFv and the second gene 120 that encodes for CD38. In another exemplary embodiment, the genetic cassette 100 is directed toward transduction of HSCs and includes the first gene 110 that encodes for anti-CD34scFv and the second gene 120 that encodes for CD117. In another exemplary embodiment, the genetic cassette 100 is directed toward transduction of HSCs and includes the first gene 110 that encodes for anti-CD34scFv and the second gene 120 that encodes for CD71. In another exemplary embodiment, the genetic cassette 100 is directed toward transduction of HSCs and includes the first gene 110 that encodes for anti-CD34scFv, the second gene 120 that encodes for CD90 and CD38. In another exemplary embodiment, the genetic cassette 100 is directed toward transduction of HSCs and includes the first gene 110 that encodes for anti-CD34scFv, the second gene 120 that encodes for CD90 and CD117. In another exemplary embodiment, the genetic cassette 100 is directed toward transduction of HSCs and includes the first gene 110 that encodes for anti-CD34scFv, the second gene 120 that encodes for CD90 and CD71. In another exemplary embodiment, the genetic cassette 100 is directed toward transduction of HSCs and includes the first gene 110 that encodes for anti-CD34scFv, the second gene 120 that encodes for CD38 and CD117. In another exemplary embodiment, the genetic cassette 100 is directed toward transduction of HSCs and includes the first gene 110 that encodes for anti-CD34scFv, the second gene 120 that encodes for CD38 and CD71. In another exemplary embodiment, the genetic cassette 100 is directed toward transduction of HSCs and includes the first gene 110 that encodes for anti-CD34scFv, the second gene 120 that encodes for CD117 and CD71. In another exemplary embodiment, the genetic cassette 100 is directed toward transduction of HSCs and includes the third gene 130 that encodes for VSV-G, Gag-pol and Rev. In another exemplary embodiment, the genetic cassette 100 is directed toward transduction of HSCs and includes the first gene 110 that encodes for anti-CD34scFv, the third gene 130 that encodes for VSV-G, Gag-pol and Rev. In another exemplary embodiment, the genetic cassette 100 is directed toward transduction of HSCs and includes the first gene 110 that encodes for anti-CD34scFv, the second gene 120 that encodes for CD90, the third gene 130 that encodes for VSV-G, Gag-pol and Rev. In another exemplary embodiment, the genetic cassette 100 is directed toward transduction of HSCs and includes the first gene 110 that encodes for anti-CD34scFv, the second gene 120 that encodes for CD38, the third gene 130 that encodes for VSV-G, Gag-pol and Rev. In another exemplary embodiment, the genetic cassette 100 is directed toward transduction of HSCs and includes the first gene 110 that encodes for anti-CD34scFv, the second gene 120 that encodes for CD117, the third gene 130 that encodes for VSV-G, Gag-pol and Rev. In another exemplary embodiment, the genetic cassette 100 is directed toward transduction of HSCs and includes the first gene 110 that encodes for anti-CD34scFv, the second gene 120 that encodes for CD71, the third gene 130 that encodes for VSV-G, Gag-pol and Rev.
[0039] Fig. 2 depicts an exemplary method 200 to produce the plurality of particles from at least one producer cell (or a population of producer cells thereof) and then transduce at least one pre-defined host cell (or a population of pre-defined host cells thereof). In an exemplary embodiment, as shown in Fig. 2a, the producer cell 300 is HEK293T cells provided with the genetic cassette 100 (as described above) that produces the particles 400. The genetic cassette 100 enables the producer cells 300 to display the scfV(s) 310 and/or the ligand(s) (not shown) on an outer membrane of the producer cell 300. In the depicted embodiment, the producer cell 300 displays the scFVs 310 on an outer membrane of the producer cell 300. The one or more scFvs 310 is at least one of anti-CD3scFv and anti-CD34scFV. The ligand is at least one of CD38, CD70, CD71, CD80, CD90, CD117, CD252, and CD275. When the particles 400 are produced by the producer cell 300, the particle 400 buds off from the producer cell 300 by taking a portion of the outer membrane of the producer cell 300. In other words, the particles 400 produced by the producer cells 300 are encapsulated by a portion of the outer membrane of the producer cells 300 having the scFv(s) 410 and/or the ligands 420.
