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:
CRISPR-MEDIATED GENE EDITING TO INDUCE FETAL HEMOGLOBIN

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

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 CRISPR mediated gene editing system. More particularly, the present disclosure relates to a gene editing system for fetal hemoglobin induction.
BACKGROUND OF INVENTION
[002] A blood monogenic disorder specifically refers to genetic disorders that affect the blood and are caused by mutations in genes involved in the production or function of blood cells. Majorly, the blood monogenic disorder includes sickle cell anemia (SCD) and beta-thalassemia.
[003] SCD is a monogenic condition that is prevalent and affects millions of people. Over 100,000 persons in the US and roughly 42,000 in Europe are thought to be impacted. Sickle cell anemia, the most severe and common form of sickle cell disease (SCD), is an autosomal recessive condition caused by homozygous mutations in which the glutamic acid at position 6 in ß-globin is replaced with valine, leading in deoxyhemoglobin polymerization and sickling of red blood cells (RBCs).
[004] Another prevalent clinical hereditary hemolytic anemia is beta-thalassemia, also known by its abbreviation, ß thalassemia. An estimated 80 million to 90 million people worldwide carry the ß-thalassemia gene, and every year, at least tens of thousands of babies are born with severe ß-thalassemia, posing a threat to global public health.
[005] Conventionally, available treatments for the blood monogenic disorders include periodic blood transfusions, medications (such as administration of Hydroxyurea along with iron chelation therapy), and bone marrow transplantation.
[006] However, there is no permanent cure for these blood monogenic disorders. Treatment using Hydroxyurea medication coupled with regular blood transfusions is only a way to manage the disease and not a permanent cure. Although Bone marrow transplantation offers a possible cure but a human leukocyte antigen (HLA) matched donor availability is extremely limiting.
[007] Hence, there arises a need to develop an alternative that overcomes the drawbacks of the conventionally available treatments.
SUMMARY
[008] 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.
[009] In an embodiment, the present disclosure relates to a gene editing system to induce expression of fetal hemoglobin (HbF) in hematopoietic stem cells (HSCs). The gene editing system includes a guide RNA (gRNA) extending between a 5’end and a 3’ end, and an endonuclease. The gRNA includes at least one Crispr RNA (crRNA) disposed towards the 5’ end of the gRNA and a trans-activating Crispr RNA (tracrRNA) disposed towards the 3’ end of the gRNA. The crRNA is encoded by at least one of SEQ ID NO. 1 – 6. The crRNA is configured to bind a target strand at a pre-defined locus of a genomic DNA (gDNA). The endonuclease is coupled to the gRNA via the tracrRNA. The endonuclease is configured to introduce a double-strand break (DSB) in the gDNA.
[0010] In another embodiment, the present disclosure relates to a hematopoietic stem cell (HSC) having a genomic DNA (gDNA) with a BCL11A gene. The HSC includes at least one of indel mutations, insertions, deletions, and point mutations in Exon 4 (Exons and introns are mapped as per NM_022893.4 and NP_075044.2) of the BCL11a gene (Nucleotide ID: ENST00000642384.1) present on the chromosome 2 of a gDNA introduced by a gene editing system as described above.
[0011] In yet another embodiment, the present disclosure relates to a composition including at least a population of the CD34+ hematopoietic stem cells (CD34+ HSCs) having their respective genomic DNA (gDNA) modified using the gene editing system as described above. The modification is at least one of indel mutations, insertions, deletions, and point mutations in Exon 4 (Exons and introns are mapped as per NM_022893.4 and NP_075044.2) of the BCL11a gene (Nucleotide ID: ENST00000642384.1) present on the chromosome 2 of the gDNA.
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 a gene editing system 100 according to an embodiment of the present disclosure.
[0014] Figs. 2 - 9 depict experimental observations in accordance with one or more exemplary embodiments of the present disclosure.
DETAILED DESCRIPTION OF THE ACCOMPANYING DRAWINGS
[0015] 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.
