Engineered Cells for Increased Collagen Production
20240043818 ยท 2024-02-08
Inventors
- Tim John MASTOVICH (Boston, MA, US)
- Michael FITZGERALD (Santa Clara, CA, US)
- Jessie Lyn GIFFORD (Boston, MA, US)
- Andrew C. HOROWITZ (Mansfield, MA, US)
- Megan Jayne POWELL (Malden, MA, US)
- Jeffrey William RUBERTI (Lexington, MA, US)
Cpc classification
A61L2430/40
HUMAN NECESSITIES
C12N2310/20
CHEMISTRY; METALLURGY
C07K14/78
CHEMISTRY; METALLURGY
C12N9/22
CHEMISTRY; METALLURGY
A61K8/65
HUMAN NECESSITIES
A61L2430/16
HUMAN NECESSITIES
C12N2800/80
CHEMISTRY; METALLURGY
C12N15/11
CHEMISTRY; METALLURGY
C12N15/113
CHEMISTRY; METALLURGY
A61L2430/02
HUMAN NECESSITIES
A61K38/39
HUMAN NECESSITIES
C12N15/1136
CHEMISTRY; METALLURGY
C12P21/02
CHEMISTRY; METALLURGY
International classification
C12N9/22
CHEMISTRY; METALLURGY
C12N15/11
CHEMISTRY; METALLURGY
C12P21/02
CHEMISTRY; METALLURGY
A61K38/39
HUMAN NECESSITIES
A61K8/65
HUMAN NECESSITIES
Abstract
Cells for increased collagen production are engineered by a novel CRISPR cellular engineering process. The process can be carried out using human cells or even patient-harvested cells. Collagen produced by the cells has a low risk of immunogenicity when implanted into human patients compared to collagen produced by non-human cells. Cell culture media including chemical additives are also provided to have a further positive effect on collagen production.
Claims
1. An engineered cell capable of enhanced collagen biosynthesis, wherein the cell has been engineered to perform CRISPR-based activation (CRISPRa) of a targeted gene related to collagen biosynthesis by the cell, wherein the cell expresses an endonuclease deficient Cas9 (dCas9) protein fused to a transcriptional activator protein (dCas9-activator) and a guide RNA (gRNA) specific for the targeted gene, wherein the engineered cell is capable of at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, at least 200-fold, or at least 300-fold higher collagen biosynthesis compared to a non-engineered cell of the same type.
2. The engineered cell of claim 1, wherein the targeted gene is selected from the group consisting of COL1A1, COL1A2, TGF-1, TGF-2, TGF-3, COL2A1, COL3A1, COL4A1, COL4A2, COL4A3, COL4A4, COL4A5, COL4A6, COL5A1, COL5A2, COL5A3, ADAMTS2, ADAMTS3, ADAMTS4, ADAMTS5, ADAMTS6, ADAMTS7, ADAMTS8, ADAMTS9, ADAMTS10, ADAMTS12, ADAMTS13, ADAMTS14, ADAMTS15, ADAMTS16, ADAMTS17, ADAMTS18, ADAMTS19, ADAMTS20, TLL1, TLL2, and BMP1.
3. The engineered cell of claim 2, wherein the gRNA expressed by the cell and specific for said targeted gene comprises the nucleotide sequence of any of SEQ ID NOS:1-156.
4. The engineered cell claim 1, wherein the cell has been engineered to perform CRISPRa of one or more further targeted genes selected from the group consisting of prolyl-3-hydroxylase family genes, prolyl-4-hydroxylase family genes, lysyl hydroxylase family genes, GLT25D1, GLT25D2, Grp78, Grp94, protein disulfide isomerase (PDI) family genes, calreticulin, calnexin, CypB, PPlase family genes, cyclophilins, FK506 binding protein (FKBP) genes, cyclophilin B (CypB), HSP47, TANG01, SEC13, SEC31, and Sedlin.
5. The engineered cell of claim 1 that expresses gRNAs specific for two or more of said targeted genes, and each of the two or more targeted genes is activated.
6. The engineered cell of claim 5, wherein one or more collagen genes and one or more TGF genes are targeted; wherein the one or more collagen genes are selected from the group consisting of COL1A1, COL1A2, COL2A1, COL3A1, COL4A1, COL4A2, COL4A3, COL4A4, COL4A5, COL4A6, COL5A1, COL5A2, and COL5A3; wherein the one or more TGF- genes are selected from the group consisting of TGF-1, TGF-2, and TGF-3; and wherein the cell expresses gRNAs specific for said one or more collagen genes and said one or more TGF- genes.