[0040] The method 200 commences at step 201 by ligating the genetic cassette 100 to a pre-defined vector to obtain a modified plasmid. The vector is at least one of pLYS1-FLAG-MitoGFP-HA (or pLenti) plasmid (procured from Addgene), GLV2-CMV-[ORF]-PGK-Puro-P2A-EGFP plasmid (procured from GenScript), etc. In an exemplary embodiment, the genetic cassette 100 is ligated to pLYS1-FLAG-MitoGFP-HA plasmid encoded by SEQ ID No. 20. In an exemplary embodiment, the genetic cassette 100 is ligated to the pLenti plasmid using a ligase enzyme, for example, T4 DNA ligase (procured from New England Biolabs) following the manufacturer’s protocol. In an exemplary embodiment, before ligating the genetic cassette 100 to the pLenti plasmid, the genetic cassette 100 and the pLenti plasmid is digested with Nhe1 and EcoR1 restriction enzymes (procured from New England Biolabs) by following the manufacturer’s protocol.
[0041] Additionally or optionally, a genetic insert may be ligated to the vector using the ligase enzyme. The genetic insert is a polynucleotide sequence that is to be packaged in the particles and delivered within a host cell as required. Thus, the sequence of the genetic insert may vary with the requirements/application.
[0042] Either one modified plasmid (and copies thereof) is made with one genetic cassette 100 (and copies thereof), where the genetic cassette 100 has all the one or more first genes 110, the second genes 120 and/or the third genes 130. Else, two or more plasmids (and copies thereof) are made with two or more genetic cassette 100 (and copies thereof), where each of the genetic cassette 100 has different first gene 110, second gene 120 and/or third gene 130.
[0043] The first gene 110 encoding for anti-CD3scFv and/or anti-CD34scFV are synthetically synthesized (from Genscript). The second gene 120 encoding for CD70, CD80, CD252, CD275, CD38, CD71, CD90 and/or CD117 are isolated through complementary DNA (cDNA) synthesis and polymerase chain reaction (PCR) from, for example, Jurkat cell line (a T-cell line derived from a T-cell leukemia) and Raji cell line (a B-cell line derived from a Burkitt's lymphoma).
[0044] At step 203, the modified plasmid(s) obtained from step 201 are transfected to the plurality of producer cells 300. In an exemplary embodiment, as shown in Fig. 2a, the producer cells 300 are HEK293T cells.
[0045] Additionally or optionally, the modified plasmid(s) are amplified to increase their number before transfecting the producer cells 300 with the modified plasmid(s). In an exemplary embodiment, the modified plasmids are cloned in Escherichia coli (E. coli) cells to increase their number. Alternatively, the modified plasmid(s) or portions thereof are amplified using polymerase chain reaction (PCR).
[0046] At step 205, the producer cell 300, using the genetic cassette 100, produces the plurality of lentiviral particles 400 (as shown in Fig. 2a). Depending upon the sequence of the genetic cassette 100, the lentiviral particles (or particles) 400 are pseudotyped with at least one of the plurality of scFvs 410 and/or the plurality of ligands 420. The scFv 410 may be at least one of anti-CD3scFv and anti-CD34scFV. The ligands 420 may be at least one of CD38, CD70, CD71, CD80, CD90, CD117, CD252, and CD275.
[0047] In an exemplary embodiment, the particles 400 displaying anti-CD3scFv, CD70, CD80, CD252, and CD275 on an outer membrane of the particles 400 has a binding affinity towards T cells (an exemplary host cell). In another exemplary embodiment, the particles 400 displaying CD34scFv, CD38, CD71, CD90, CD117 on the outer membrane of the particles 400 has a binding affinity towards HSCs (an exemplary host cell).
[0048] At step 207, the pre-defined host cell is transduced by the particles 400. A pre-defined amount of the host cells is transduced by adding a pre-defined amount of the particles 400 in a pre-defined ratio. In an exemplary embodiment, the particles 400 are added to the host cells at a ratio of 1:1. After adding the particles 400 to the pre-defined host cells, the host cells are incubated for a pre-defined time period at a pre-defined temperature and environment. The pre-defined time period ranges from 44 hours to 52 hours. The pre-defined temperature ranges from 35 °C to 37 °C. In an exemplary embodiment, the host cells along with the particles 400 are incubated for 48 hours at 37 °C, 5% CO2.