[0016] 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.
[0017] 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.
[0018] 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.
[0019] The present disclosure discloses a gene editing system (or system) to induce expression of high levels of fetal hemoglobin (HbF) in hematopoietic stem cells (HSCs). The HSCs are origin/immature cells that differentiate/develop into different types of blood cells or the like. The HSCs may either be allogenic or autologous. In an exemplary embodiment, the HSCs include autologous HSCs having cluster of differentiation 34 cell surface antigens, i.e., CD34+ HSCs. The ability of the system of the present disclosure to modify autologous HSCs provides accessibility to a larger cohort of patients suffering from sickle cell disease and possibly also transfusion dependent Beta thalassemia as a potential therapeutic alternative. Further, the system allows nonviral nucleofection of the HSCs to alter the genomic Deoxyribonucleic acid (gDNA) of the HSCs using techniques, for example, electroporation.
[0020] The system of the present disclosure creates at least one mutation in the gDNA of the HSCs such as indel mutations, insertions, deletions, point mutations, etc. The mutation(s) introduced in the gDNA of the HSCs induces the production of HbF. The HbF, in adults, compensates for the abnormal/diseased hemoglobin thereby alleviating symptoms of sickle cell disease, ß-thalassemia, or the like.
[0021] Although the gene editing system of the present disclosure is described with the examples of CD34+ HSCs, other precursor cells capable of differentiating into blood cells are within the scope of the teachings of the present disclosure.
[0022] The system of the present disclosure is directed to a pre-determined locus present on one of the chromosomes of human gDNA. In an exemplary embodiment, the system is directed to at least a portion of the BCL11a gene located on chromosome two and adjacent regions.
[0023] The BCL11a is a transcriptional repressor of gamma globin genes, i.e., the BCL11a repress production of HbF in adults. Thus, disruption of the BCL11a gene to repress BCL11a induces production of the HbF in adults thereby alleviating symptoms of sickle cell disease, ß-thalassemia, or the like.
[0024] Now referring to the figures, Fig. 1 depicts an exemplary embodiment of a system 100 of the present disclosure. In an exemplary embodiment, the system 100 is a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) based ribonucleoprotein (RNP) complex. The system 100 includes a guide ribonucleic acid (gRNA) 110 and an endonuclease 120. The gRNA 110 is a polynucleotide sequence including at least one Crispr RNA (crRNA) 110a and a trans-activating Crispr RNA (tracrRNA) 110b. The gRNA 110 has a 5’ end and a 3’end, and extends therebetween (as shown in Fig. 1).
[0025] The crRNA 110a is disposed towards the 5’ end of the gRNA 110. The crRNA 110a has a pre-defined length ranging from 17 nucleotides to 20 nucleotides. The crRNA 110a forms base-pair (or binds) with a pre-defined locus of a genomic deoxyribonucleic acid (gDNA) 10 of, for example, a hematopoietic stem cell(s) (or CD34+ HSCs). Fig. 1 depicts only one of the two strands of the gDNA 10, i.e., a target strand. The complementary strand of the target strand of the gDNA 10 is not depicted in the figures. At least a portion of the target strand of the gDNA 10 binds/interacts and forms base pair with at least a portion of the crRNA 110a (and the gRNA 110). In an exemplary embodiment, the crRNA 110a forms base-pair with a portion of the BCL11a gene (Nucleotide ID: ENST00000642384.1) on chromosome two or adjacent regions thereof of the gDNA 10. In an exemplary embodiment, the crRNA 110a base pairs with a portion of the Exon four (Exons and introns are mapped as per NM_022893.4 and NP_075044.2) of the BCL11a gene on chromosome two of the gDNA 10. The crRNA 110a is encoded by at least one of SEQ ID No. 1-6.
[0026] In an embodiment, the crRNA 110a encoded by SEQ ID No. 1 binds with at least a portion of a negative strand of chromosome 2 from 60462304 to 60462323, which is part of Exon four of the BCL11a gene of the gDNA 10.