7. The engineered cell of claim 6, wherein the collagen genes are selected from COL1A1 and COL1A2 and the TGF- genes are selected from TGF-1 and TGF-3, wherein the COL1A1 gRNA comprises the nucleotide sequence of any of SEQ ID NOS:1-4 and the COL1A2 gRNA comprises the nucleotide sequence of any of SEQ ID NOS:5-8, and wherein the TGF-1 gRNA comprises the nucleotide sequence of any of SEQ ID NOS:1 3-16, and the TGF-3 gRNA comprises the nucleotide sequence of any of SEQ ID NOS:9-12.
8. The engineered cell of claim 6, wherein one or more propeptidase genes are further targeted, wherein the propeptidase genes are selected from the group consisting of ADAMTS2, ADAMTS3, ADAMTS4, ADAMTS5, ADAMTS6, ADAMTS7, ADAMTS8, ADAMTS9, ADAMTS10, ADAMTS12, ADAMTS13, ADAMTS14, ADAMTS15, ADAMTS16, ADAMTS17, ADAMTS18, ADAMTS19, ADAMTS20, TLL1, TLL2, and BMP1.
9. The engineered cell of claim 8, wherein the propeptidase genes ADAMTS2 and BMP-1 are targeted, and wherein the ADAMTS2 gRNA comprises the nucleotide sequence of any of SEQ ID NOS:69-72 and the BMP-1 gRNA comprises the nucleotide sequence of any of SEQ ID NOS:153-156.
10. The engineered cell of claim 1, wherein said transcriptional activator is selected from the group consisting of VP64, 65, Rta, VPR (a combination of VP64, 65, and Rta), MS2, HSF1, SAM (a combination of MS2, 65, and HSF-1), and SunTag.
11. The engineered cell of claim 10, wherein the dCas9-activator is dCas9-VPR.
12. The engineered cell of claim 1, wherein the cell has been transfected to express said dCas9-activator and said gRNA or gRNAs.
13. The engineered cell of claim 1, wherein the cell has been transduced to express said dCas9-activator and said gRNA or gRNAs.
14. The engineered cell of claim 1, wherein the cell is derived from a cell type selected from the group consisting of fibroblasts, mesenchymal cells, myofibroblasts, osteoblasts, chondrocytes, and induced pluripotent stem cells.
15. The engineered cell of claim 14, wherein the cell is derived from a human corneal fibroblast.
16. The engineered cell of claim 1, wherein the cell is derived from a cell obtained from a mammalian subject in need of collagen administration.
17. A cell culture comprising the engineered cell of claim 1.
18. The cell culture of claim 17 that is immortalized.
19. A method for engineering a cell to provide enhanced collagen biosynthesis, the method comprising the steps of: (a) providing the cell, a first nucleic acid molecule encoding a dCas9-activator, and a second nucleic acid molecule specific for a target gene related to collagen biosynthesis; and (b) transfecting or transducing the cell with said first and second nucleic acid molecules; whereby the cell becomes capable of expressing said dCas9-activator and said gRNA, and the target gene is activated.
20. The method of claim 19, wherein the target gene is selected from the group consisting of COL1A1, COL1A2, TGF-1, TGF-2, TGF-3, COL2A1, COL3A1, COL4A1, COL4A2, COL4A3, COL4A4, COL4A5, COL4A6, COL5A1, COL5A2, COL5A3, ADAMTS2, ADAMTS3, ADAMTS4, ADAMTS5, ADAMTS6, ADAMTS7, ADAMTS8, ADAMTS9, ADAMTS10, ADAMTS12, ADAMTS13, ADAMTS14, ADAMTS15, ADAMTS16, ADAMTS17, ADAMTS18, ADAMTS19, ADAMTS20, TLL1, TLL2, and BMP1
21. The method of claim 20, wherein the gRNA comprises the nucleotide sequence of any of SEQ ID NO:1 to SEQ ID NO:156.
22. The method of claim 19, wherein the cell is engineered to perform CRISPRa of one or more further targeted genes selected from the group consisting of prolyl-3-hydroxylase family genes, prolyl-4-hydroxylase family genes, lysyl hydroxylase family genes, GLT25D1, GLT25D2, Grp78, Grp94, protein disulfide isomerase (PDI) family genes, calreticulin, calnexin, CypB, PPlase family genes, cyclophilins, FK506 binding protein (FKBP) genes, cyclophilin B (CypB), HSP47, TANG01, SEC13, SEC31, and Sedlin.
23. The method of claim 19, wherein the cell is transfected with two or more second nucleic acid molecules, each specific for a different target gene, whereby each of the target genes is activated.
24. The method of claim 23, wherein one or more collagen genes and one or more TGF genes are targeted; wherein the one or more collagen genes are selected from the group consisting of COL1A1, COL1A2, COL2A1, COL3A1, COL4A1, COL4A2, COL4A3, COL4A4, COL4A5, COL4A6, COL5A1, COL5A2, and COL5A3; wherein said TGF- genes are selected from the group consisting of TGF-1, TGF-2, and TGF-3; and wherein the cell expresses gRNAs specific for said one or more collagen genes and said one or more TGF- genes.