[0049] In an exemplary embodiment, as shown in Fig. 2b, the particles 400 pseudotyped with anti-CD3scFv (scFv 410) is configured to transduce a T cell 500 (an exemplary host cell) displaying CD3 510 (an exemplary cell-surface ligand) on an outer membrane. The anti-CD3scFv of the particle 400 binds with CD3 510 of the T cell 500 thereby enabling the particle 400 to easily transduce the T cell 500.
[0050] In an exemplary embodiment, as shown in Fig. 2c, the particles 400 pseudotyped with anti-CD34scFv (scFv 410) is configured to transduce an HSC 600 (an exemplary host cell) displaying CD34 610 (an exemplary cell-surface ligand) on an outer membrane. The anti-CD34scFv of the particle 400 binds with CD34 610 of the HSC 600 thereby enabling the particle 400 to easily transduce the HSC 600.
[0051] The scFvs 410 and/or the ligands 420 of the particles 400 helps the particles 400 to easily and efficiently transduce the host cells (i.e., the T cell 500 and the HSC 600), even when the host cells are in their quiescent stage (i.e., non-dividing stage). Thus, the particles 400 of the present disclosure can transduce host cells prior to their activation thereby reducing ex vivo manipulation time of the host cells.
[0052] The present disclosure will now be explained with the help of the following examples:
[0053] Example 1: Isolating the second gene 120
[0054] The second gene 120 encoding for CD80, CD252, and CD275 were isolated through PCR amplification from, for example, Jurkat cell line (a T-cell line derived from a T-cell leukemia) and Raji cell line (a B-cell line derived from a Burkitt's lymphoma). Ribonucleic acid (RNA) fractions of the respective cell lines were isolated using TRIzol reagent (procured from Invitrogen) by following the manufacturer’s protocol. 10 ng of the RNA fraction was used as a template to synthesize the complementary DNA (cDNA) of the second gene 120 in a thermal cycler (procured from BioRad) using Verso cDNA synthesis kit (procured from Thermo Scientific) by following manufacturer’s protocol. The RNA primers (OligoDt and Random hexamer (1:3 ratio) kit, procured from Thermoscientific) used for the corresponding cDNA is tabulated in table A below.
Second gene 120 Forward primer Reverse primer
CD80 SEQ ID No. 21 SEQ ID No. 22
CD252 SEQ ID No. 23 SEQ ID No. 24
CD275 SEQ ID No. 25 SEQ ID No. 26
(table A)
[0055] The cDNA obtained were the second gene 120.
[0056] Example 2: Cloning the first gene 110 and the second gene 120
[0057] 10 µg of a vector (pLYS1-FLAG-MitoGFP-HA) and 5 µg of the respective first gene 110 and second gene 120 obtained were digested using Nhe1 and EcoR1 nucleases (procured from New England BioLabs) for 3hours at 37 °C to obtain a digested mixture. The composition of the digested mixture includes 8 µL of rCutSmart buffer, 10 µg of vector, 5 µg of the first gene(s) 110, and/or the second gene(s) 120, 1 µL of Nhe1, 1 µL of EcoR1, and 60 µL of nuclease free water.
[0058] After digestion, the digested mixture was purified using Nucleopore extraction and PCR clean up kit (procured from Genetix Biotech Asia Pvt. Ltd.) by following the manufacturer’s protocol.
[0059] After purification, the respective first gene 110 and the second gene 120 were ligated to the vector separately using T4 DNA ligase (procured from New England BioLabs) for 2 hours at room temperature by following manufacturer’s protocol to obtain the modified plasmids.
[0060] The modified plasmids were individually used to transform Escherichia coli DH5a cells (E. coli cells) using for example, CaCl2-MgCl2 technique. The transformed E. coli cells (or clones) were plated on ampicillin (100 µg/mL) agar plates and incubated at 37 °C for 14hrs. Positive colonies of the clones were picked up from the plates and confirmed using colony polymerase chain reaction (PCR). The PCR was set up in a thermal cycler (procured from BioRad) using PCR Master Mix 2x (procured from Thermo Scientific) and forward and reverse primers (tabulated in table B below) by following manufacturer’s protocol.