[0027] In an embodiment, the crRNA 110a encoded by SEQ ID No. 2 binds with at least a portion of a positive strand of chromosome 2 from 60462265 to 60462284, which is part of Exon four of the BCL11a gene of the gDNA 10.
[0028] In an embodiment, the crRNA 110a encoded by SEQ ID No. 3 binds with at least a portion of a positive strand of chromosome 2 from 60462273 to 60462292, which is part of Exon four of the BCL11a gene of the gDNA 10.
[0029] In an embodiment, the crRNA 110a encoded by SEQ ID No. 4 binds with at least a portion of a positive strand of chromosome 2 from 60462267 to 60462286, which is part of Exon four of the BCL11a gene of the gDNA 10.
[0030] In an embodiment, the crRNA 110a encoded by SEQ ID No. 5 binds with at least a portion of a negative strand of chromosome 2 from 60460720 to 60460742, which is part of Exon four of the BCL11a gene of the gDNA 10.
[0031] In an embodiment, the crRNA 110a encoded by SEQ ID No. 6 binds with at least a portion of a negative strand of chromosome 2 from 60460669 to 60460691, which is part of Exon four of the BCL11a gene of the gDNA 10.
[0032] The tracrRNA 110b is disposed towards the 3’end of the gRNA 110. The tracrRNA 110b forms a binding scaffold for coupling the crRNA 110a (and the gRNA 110) to the endonuclease 120. In other words, the endonuclease 120 is coupled to the crRNA 110a (and the gRNA 110) via the tracrRNA 110b. In an exemplary embodiment, the tracrRNA 110b is encoded by SEQ ID No. 7.
[0033] The gRNA 110 is a polynucleotide, i.e., the gRNA 110 includes a plurality of nucleotides. One or more of the nucleotides of the gRNA 110 may have one or more chemical modifications selected from at least one of 2'-O-methyl (OMe) analogs and 3' phosphorothioate inter-nucleotide (PS) linkages. In an exemplary embodiment, from the 5’ end to the 3’ end, the first and last three nucleotides of the gRNA 110 are modified with OMe analogs. In another exemplary embodiment, from the 5’ end to the 3’ end, the first three and last two nucleotides of the gRNA 110 are provided with PS linkages.
[0034] In yet another exemplary embodiment, the gRNA 110 includes six nucleotides modified with OMe analogs and five nucleotides modified with PS linkages. The six nucleotides modified with the OMe analogs include, from the 5’ end to the 3’ end, the first and last three nucleotides of the gRNA 110. And, the five nucleotides modified with the PS linkages include, from the 5’ end to the 3’ end, the first three and last two nucleotides of the gRNA 110. Chemical modifications of the one or more nucleotides of the gRNA 110 provide stability and protection from exonucleases thereby increasing the half-life of the gRNA 110 in vivo. The chemical modifications also improve editing efficiency of the system 100, reduces risk of innate immune response and reduces off-target activity.
[0035] The endonuclease 120 of the system 100 is a protein/enzyme that behaves as a non-specific endonuclease. In an exemplary embodiment, the endonuclease 120 includes a CRISPR-associated protein 9 (Cas9) (Protein sequence ID No. NP_269215.1) from Streptococcus pyogenes. In another exemplary embodiment, the endonuclease 120 is high-fidelity Cas9 (HiFi Cas9). The HiFi Cas9 includes the Cas9 protein with a single point mutation, i.e., p.R691A. The tracrRNA 110b includes at least one complementary "direct repeat" sequence region that binds with the endonuclease 120, thereby coupling the endonuclease 120 to the gRNA 110.