25. The method of claim 24, wherein the collagen genes are selected from COL1A1 and COL1A2 and the TGF- genes are selected from TGF-1 and TGF-3, wherein the COL1A1 gRNA comprises the nucleotide sequence of any of SEQ ID NOS:1-4 and the COL1A2 gRNA comprises the nucleotide sequence of any of SEQ ID NOS:5-8, and wherein the TGF-1 gRNA comprises the nucleotide sequence of any of SEQ ID NOS:13-16, and the TGF-3 gRNA comprises the nucleotide sequence of any of SEQ ID NOS:9-12.
26. The method of claim 24, wherein one or more propeptidase genes are further targeted, wherein the propeptidase genes are selected from the group consisting of ADAMTS2, ADAMTS3, ADAMTS4, ADAMTS5, ADAMTS6, ADAMTS7, ADAMTS8, ADAMTS9, ADAMTS10, ADAMTS12, ADAMTS13, ADAMTS14, ADAMTS15, ADAMTS16, ADAMTS17, ADAMTS18, ADAMTS19, ADAMTS20, TLL1, TLL2, and BMP1.
27. The method of claim 26, wherein the propeptidase genes ADAMTS2 and BMP-1 are targeted, and wherein the ADAMTS2 gRNA comprises the nucleotide sequence of any of SEQ ID NOS:69-72 and the BMP-1 gRNA comprises the nucleotide sequence of any of SEQ ID NOS:153-156.
28. The method of claim 19, wherein the dCas9-activator is dCas9-VPR.
29. The method of claim 19, wherein the cell is derived from a cell type selected from the group consisting of fibroblasts, myoblasts, osteoblasts, chondrocytes, and induced pluripotent stem cells.
30. The method of claim 19, wherein the cell is derived from a human corneal fibroblast.
31. The method of claim 19, wherein the cell is transduced using a lentiviral vector in step (b).
32. The method of claim 19, wherein step (a) includes obtaining a sample from a mammalian subject in need of collagen administration, or from a different mammalian subject of the same species, and deriving the provided cell from the sample.
33. A kit for engineering a cell to enhance biosynthesis of collagen by the cell, the kit comprising: (i) a first nucleic acid molecule encoding a dCas9-activator protein; (ii) a second nucleic acid molecule comprising or encoding a crRNA specific for a target gene related to collagen biosynthesis; and (iii) optionally one or more reagents for transfecting or transducing a cell with the first and second nucleic acid molecules.
34. The kit of claim 33, wherein the target gene is selected from the group consisting of COL1A1, COL1A2, TGF-1, TGF-2, TGF-3, COL2A1, COL3A1, COL4A1, COL4A2, COL4A3, COL4A4, COL4A5, COL4A6, COL5A1, COL5A2, COL5A3, ADAMTS2, ADAMTS3, ADAMTS4, ADAMTS5, ADAMTS6, ADAMTS7, ADAMTS8, ADAMTS9, ADAMTS10, ADAMTS12, ADAMTS13, ADAMTS14, ADAMTS15, ADAMTS16, ADAMTS17, ADAMTS18, ADAMTS19, ADAMTS20, TLL1, TLL2, and BMP1.
35. The kit of claim 34, wherein the crRNA comprises the nucleotide sequence of any of SEQ ID NOS:1-156.
36. The kit of claim 33, wherein two or more second nucleic acid molecules are provided, each comprising or encoding a crRNA specific for a different target gene.
37. The kit of claim 36, wherein the two or more second nucleic acid molecules comprise or encode crRNAs specific for one or more collagen genes and one or more TGF genes; wherein the one or more collagen genes are selected from the group consisting of COL1A1, COL1A2, COL2A1, COL3A1, COL4A1, COL4A2, COL4A3, COL4A4, COL4A5, COL4A6, COL5A1, COL5A2, and COL5A3; and wherein said TGF- genes are selected from the group consisting of TGF-1, TGF-2, and TGF-3.
38. The kit of claim 37, wherein the collagen genes are selected from COL1A1 and COL1A2 and the TGF- genes are selected from TGF-1 and TGF-3, wherein the COL1A1 gRNA comprises the nucleotide sequence of any of SEQ ID NOS:1-4 and the COL1A2 gRNA comprises the nucleotide sequence of any of SEQ ID NOS:5-8, and wherein the TGF-1 gRNA comprises the nucleotide sequence of any of SEQ ID NOS:13-16, and the TGF-3 gRNA comprises the nucleotide sequence of any of SEQ ID NOS:9-12.
39. The kit of claim 37, wherein the two or more second nucleic acids further comprise or encode crRNAs specific for one or more propeptidase genes, wherein the propeptidase genes are selected from the group consisting of ADAMTS2, ADAMTS3, ADAMTS4, ADAMTS5, ADAMTS6, ADAMTS7, ADAMTS8, ADAMTS9, ADAMTS10, ADAMTS12, ADAMTS13, ADAMTS14, ADAMTS15, ADAMTS16, ADAMTS17, ADAMTS18, ADAMTS19, ADAMTS20, TLL1, TLL2, and BMP1.