Gene Forward primer Reverse primer
CD3 scFv SEQ ID No. 27 SEQ ID No. 28
CD80 SEQ ID No. 29 SEQ ID No. 30
CD252 SEQ ID No. 31 SEQ ID No. 32
CD275 SEQ ID No. 33) SEQ ID No. 34
(table B)
[0061] The positive colonies were further confirmed by isolating the modified plasmids and subjecting the modified plasmids to restriction digestion and sanger sequencing (from CMV promoter onwards). The plasmids were digested using the following nucleases (procured from New England BioLabs) as tabulated in table C below by following the manufacturer’s protocol:
Modified plasmid name Restriction enzyme used Size of the digested fragments
pLenti-CD3scFv Kpn1 6530 bp and 1715 bp
pLenti-CD80 Kpn1 6858 bp and 1401 bp
pLenti-CD252 Kpn1 6543 bp and 1401 bp
pLenti-CD275 Nco1 5111 bp and 3119 bp
(table C)
[0062] The plasmids after their digestion were loaded on an Agarose gel along with a 1Kb DNA ladder (procured from GeneDirex) and subjected to electroporation. The picture of the agarose gels is depicted in Figs. 3a and 3b. The fragment size determined from the picture of the agarose gels confirmed the successful cloning of the first gene 110 and the second gene 120.
[0063] Example 3: Transfection of the producer cells (HEK293T, procured from American Type Culture Collection)
[0064] 0.7 x 106 HEK293T cells were seeded in a treated T25 flask with 4 mL of Dulbecco's Modified Eagle Medium (DMEM) high glucose medium (procured from Gibco), without any antibiotic. The T25 flask was then incubated at 37 °C, 5% CO2 for 48 hours or until the confluency had reached 70%. Thereafter, the DMEM medium in the T25 flask was replaced with 1.5 mL of Opti-MEM (procured from Gibco) and incubated at 37 °C for 30 minutes.
[0065] A transfection mix was prepared by making two solutions. The first solution was prepared by adding the composition provided in table D below in 0.25 mL of Opti-MEM. The first solution was incubated at room temperature for 10 mins.
DNA Amount (µg)
Modified plasmid obtained from Example 2 above 3.4
Rev 1.6
VSV-G 0.75
Gag-pol 1.2
Total 6.95
(table D)
[0066] The second solution was prepared by adding 20.85 µg Polyethylenimine (PEI) (procured from Sigma-Aldrich) in 0.25 mL of Opti-MEM. The second solution was incubated at room temperature for 10 mins. The first solution and the second solution were mixed together to obtain the transfection mix. The transfection mix was incubated at room temperature for 20 minutes.
[0067] The transfection mix was slowly added to the T25 flask containing the HEK293T cells. Thereafter, the T25 flask was incubated at 37 °C, 5% CO2 for 6 hours. After incubation, the media in the T25 flask was replaced with 4 mL of fresh DMEM supplemented with 10% fetal bovine serum (FBS) (procured from Gibco), 1 µM sodium pyruvate (procured from Gibco), 1 µM non-essential amino acids (procured from Gibco). Then the T25 flask was incubated at 37 °C, 5% CO2 for 48 hours. The T25 flask contained the transduced HEK293T cells.
[0068] Example 4: Selecting and confirming the transduced producer cells
[0069] In a 24 well cell culture plate, the transduced HEK293T cells obtained from Example 3 above were seeded at a density of 0.05 x 106 cells/well in 500 µL of complete DMEM medium. The composition of the complete DMEM medium includes 10% (w/v) fetal bovine serum (FBS, heat inactivated and filtered through 0.22 µm filter, procured from Gibco), 1mM sodium pyruvate (procured through Gibco), 1mM MEM non-essential amino acid (NEAA, procured from Gibco) and Dulbecco's Modified Eagle Medium (DMEM, procured from Gibco). The HEK293T cells were incubated for 24 hours at 37 °C, 5% CO2. After the HEK293T cells adhered to the well, the complete DMEM medium was refreshed and supplemented with 0.8 µg/mL of Puromycin. The HEK293T cells were incubated for 48 hours at 37 °C, 5% CO2. In the presence of Puromycin, only the transduced HEK293T cells were left alive and the untransduced HEK293T cells died. The microscopic image of alive transduced HEK293T cells and dead untransduced HEK293T cells are depicted in Fig. 4a and 4b respectively.