[0036] The endonuclease 120 includes at least two nuclease domains for cleaving each of the two strands of the gDNA 10 and introduce at least one double strand break (DSB) in the gDNA 10. The specificity of the nuclease activity of the endonuclease 120 is governed by the binding of the crRNA 110a with at least a portion of the gDNA 10. In an exemplary embodiment, the endonuclease 120 includes a HNH domain to cleave the target strand of the gDNA 10 and a RuvC domain to cleave the complementary strand of the gDNA 10. Depending on the endonuclease 120, the cleavage of the gDNA 10 by the endonuclease 120 is either close to or at a portion of the target strand where the gRNA 110 base pairs (or binds) with the gDNA 10. In an exemplary embodiment, the Cas9 endonuclease 120 cleaves the gDNA 10 at a portion of the target strand where the gRNA 110 base pairs (or binds) with the gDNA 10.
[0037] Although the system 100 of the present disclosure is described with the examples of Cas9 endonuclease 120, other functionally equivalent endonucleases are within the scope of the teachings of the present disclosure.
[0038] In an exemplary embodiment, the system 100 of the present disclosure is introduced within a population of autologous CD34+ HSCs via ex vivo electroporation. The crRNA 110a of the gRNA 110 binds (or forms base pairs) with a portion of the Exon 4 of the BCL11a gene present on the chromosome 2 of the gDNA 10. The endonuclease 120 cleaves the two strands of the gDNA 10 to introduce one DSB. The CD34+ HSCs may repair the DSB of the gDNA 10 via at least one of a non-homologous end joining technique or homology directed repair technique. The repair of the DSB introduces a mutation (one of indel, insertion, deletion) in the BCL11a gene of the gDNA 10 thereby repressing the expression of the BCL11a gene. Repression of the BCL11a gene at least partially removes regulation of expression of the gamma globin genes thereby inducing expression of the gamma globin genes and producing high levels of HbF. The HbF may reduce the symptoms of abnormal hemoglobin.
[0039] Although the present disclosure describes the system 100 being introduced via electroporation, other functionally equivalent technique to introduce the system 100 inside the HSCs are within the scope of the teachings of the present disclosure.
[0040] A hematopoietic stem cell (CD34+ HSC), having the gDNA 10 with a BCL11A gene, includes at least one of indel mutations, insertions, deletions, and point mutations in Exon 4 (Exons and introns are mapped as per NM_022893.4 and NP_075044.2) of the BCL11a gene (Nucleotide ID: ENST00000642384.1) present on the chromosome 2 of the gDNA 10 introduced by the system 100.
[0041] In an exemplary embodiment, a composition including a population of the CD34+ HSCs having their gDNA 10 modified using the system 100 is administered to a subject having abnormal hemoglobin.
[0042] The ability of the gene editing system 100 to modify the autologous HSCs provides accessibility to a larger cohort of patients of sickle cell disease and possibly also transfusion-dependent Beta thalassemia as a potential therapeutic alternative.
[0043] The present disclosure will now be explained with the help of the following examples:
[0044] Example 1: Isolation of peripheral blood mononuclear cells (PBMCs)
[0045] A whole blood sample was drawn from an individual suffering from sickle cell disease (SCD). The whole blood sample was diluted with equal volume of phosphate buffer saline (PBS) to obtain a whole blood mixture. The whole blood mixture was carefully layered on a Ficoll-Paque separation medium in a conical tube. The conical tube was centrifuged at 600 x g for 45 minutes at room temperature with no brake. The tube was recovered from the centrifuge. The tube had three layers, a plasma layer, a Ficoll layer and a PBMC layer disposed therebetween. The PBMC layer was separated and collected into a fresh conical tube. The PBMC layer was diluted with PBS in a ratio of 1:2 and the conical tube was centrifuged at 300 x g for 10 minutes at room temperature to wash the PBMCs. Thereafter, the PBMCs were suspended at 107 cells/mL in MACS buffer to obtain a cell suspension. The composition of the MACS buffer was PBS, 0.5% bovine serum albumin (BSA), and 2mM Ethylenediaminetetraacetic acid (EDTA).