40. The kit of claim 39, wherein the two or more second nucleic acid molecules comprise or encode crRNAs specific for propeptidase genes ADAMTS2 and BMP-1, and wherein the ADAMTS2 gRNA comprises the nucleotide sequence of any of SEQ ID NOS:69-72 and the BMP-1 gRNA comprises the nucleotide sequence of any of SEQ ID NOS:153-156.
41. The kit of claim 33, wherein the second nucleic acid molecules further comprise or encode crRNAs specific for one or more genes selected from the group consisting of prolyl-3-hydroxylase family genes, prolyl-4-hydroxylase family genes, lysyl hydroxylase family genes, GLT25D1, GLT25D2, Grp78, Grp94, protein disulfide isomerase (PDI) family genes, calreticulin, calnexin, CypB, PPlase family genes, cyclophilins, FK506 binding protein (FKBP) genes, cyclophilin B (CypB), HSP47, TANG01, SEC13, SEC31, and Sedlin.
42. The kit of claim 33, wherein the dCas9-activator is dCas9-VPR.
43. A medical device comprising the engineered cell of claim 1.
44. The medical device of claim 43 that is implantable in a subject's body.
45. A method of producing collagen, the method comprising the steps of: (a) providing the cell culture of claim 17; (b) growing the cell culture in a bioreactor under conditions in which collagen is biosynthesized by the cells of the cell culture; and (c) harvesting and purifying collagen from the bioreactor.
46. The method of claim 45, wherein step (b) is performed in the presence of a modulator of collagen biosynthesis.
47. The method of claim 46, wherein the modulator is selected from the group consisting of acetaldehyde, ascorbate, hyaluronic acid, -aminopropionitrile, transforming growth factor beta (TGF-), insulin-like growth factor 1 (IGF-1), glutamine, and combinations thereof.
48. The method of claim 47, wherein the modulator is a combination of ascorbate and p-aminopropionitrile or a combination of ascorbate, acetaldehyde, and -aminopropionitrile.
49. The method of claim 47, wherein the modulator is -aminopropionitrile, and wherein crosslinking of collagen is reduced or prevented compared to absence of -aminopropionitrile.
50. The method of claim 45, wherein step (b) is performed in the presence of application of mechanical strain to the cells.
51. The method of claim 50, wherein mechanical strain is induced using cells adhered to a substrate, beads, or a scaffold.
52. The method of claim 45, further comprising, between steps (b) and (c): (b1) concentrating the biosynthesized collagen in the cell growth medium, whereby propeptide cleavage of the biosynthesized collagen is enhanced.
53. The method of claim 45, wherein the collagen produced is a type selected from the group consisting of collagen types I-V.
54. The method of claim 53, wherein the collagen is type I collagen.
55. A method of treating a mammalian subject having a medical condition characterized by insufficiency of collagen, the method comprising: (a) performing the method of claim 32 and thereby obtaining collagen produced by cells derived from the mammalian subject, or a different mammalian subject of the same species; and (b) administering the collagen to the subject.
56. The method of claim 55, wherein a medical device is used to administer the collagen.
57. The method of claim 55, wherein the medical device is selected from the group consisting of a burn/wound covering or dressing, an osteogenic and/or bone filling material, a device having an antithrombogenic surface, a device having a therapeutic enzyme immobilization surface, a collagen patch, a closure graft, an implant operative to provide collagen, a corneal implant, a bandage contact lens, a collagen-based membrane, and a collagen-based drug delivery device.
58. The method of claim 55, wherein the medical condition is selected from the group consisting of a wound, a torn ligament or tendon, a bone fracture, damaged cartilage, an eye condition, a condition requiring cosmetic treatment or surgery, a dermatological condition, skin wrinkles or scars, and a burn.
59. A method of performing a cosmetic treatment to a human subject, the method comprising: (a) performing the method of claim 32, thereby obtaining collagen produced by cells derived from the mammalian subject or other subject of the same mammalian species; and (b) administering the collagen obtained in step (a) to the subject.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION
[0117] The present technology provides novel human cells engineered to increase collagen production. The cells are produced utilizing a CRISPR activation (CRISPRa) cellular engineering process to induce rapid collagen production from a variety of cell types, including human cells. Genetic modifications are introduced into cells that naturally produce a desired type of collagen using CRISPRa to increase the expression of one or more genes responsible for the transcription, translation, and/or post-translational processing of collagen. Collagen production from the engineered cells can be further stimulated by growing the engineered cells in the presence of one or more chemical additives in the cell culture medium to achieve even greater rates of collagen production. The synthesized collagen can be isolated, purified, and then used in collagen patches, gels, or other forms for soft tissue repairs.