[0070] The HEK293T cells were incubated in Puromycin for 2 weeks (with periodic media refreshments) and then seeded over a sterile coverslip (12 mm x 12 mm) at a cell density of 0.05 x 106 per well having 500 µL of complete DMEM. The HEK293T cells were incubated for 24 hours at 37 °C, 5% CO2. Thereafter, the HEK293T cells were washed with 1X phosphate buffer saline (PBS, procured from Gibco) and fixed with 4% paraformaldehyde (procured from Sigma Aldrich) for 20 mins at 37 °C. The HEK293T cells were again washed with 1X PBS and stained with respective antibodies for 1 hour at room temperature. In other words, the antibodies were bound to the respective scFvs and ligands present on the outer membrane of the HEK293T cells. The antibodies used for staining the HEK293T cells are tabulated below in table E:
Antibody against Primary antibody Secondary antibody
Anti-CD3scFv 2 µg/mL of Rb pAb to Myc tag (ab9106, procured from Abcam) 1 µg/mL of Goat Anti Rb IgG H&L (Alexa Fluor @488) (procured from abcam)
CD80 2 µg/mL of CD80 (L 307.4) PE (procured from BD Biosciences)
CD252 2 µg/mL of PE-Mouse Anti-Human OX40 Ligand (CD252) (Cat. no. BD 55816, procured from BD biosciences)
CD 275 2 µg/mL of Purified Mouse anti-Human CD275 (Cat. no. BD 552501, procured from BD biosciences) 1 µg/mL of Goat Anti-Mouse IgG H&L (Alexa Flour @488) (procured from abcam)
(table E)
[0071] After staining the HEK293T cells with the antibodies, the HEK293T cells were washed with 1X PBS and mounted on a microscope slide using DAPI+Mountant (procured from Thermo Scientific). The microscopic slides having the HEK293T cells were observed under a confocal microscope (procured from Leica TCS) and floid microscope (procured from Thermo Scientific). The images from microscopy of the HEK293T cells are depicted in Fig. 5.
[0072] The images from microscopy confirmed that the HEK293T cells were displaying the antiCD3scFv, CD80, CD252, and CD275 on the cell surface.
[0073] Example 5: Titer estimation
[0074] Then the T25 flask containing the HEK293T obtained from Example 3 above was incubated at 37 °C, 5% CO2 for 48 hours to 72 hours. The supernatant from the T25 flask was collected after 48 hours and 72 hours in a falcon tube. The falcon tube was then centrifuged at 500g for 10 minutes. The supernatant was from the falcon tube was filtered using a 0.45 µm Polyethersulfone (PES) filter. The flow through from the filter was collected and stored at -80 °C. The titer of the lentiviral particles 400 in the flow through was estimated in the range of 2 X 105 TU/mL to 4 X 105 TU/mL.
[0075] Example 6: Transduction efficiency in T cells using the particles of the lentiviral vector obtained from example 5
[0076] 8mL of blood was drawn from an individual and diluted with 8mL of 1X Dulbecco's Phosphate-Buffered Saline (DPBS, procured from Gibco). The diluted blood was carefully layered on top of Ficoll-paque reagent (procured from Cytiva) in a centrifuge tube without mixing. The centrifuge tube was centrifuged at 300g for 45 mins with brakes off. The mononuclear cells were harvested present in the upper plasma layer at the Ficoll-paque reagent - plasma interface using a pipette. The mononuclear cells were washed using twice the volume of PBS and centrifuging at 300g for 10 mins.
[0077] From the mononuclear cells, primary T cells were isolated using CD4+/CD8+ microbeads (procured from Miltenyi) by following manufacturer’s protocol. The primary T cells were activated via CD3/CD28 microbeads using T Cell TransAct (produced from Miletenyi) and following manufacturer’s protocol. After 24 hours of activation, in a 12 well plate, 1.6 x 105 primary T cells were seeded in 500 µL of complete DMEM media. The primary T cells were transduced by adding 105 lentiviral particles 400 (500 µL) containing 2nd Gen CAR-GFP (procured from Addgene). The 12 well plate was then incubated 37 °C, 5% CO2. After 48 hours, the medium in the 12 well plate was replaced with fresh complete DMEM medium. After 96 hours of transduction, the T cells were analyzed for GFP expression. The T cells were collected in microcentrifuge tubes and centrifuged for 10 mins at 400g. The supernatant was discarded and washed with 1X PBS. The pellet was then resuspended in 500 µL of PBS and analyzed using BD Lyric flow cytometer (procured from BD Biosciences). The percentage of GFP expression is depicted in Fig. 6. The first bar depicts the expression of GFP when conventional particles of lentiviral vector was used. The remaining bars depict GFP expression for the particles of lentiviral vectors of the present disclosure. It was evident from the plot depicted in Fig. 6 that the particles of the lentiviral vector of the present disclosure provided better transduction efficiency compared to the conventional particles of the lentiviral vectors.