[0046] Example2: CD34+ cell enrichment
[0047] 20 µL of CD34 MicroBeads (procured from Miltenyi) was added to the cell suspension obtained from Example 1 above for every 107 cells. In other words, for every mL of the cell suspension, 20 µL of CD34 MicroBeads was added to obtain a mixture. The mixture was incubated at 4°C for 15-30 minutes with occasional gentle mixing. Equal volume of MACS buffer was added to the mixture and the conical tube was centrifuged at 300 x g for 10 minutes at 4°C to wash the PBMCs. Thereafter, the PBMCs were resuspended at 107 cells/mL in MACS buffer.
[0048] A MACS column (procured from Miltenyi) was placed in a magnetic field of a MACS separator (procured from Miltenyi). The cell suspension was loaded onto the MACS column. The magnetic field arrested the movement of the MACS microbead bound to CD34+ cells while allowing the other cell types to flow through the MACS column. The MACS column was then washed using 300 µL to 500 µL of MACS buffer to remove any unbound cells. Thereafter, the MACS column was removed from the magnetic field and the flow through/eluent having the CD34+ cells were collected. About 1 mL to 5 mL of MACS buffer was added to the collected eluent in a conical tube and centrifuged at 300 x g for 10 minutes at 4°C to wash the CD34+ cells. Thereafter, the CD34+ cells were resuspended at 107 cells/mL in a HSPC culture medium (procured from Stem cell technologies) to obtain a CD34+ cell culture.
[0049] A few of the CD34+ cells were separated and labelled with anti-human mice CD34-PE conjugated antibodies (procured from BD Biosciences) and analyzed using flow cytometry (BD Accuri C6 Plus procured from BD Biosciences) and microscopy (CKX53SF procured from Olympus) to assess the purity and viability of the CD34+ cell population in the CD34+ cell culture. The flow cytometry data is depicted in Fig. 2. Figs. 3 and 3a depicts the CD34+ cells under microscopy at 10x and 20x magnification respectively. Both the flow cytometry data as well as the microscopy images confirmed that the CD34+ cell culture has a rich population of more than 99% CD34+ cells.
[0050] Example 3: Nucleofection
[0051] The system 100 of the present disclosure was introduced within the CD34+ cells obtained from Example 2 above via ex vivo electroporation (by following the DZ100 method in a Lonza 4D-Nucleofector apparatus). Thereafter, the CD34+ cells were cultured at 37°C, under 5% CO2 and analyzed after periodic intervals of incubation.
[0052] Fig. 4 depicts the CD34+ cells, 18 hours after nucleofection. Fig. 4a depicts the CD34+ cells, 10 days after nucleofection/differentiation. Group WT represents a wild-type CD34+ cell population that were not being modified with the system 100 of the present disclosure. Group A represents the population of CD34+ cells being modified with the system 100 having crRNA 110a encoded by SEQ ID No. 1. Group B represents the population of CD34+ cells being modified with the system 100 having crRNA 110a encoded by SEQ ID No. 2. Group C represents the population of CD34+ cells being modified with the system 100 having crRNA 110a encoded by SEQ ID No. 3. Group D represents the population of CD34+ cells being modified with the system 100 having crRNA 110a encoded by SEQ ID No. 4.