[0118] The present inventors have achieved dramatically increased collagen production by applying cellular engineering to certain bottlenecks in collagen biosynthesis. Collagen has a unique protein structure. Collagen consists of three amino acid chains which form a triple helix. The primary amino acid sequence found in collagen is glycine-X-hydroxyproline or glycine-proline-X, where X is any other amino acid. The significant amount of glycine (every third amino acid) allows the helix to form in a tight configuration making the molecule structurally resistant to stress (Lodish, et al., 2000). Type I collagen molecules are 300 nm long and 1.5 nm in diameter. Each collagen molecule is composed of a characteristic right-handed triple helix which is composed of two alpha 1 chains and one alpha 2 chain. Each chain contains 1050 sequential amino acids. Once the individual collagen molecules are formed, they pack together side by side to form fibers with a diameter of about 10-300 nm and about a 67 nm gap between the head and tail of adjacent molecules (Schleip, 2012). N-terminal to C-terminal covalent bonds stabilize interactions of collagen molecules that are located adjacent to one another. The periodic pattern of molecule packing creates striations which are visible by electron microscopy. The links between molecules facilitate collagen packing stability to form strong fibrils. In addition to type I collagen, Table 1 lists examples of other collagen types and their features.
TABLE-US-00001 TABLE 1 Collagen Types and Key Features. Collagen Type Features I Most abundant collagen in human body: found in tendons, ligaments, skin, artery walls, cornea, endomysium, fibrocartilage, intestines, bones, uterus, and teeth. II Hyaline cartilage; Found in vitreous humor of the eye, tendons, liver, and cartilage. III Reticular fibers; Found in artery walls, skin, intestines, liver, scar tissue, and uterus. IV Functions in kidney filtration system; Found in basal lamina, the epithelium secreted layer of the basement membrane, placenta, and amniotic membrane. V Interstitial tissue; Found in cornea, ligaments, and intestines. VI Interstitial tissue; Found in ligaments, vasculature, skin, and cartilage. VII Forms anchoring fibrils; Found in epidermal/dermal junctions. VIII Component of some endothelial cells. IX FACIT collagen. X Mineralizing and hypertrophic cartilage. XI Cartilage. XII FACIT collagen. XIII MACIT collagen. XIV FACIT collagen (undulin). XVI FACIT collagen. XVII Transmembrane collagen. XVIII Source of endostatin. XIX FACIT collagen. XX FACIT collagen. XXI FACIT collagen. XXIII MACIT collagen. XXV MACIT collagen. XXIX Epidermal collagen.
[0119] Collagen synthesis occurs primarily in fibroblasts through a process that spans both the intracellular and extracellular space. Type I collagen production is primarily controlled by two genes: Collagen type I alpha 1 (COL1A1), a strip of 17,533 base pairs on chromosome 17 that occurs after the 50,184,096th base pair, and Collagen type I alpha 2 (COL1A2), a strip of 36,671 base pairs on chromosome 7 that occurs after the 94,394,561st base pair (NIH, 2019).
[0120] Most genes responsible for collagen production contain an exon-intron pattern with an average number of exons ranging from 3 to 117. Depending on the type of cell and collagen, there are multiple transcription initiation sites and exon splicing mechanisms which result in different mRNA species (Gelse, et al., 2003). Specifically for type I collagen production, the pro-alpha 1 and pro-alpha 2 chain genes are transcribed from the COL1A1 and COL1A2 genes, respectively. During this phase of collagen production, the pre-mRNA undergoes both splicing and capping. The cellular transcription activity depends on cell type and is regulated by numerous growth factors and cytokines. Some of these growth factors include members of the Transforming Growth Factor Beta (TGF-) family, fibroblast-growth factors, and insulin-like growth factors (Gelse, et al., 2003). The efficacy of these growth factors depends on the cell type.
[0121] Once translation has occurred, the collagen is in a pre-pro-polypeptide chain phase, and it moves to the lumen of the RER for post translational modifications (Wu & Crane, 2019; Lodish, et al., 2000). These molecules intrude into the lumen by the assistance of receptors that recognize the signal recognition domain of the collagen molecules (Gelse, et al., 2003). Three major modifications are made to convert this chain to procollagen. The first modification is the removal of the signal peptide on the N-terminal of the peptide chain by the enzyme signal peptidase. Efficient cleavage by the signal peptidase requires smaller amino acids (i.e., alanine, glycine, serine) just before the cleavage site, so that the signal peptidase 1 (SPase 1) can properly cleave the terminal (Tuteja, 2005).