[0078] The scope of the invention is only limited by the appended patent claims. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the teachings of the present invention is/are used. ,CLAIMS:WE CLAIM
1. A genetic cassette (100) comprising:
a. one or more promoters (103);
b. one or more genes disposed downstream of the one or more promoters (103), the one or more genes including at least one of:
i. one or more first genes (110) encoding for a single chain variable fragment (scFv) each, the scFv is at least one of anti-CD3scFv and anti-CD34scFv; and
ii. one or more second genes (120) encoding for a ligand each, the ligand is at least one of CD38, CD70, CD71, CD80, CD90, CD117, CD252, and CD275.
2. The genetic cassette (100) as claimed in claim 1, wherein the one or more promoters (103) is at least one of cytomegalovirus (CMV) promoter encoded by SEQ ID No. 3, or human phosphoglycerate kinase 1 (hPGK) promoter encoded by SEQ ID No. 4.
3. The genetic cassette (100) as claimed in claim 1, wherein the genetic cassette (100) extends between a 5’ end and a 3’ end flanked by:
a. a 5’ long terminal repeat region (101) encoded by SEQ ID No. 1, and
b. a 3’ long terminal repeat region (101a) encoded by SEQ ID No. 2.
4. The genetic cassette (100) as claimed in claim 1, wherein the first gene (110) encoding for anti-CD3scFv is encoded by at least one of SEQ ID No. 5-7.
5. The genetic cassette (100) as claimed in claim 1, wherein the first gene (110) encoding for anti-CD34scFv is encoded by SEQ ID No. 8.
6. The genetic cassette (100) as claimed in claim 1, wherein the second gene (120) encoding for CD38 is encoded by SEQ ID No. 9.
7. The genetic cassette (100) as claimed in claim 1, wherein the second gene (120) encoding for CD70 is encoded by SEQ ID No. 10.
8. The genetic cassette (100) as claimed in claim 1, wherein the second gene (120) encoding for CD71 is encoded by SEQ ID No. 11.
9. The genetic cassette (100) as claimed in claim 1, wherein the second gene (120) encoding for CD80 is encoded by SEQ ID No. 12.
10. The genetic cassette (100) as claimed in claim 1, wherein the second gene (120) encoding for CD90 is encoded by SEQ ID No. 13.
11. The genetic cassette (100) as claimed in claim 1, wherein the second gene (120) encoding for CD117 is encoded by SEQ ID No. 14.
12. The genetic cassette (100) as claimed in claim 1, wherein the second gene (120) encoding for CD252 is encoded by SEQ ID No. 15.
13. The genetic cassette (100) as claimed in claim 1, wherein the second gene (120) encoding for CD275 is encoded by SEQ ID No. 16.
14. The genetic cassette (100) as claimed in claim 1, wherein the one or more genes of the genetic cassette (100) includes at least one third gene (130) including:
a. a envelop component (VSV-G) being encoded by SEQ ID No. 17,
b. a first packaging plasmid component (Gag-pol) encoded by SEQ ID No. 18, and
c. a second packaging plasmid component (Rev) encoded by SEQ ID No. 19.
15. A modified plasmid comprising:
a. a vector; and
b. a genetic cassette (100) as claimed in any of the preceding claims 1-14 ligated to the vector.
16. The modified plasmid as claimed in claim 15, wherein the vector is at least one of pLYS1-FLAG-MitoGFP-HA plasmid, and GLV2-CMV-[ORF]-PGK-Puro-P2A-EGFP.
17. A producer cell (300) comprising a modified plasmid as claimed in any of the preceding claims 15 and 16.
18. The producer cell (300) as claimed in claim 17, wherein the producer cell (300) is a human embryonic kidney 293T (HEK293T) cell.
19. The producer cell (300) as claimed in claim 17, wherein the producer cell (300) includes an outer membrane displaying at least one of the single chain variable fragment (scFv) (310), and the ligand.
20. A lentiviral particle (400) produced by a producer cell (300) as claimed in any of the preceding claims 17-19, the lentiviral particle (400) encapsulated within a portion of an outer membrane of the producer cell (300).
21. The lentiviral particle (400) as claimed in claim 20, wherein the lentiviral particle (400) is configured to transduce at least one of a T cell (500) and a hematopoietic stem cell (HSC) (600).