[0053] The total number of differentiated cells in all the groups were periodically counted. The cell count data is depicted in the following table 1:
Group Day 0 Day 2 Day 4 Day 6 Day 8 Day 10
Group WT 8.53 x 105 2.25 x 106 3.44 x 106 3.31 x 106 1.21 x 106 2.42 x 106
Group A 7.48 x 105 1.93x 106 2.14 x 106 2.52 x 106 1.84 x 106 2.22 x 106
Group B 4.71 x 105 5.02 x 105 NA NA NA NA
Group C 7.80 x 105 2.32 x 106 2.59 x 106 2.66 x 106 1.61x106 1.97 x 106
Group D 5.96 x 105 2.19 x 106 1.91 x 106 1.94 x 106 1.11 x 106 1.11 x 106
(Table 1)
[0054] Example 4: Genomic DNA (gDNA) 10 isolation
[0055] The gDNA 10 was isolated using DNeasy Blood and Tissue kit (procured from Qiagen, #69506) from all the groups of CD34+ cells obtained in Example 3 above. The DNeasy Blood and Tissue kit includes a lysis buffer (buffer AL), a DNeasy mini spin column, wash buffer 1 (buffer AW1), wash buffer 2 (buffer AW2), and elution buffer (buffer AE). 5 x 106 cells from Group A were separately centrifuged at 300 x g and resuspended in 200 µL of PBS and 20 µL of proteinase K in a micro-centrifuge tube. 200 µL of the lysis buffer (buffer AL) was added to the micro-centrifuge tube and mixed thoroughly by vortexing the micro-centrifuge tube. 200 µL of 96-100% ethanol was added to the micro-centrifuge tube and mixed thoroughly by vortexing the micro-centrifuge tube. The contents of the micro-centrifuge tube were loaded onto the DNeasy mini spin column placed in a 2 mL collection tube. The DNeasy mini spin column was centrifuged at more than 6,000 x g for 1 min. The eluent/flow-through and the collection tube was discarded. The DNeasy mini spin column was then placed in a new 2mL collection tube. 500 µL of the wash buffer 1 (buffer AW1) was added to the DNeasy mini spin column and centrifuged at more than 6,000 x g for 1 min. The eluent/flow-through and the collection tube was discarded. The DNeasy mini spin column was then placed in a new 2mL collection tube. 500 µL of the wash buffer 2 (buffer AW2) was added to the DNeasy mini spin column and centrifuged at more than 20,000 x g for 3 min. The eluent/flow-through and the collection tube was discarded. The DNeasy mini spin column was then placed in a new micro-centrifuge tube. 200 µL of the elution buffer (buffer AE) was added to the center of the DNeasy mini spin column and incubated for 1 min at room temperature. Thereafter, the DNeasy mini spin column along with the micro-centrifuge tube was centrifuged at more than 6,000 x g for 1 min. The DNeasy mini spin column was discarded and the micro-centrifuge having the gDNA 10 was refrigerated for storage. The above steps were repeated for Group B, Group C and Group D.
[0056] The gDNA 10 of all the groups were loaded in the wells of an agarose gel along with a 1kb standard DNA ladder (procured from New England Biolabs). The pMAX GFP was loaded in one of the wells of the agarose gel as a positive control. The agarose gel was run and the then viewed under UV light as shown in Fig. 5.
[0057] Example 5: T7 endonuclease assay
[0058] The gDNA 10 isolated in Example 4 above was subjected to PCR amplification to amplify a portion of Exon 4 of the BCL11A gene. The primers used in the amplification reaction includes a forward primer encoded by SEQ ID No. 8 and a reverse primer encoded by SEQ ID No. 9.
[0059] A PCR reaction mixture was separately made for the gDNA 10 isolated from each of the groups (Group WT, Group A, Group C, Group D). The reaction mixture had 100 ng of gDNA 10, 10 µM forward primer, 10 µM reverse primer, 200 nM dNTPs, and 2 U of Taq polymerase (procured from New England Biolabs). The thermal cycler (T100 procured from BioRad) was set up for 30 cycles of denaturation at 98°C for 10 secs, annealing at 53°C for 30 secs, extension at 72°C for 25 secs to obtain the amplicons.
[0060] In addition to the groups described above, a non-template control (Group NTC) was also amplified separately as a negative control. The Group NTC had genetic material without any binding site for the primers.
[0061] The resultant amplicons were loaded on an agarose gel along with a 100bp standard DNA ladder (procured from New England Biolabs). The agarose gel was run and then viewed under UV light as shown in Fig. 6. It was evident from the agarose gel depicted in Fig. 6 that the size of the amplicon was approximately 840bp. And, the Group NTC did not provide any amplicon as intended.