[0122] The second modification is the hydroxylation, or addition of hydroxyl groups (OH), of lysine and proline residues by hydroxylase enzymes (
[0123] The third modification is the glycosylation of hydroxylysine with glucose and galactose. During this modification, glucosyl and galactosyl residues are placed on the hydroxyl groups of hydroxylysine. Hydroxylation of specific proline and lysine residues (non-hydroxylated) in the middle of the chain are catalyzed by hydroxylysyl galactosyltransferase and galactosylhydroxylysylglucosyltransferase enzymes bound to the endoplasmic reticulum membrane. Oligosaccharides are also bound to asparagine residues in the C-terminal propeptide of procollagen (Lodish, et al., 2000).
[0124] After these three post-translational modifications are made, the glycosylated and hydroxylated chains assemble into a triple helix by folding, much like a zipper, as intrachain disulfide bonds are zipped together. The helix consists of two alpha 1 (I) chains and one alpha 2 (I) chain subunits. This assembly consists of three left-handed helices configured in a 1050 amino acid long right-handed coil, which forms from the C-terminus to N-terminus in the endoplasmic reticulum before further post-translational changes take place. C-propeptides also play a role in the assembly of the peptide chains into a collagen monomer (Gelse, et al., 2003).
[0125] After processing and procollagen assembly, the triple-helical molecule moves to the Golgi apparatus for final modifications and packaging inside the tubular portion of the complex known as vesicular tubular clusters (Wu & Crane, 2019; Bonfanti, et al., 1998). Within these clusters, the procollagen aggregates and is packaged within the Golgi compartment into secretory vesicles and released for transportation to the extracellular space.
[0126] Outside of the cell, collagen peptidase enzymes cleave the unraveled propeptides on the N-terminal and C-terminal to remove the ends of the molecule and convert the molecule to tropocollagen. The protease that performs the propeptide cleavage is procollagen C-proteinase. The tropocollagen terminates on both ends with telopeptides, which can be an issue in regard to antigenicity and immunogenicity (Stuart, et al., 1982; Lynn, et al., 2004). Collagen molecules have telopeptides on either side of their chains. The telopeptides do not form the typical triple helical formation and contain the amino acid hydroxylysine. Hydroxylysine residues form crosslinks at the C-terminal of one molecule and the N-terminal of two adjacent molecules (collplant.com/technology; Lodish, et al., 2000). These telopeptides also can be a source of immunogenicity if the collagen is transplanted into another species, or even intraspecies (Stuart, et al., 1982; Lynn, et al., 2004; Uchio, et al., 2000). The triple helical region of collagen is conserved across species. Although variations in amino acid sequences within the helix differ by less than a few percent between species, up to fifty percent of the amino acid sequence in the telopeptides can differ between species (Lynn, et al., 2004). Due to this high interspecies variation in this region of the molecule, telopeptides are thought to be the primary contributing factor to immune responses post collagen implantation.
[0127] The final extracellular step is fibrillogenesis. Fibril-forming collagen molecules spontaneously self-assemble into ordered fibrillar structures. Long thin collagen fibrils are formed when lysyl oxidase covalently bonds lysine and hydroxylysine molecules. This behavior is encoded in the collagen structure. Fibril orientation depends on the type of tissue (Gelse, et al., 2003). Each fibril has a diameter of about 100 nm after the molecules are packed together side by side, although fibril diameter can range from 25-500 nm.
[0128] Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated protein-coding genes (Cas) are a group of proteins used by the immune system of prokaryotes. CRISPR associated protein 9 (Cas9) can cut DNA, and is a highly efficient DNA targeting enzyme that has been modified for gene editing research applications. The CRISPR-Cas9 system is made up of four main parts: the Cas9 enzyme, guide RNA (gRNA), protospacer adjacent motif (PAM) sequence, and matching host DNA (matching genomic sequence). Cas9 is an endonuclease enzyme that utilizes an approximately 20 base pair section of guiding RNA to recognize, unzip, and induce double-strand breaks in DNA (Biolabs, 2019). Guide RNA (gRNA) directs the CRISPR-Cas9 system where to go in the genome and can result in the process of CRISPR-Cas9 cutting the host DNA, and then letting natural DNA repair processes incorporate an inserted gene of interest into the host's genome at a very particular point in the host genome (as defined by the gRNA). The gRNA has two main components, CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) (
[0129] The matching host DNA is a 18-23 base pair sequence that is in or near the promoter region of the gene of interest. This sequence must be immediately followed by an NGG (representing any base pair followed by two guanines) or PAM sequence. In order to determine what base pairs should be targeted by the gRNA in a given gene, computational biology tools have been developed to find PAM sequences and base pair sequences of interest (CRISPR Guide Design Software, Pelligrini, 2016). These base pairs are ranked for how well the Cas9 system is able to bind to that sequence without accidentally attaching to other similar sequences in the genome. Additionally, manufacturers, like Dharmacon, have proprietary software that is used to compute highly specific binding sites for gRNA.