[0062] The amplicons obtained above from the Group WT was separately added to the amplicons obtained from the Groups A, C, and D in a ratio of 1:1 by weight of the respective amplicons to obtain respective amplicon mixtures. The respective amplicon mixtures of Groups, A, C, D and the amplicons of the Group WT were kept inside the thermal cycler, and the thermal cycler was programed for 1 cycle of denaturation at 95°C for 5 mins, and then gradually cooled to 25°C - 37°C to obtain a heteroduplex DNA (hDNA) of the respective groups. The hDNA forms only if there were any mis-match in base pair formation between the amplicons of the Groups A, C, D and the amplicons of Group WT due to in/del mutation(s) in the amplicons.
[0063] A T7 endonuclease mixture was prepared by adding 10 U of T7 endonuclease I (procured from New England Biolabs, #M0302L) in 2 µl of 1X reaction buffer (NEBBuffer™ 2 procured from New England Biolabs). 200ng of the hDNA obtained above for respective groups were separately added to the T7 endonuclease mixture and incubated at 37°C for 15-30 minutes. During incubation, the T7 endonuclease I enzyme cleaves all the mis-matched base-pairs in the hDNA. The activity of the T7 endonuclease I was stopped by heating the T7 endonuclease mixture at 80°C for 5 minutes. The hDNA treated with the T7 endonuclease I enzyme was loaded on an agarose gel along with a 1kb standard DNA ladder (procured from New England Biolabs). The agarose gel was run and then viewed under UV light as shown in Fig. 6a. The (U) notation in Fig. 6a depicts the undigested hDNA and the (D) notation in Fig. 6a depicts the digested hDNA using the T7 endonuclease I. It was evident from the Fig. 6a that the T7 endonuclease I has created two fragments in the hDNA of Group A, Group C, and Group D. The hDNA of Group A was broken into a fragment of 566bp and a fragment of 274bp. No fragmentation was observed for Group C. The hDNA of Group D was broken into a fragment of 554bp and a fragment of 286bp. Thus, Figs. 6 and 6a confirmed that the system 100 of the present disclosure successfully disrupted the Exon 4 of the BCL11A gene in Group A and Group D thereby removing the regulatory check from the expression of the HbF gene.
[0064] Example 6: Erythroid differentiation
[0065] CD235a and CD71 are one of the cell biomarkers expressed by the erythroid cells (i.e., red blood cells). To confirm if the HSCs differentiated into erythroid, the cells from Group D was harvested on Day 13 after nucleofection and labeled using CD235a antibodies (procured from BD Biosciences) and the CD71 antibodies (procured from BD Biosciences). The labelled cells were analyzed using flow cytometry (BD Accuri C6 Plus procured from BD Biosciences). The flow cytometry data for CD235a and CD71 is depicted in Fig. 7 and Fig. 8 respectively. It was evident from Fig. 7 that more than 99% cells from Group D expressed CD235a. And, it was evident from Fig. 8 that more than 51.2% cells from Group D expressed CD71.
[0066] Example 7: HbF expression
[0067] The HSCs from Group D were harvested on day 8 after nucleofection. The HbF, if any, produced by the cells were labelled using HbF antibody (procured from Thermo Fisher Scientific). The labelled cells were analyzed using flow cytometry (BD Accuri C6 Plus procured from BD Biosciences). The flow cytometry data for HbF is depicted in Fig. 9. It was evident from Fig. 9 that more than 9% cells from Group D produced HbF.
[0068] 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 gene editing system (100) to induce expression of fetal hemoglobin (HbF) in hematopoietic stem cells (HSCs), the gene editing system (100) comprising:
a. a guide RNA (gRNA 110) extending between a 5’end and a 3’ end, the gRNA (110) including at least one Crispr RNA (crRNA 110a) disposed towards the 5’ end of the gRNA (110) and a trans-activating Crispr RNA (tracrRNA 110b) disposed towards the 3’ end of the gRNA (110), the crRNA (110a) is encoded by at least one of SEQ ID NO. 1 – 6, the crRNA (110a) is configured to bind a target strand at a pre-defined locus of a genomic DNA (gDNA 10); and
b. an endonuclease (120) coupled to the gRNA (110) via the tracrRNA (110b), the endonuclease (120) configured to introduce a double-strand break (DSB) in the gDNA (10).