[0130] The present inventors elected to use CRISPR gene activation (CRISPRa) to engineer cells that produce much greater amounts of collagen than naturally occurring cells. In CRISPRa, a deactivated or dead Cas9 enzyme (dCas9) without endonuclease activity is used together with a guide RNA (gRNA or sgRNA) to locate to a specific gene target. The dCas9 can be fused to one or more transcriptional activator proteins. The resulting fusion protein is referred to herein as dCas9-activator. The one or more activators fused to dCas9 can be, for example, VP64 (a tetramer of the Herpes Simplex Viral Protein 16) or VPR (VP64 bound to 53 and an R transcription factor)). For example, the VPR activator can be dCas9 from S. pyrogenes fused to VP64-65-Rta. Other activators or combinations of activators can be selected according to cell type or gene to be activated. The dCas9 does not cut the bound DNA, but acts to upregulate expression of the targeted gene. The activator domain fused to the dCas9 causes transcriptional activation by recruiting transcription complexes to the promoter regions of these genes.
[0131] An important design consideration when performing CRISPRa is selecting where dCas9 binds to the gene; generally, a position on the gene's promoter is selected. While specificity of a gRNA sequence and locations of its PAM sequences can be predicted using computer algorithms, the location on the promoter and its resulting effectiveness is variable. While the promoter area for effectiveness of CRISPRa is generally 50-400 base pairs upstream of the transcription start site, the most effective location for activation varies between genes, and some locations are completely ineffective (Mohr, et al., 2016).
[0132] There are two practical ways to deliver the CRISPR-Cas9 system for performing CRISPRa (i.e., the dCas9-activator and gRNA for genes to be activated) into a cell: transfection and transduction.
[0133] Transfection is the delivery of nucleic acids (typically the mRNA corresponding to the transcribed gene) into a cell and subsequent translation of the mRNA by the host cell. When performing CRISPRa using transfection, the CRISPR-Cas9 system is usually expressed for about 24-48 hours. Transfection of cells with the CRISPRa components can be performed by microinjection, electroporation, or use of ribonucleoprotein (RNP) complexes to deliver the mRNAs. Transient expression using transfection is simpler and less expensive than transduction, and decreases the odds of off-target activation due to its short expression window. Further, the use of mammalian expression vectors allows for transfection that is less transient than traditional, non-mammalian transfection vectors.
[0134] Commercial kits are available for performing CRISPRa by transfection of cells. For example, Dharmacon (horizondiscovery.com) offers a protocol and reagents for pooled transfection of gRNA and dCas9 mRNA for culture in a 96-well plate using the DharmaFECT Duo Transfection Reagent. The protocol can be scaled up to a 48-well plate or further in order to harvest more collagen, such as for more accurate collagen quantification while engineering cells for increased collagen production. Additionally, when two or more different sets of gRNA are used to activate two or more genes simultaneously, appropriate adjustments can be made to the amount of dCas9 mRNA and the amount of transfection reagent. For example, in order to simultaneously activate expression of the COLA1, COL!A2, and TGF-3 genes in a human cell, the materials can include CRISPRa Human COL1A1 crRNA pool, CRISPRa Human COL1A2 crRNA pool, CRISPRa Human TGF-3 crRNA pool, CRISPR-Cas9 Synthetic tracrRNA, Edit-R GFP dCas9-VPR mRNA, DharmaFECT Duo Transfection Reagent, 10 mM Tris-HCl Buffer pH 7.4, and serum-free medium. Example reagents needed for a pooled transfection of human corneal fibroblasts for increased collagen production are shown below in Table 2. Only one pooled crRNA purchase is necessary as target crRNAs can be mixed and matched in one pool purchase (one pool contains four times the minimum single crRNA needed for CRISPRa), but having more than the minimum crRNA necessary can lead to better gene activation (CRISPR Guide Design Software).
TABLE-US-00002 TABLE 2 Reagents Needed for Pooled Transfection Amount needed for one Catalog CRISPRa experiment (48 well Item Name Number plate) CRISPRa Human P-010502- .54 nmol (electroporation) COL1A1 crRNA pool 010005 .0025 nmol (transfection reagent) CRISPRa Human P-004758- .54 nmol (electroporation) COL1A2 crRNA pool 010005 .0025 nmol (transfection reagent) CRISPRa Human TGF- P-012562- .54 nmol (electroporation) 1 crRNA pool 010005 .0025 nmol (transfection reagent) CRISPR-Cas9 U-002005-05 .54 nmol (electroporation) Synthetic tracrRNA .0025 nmol (transfection reagent) dCas9-VPR mRNA CAS12024 5 g (electroporation) (also in GFP and .2 g (transfection reagent) Puromycin variants) DharmaFECT 1 T-2001-01 .8 L Transfection Reagent 10 mM Tris-HCl Buffer B-006000-100 .05 mL pH 7.4
[0135] The CRISPRa Human COL1A1 crRNA pool includes a pool of individual RNA sequences complimentary to different regions of the COL1A1 promoter (see target sequences SEQ ID NOS:1-4 in Table 3).