2. The gene editing system (100) as claimed in claim 1, wherein the crRNA (110a) is configured to base pair with a portion of the Exon four (Exons and introns are mapped as per NM_022893.4 and NP_075044.2) of the BCL11a gene (Nucleotide ID: ENST00000642384.1) on chromosome two or adjacent regions thereof of the gDNA (10).
3. The gene editing system (100) as claimed in claim 1, wherein the crRNA (110a) encoded by SEQ ID NO. 1 binds with at least a portion of a negative strand of chromosome 2 from 60462304 to 60462323 of the gDNA (10).
4. The gene editing system (100) as claimed in claim 1, wherein the crRNA (110a) encoded by SEQ ID NO. 2 binds with at least a positive strand of chromosome 2 from 60462265 to 60462284 of the gDNA (10).
5. The gene editing system (100) as claimed in claim 1, wherein the crRNA (110a) encoded by SEQ ID NO. 3 binds with at least a portion of a positive strand of chromosome 2 from 60462273 to 60462292 of the gDNA (10).
6. The gene editing system (100) as claimed in claim 1, wherein the crRNA (110a) encoded by SEQ ID NO. 4 binds with at least a positive strand of chromosome 2 from 60462267 to 60462286 of the gDNA (10).
7. The gene editing system (100) as claimed in claim 1, wherein the crRNA (110a) encoded by SEQ ID NO. 5 binds with at least a portion of a negative strand of chromosome 2 from 60460720 to 60460742 of the gDNA (10).
8. The gene editing system (100) as claimed in claim 1, wherein the crRNA (110a) encoded by SEQ ID NO. 6 binds with at least a portion of a negative strand of chromosome 2 from 60460669 to 60460691 of the gDNA (10).
9. The gene editing system (100) as claimed in claim 1, wherein the gRNA (110) includes one or more nucleotides having one or more chemical modifications selected from at least one of 2'-O-methyl (OMe) analogs and 3' phosphorothioate (PS) inter-nucleotide linkages.
10. The gene editing system (100) as claimed in claim 9, wherein the first and last three nucleotides of the gRNA (110) are modified with OMe analogs.
11. The gene editing system (100) as claimed in claim 9, wherein the first three and last two nucleotides of the gRNA (110) are provided with PS linkages.
12. The gene editing system (100) as claimed in claim 1, wherein the endonuclease (120) is at least one of CRISPR-associated protein 9 (Cas9) from Streptococcus pyogenes, and high-fidelity Cas9 (HiFi Cas9).
13. The gene editing system (100) as claimed in claim 1, wherein the endonuclease (120) includes a HNH domain to cleave the target strand of the gDNA (10) and a RuvC domain to cleave a complementary strand of the gDNA (10).
14. The gene editing system (100) as claimed in claim 1, wherein the tracrRNA (110b) is encoded by SEQ ID No. 7.
15. A hematopoietic stem cell (HSC) having a genomic DNA (gDNA 10) with a BCL11A gene, the HSC comprising:
a. at least one of indel mutations, insertions, deletions, and point mutations in Exon 4 (Exons and introns are mapped as per NM_022893.4 and NP_075044.2) of the BCL11a gene (Nucleotide ID: ENST00000642384.1) present on the chromosome 2 of a gDNA (10) introduced by a gene editing system (100) as claimed in claim 1.
16. A composition comprising:
a. at least a population of the CD34+ hematopoietic stem cells (CD34+ HSCs) having their respective genomic DNA gDNA (10) modified using the gene editing system (100) as claimed in claim 1, the modification is at least one of indel mutations, insertions, deletions, and point mutations in Exon 4 (Exons and introns are mapped as per NM_022893.4 and NP_075044.2) of the BCL11a gene (Nucleotide ID: ENST00000642384.1) present on the chromosome 2 of the gDNA (10).