TABLE-US-00003 TABLE3 CRISPRTargetSequencesforPromotersofTargetedGenesRelated toCollagenBiosynthesis DNATargetSequence TargetGene SEQIDNO:1 GCTGCGAAGAGGGGAGATGT COL1A1(human) SEQIDNO:2 GGGGAGGCAGAGCTGCGAAG COL1A1(human) SEQIDNO:3 CCGGCCCCCAATTTGGGAGT COL1A1(human) SEQIDNO:4 GGAACCCTGCCCCTCGGAGA COL1A1(human) SEQIDNO:5 AAGGGCCTCCACCAATGGGA COL1A2(human) SEQIDNO:6 CGCAGAGGAGGGAGCGAATG COL1A2(human) SEQIDNO:7 GGGGAAGGGACGTGGCCACG COL1A2(human) SEQIDNO:8 GGGAGGGCGGGAGGATGCGG COL1A2(human) SEQIDNO:9 TTAACATCGTGCAGCAAAAG TGF-3(human) SEQIDNO:10 GAGGGCGCGGGACCCGGTAG TGF-3(human) SEQIDNO:11 GCAAAAGAGGCTGCGTGCGC TGF-3(human) SEQIDNO:12 CCGGGACCGGGGGACCAGGA TGF-3(human) SEQIDNO:13 GCGGCCAAGCGCCACCAAAG TGF-1(human) SEQIDNO:14 GAGCCCGCCCACGCGAGATG TGF-1(human) SEQIDNO:15 CCCCGCGGGCGGCTCAGAGC TGF-1(human) SEQIDNO:16 CCGCCCACGCGAGATGAGGA TGF-1(human) SEQIDNO:17 GAGCTCTCCCCGAACCGTTG TGF-2(human) SEQIDNO:18 ATGAGGACCGCTGTGGGTAA TGF-2(human) SEQIDNO:19 GTGGAAATGAGGACCGCTGT TGF-2(human) SEQIDNO:20 CTCGTGGTCTAAGTAACGAG TGF-2(human) SEQIDNO:21 GCCGCAGCCCCGGGTTTGGG COL2A1(human) SEQIDNO:22 GCCACTCGGCGCACTAGGGG COL2A1(human) SEQIDNO:23 CAGGCCACTCGGCGCACTAG COL2A1(human) SEQIDNO:24 CCAAGCCGGACCCCCCTCTC COL2A1(human) SEQIDNO:25 CGGCTCTCATATTTCAGAAA COL3A1(human) SEQIDNO:26 GCAGTTGTTAACTTCATAAG COL3A1(human) SEQIDNO:27 TGTGGGTTGTGTCTTCTATA COL3A1(human) SEQIDNO:28 TAACTTCTAGGACCCAGGGT COL3A1(human) SEQIDNO:29 GGGCGCGAGGGGTTGGGACG COL4A1(human) SEQIDNO:30 GACCCTGCGGCGCGGGTAAG COL4A1(human) SEQIDNO:31 GAGCGCGCGGCCCGGGAGTG COL4A1(human) SEQIDNO:32 GCGCACTGCAGCCACACTCC COL4A1(human) SEQIDNO:33 TGGAGCCGCCGCACCCGGGA COL4A2(human) SEQIDNO:34 CCGGGTGCGGCGGCTCCAAG COL4A2(human) SEQIDNO:35 GCGGACAGCTAGCTCTCGGA COL4A2(human) SEQIDNO:36 GCCCCATGGTGGCGCGCCCG COL4A2(human) SEQIDNO:37 CGTGCCCAGGAGGCGAGAAA COL4A3(human) SEQIDNO:38 TCTACCCGGGCATCGTGCCC COL4A3(human) SEQIDNO:39 GGCACGATGCCCGGGTAGAA COL4A3(human) SEQIDNO:40 AGGGACACTGCCTGGTAAGT COL4A3(human) SEQIDNO:41 CGTGCCCAGGAGGCGAGAAA COL4A4(human) SEQIDNO:42 TCTACCCGGGCATCGTGCCC COL4A4(human) SEQIDNO:43 GGGAAGTGGGGTGCGGTCGG COL4A4(human) SEQIDNO:44 GGATCCAGGGTAAGGGGTTA COL4A4(human) SEQIDNO:45 GAGTGACGCTCAGTTATTTG COL4A5(human) SEQIDNO:46 GACTGCACCGGCAACCTGCG COL4A5(human) SEQIDNO:47 GGTACGCACACCAATGAGAT COL4A5(human) SEQIDNO:48 GTCACTCCCTCGCAGGTTGC COL4A5(human) SEQIDNO:49 TTAGCGTATAGGTCTCTAAG COL4A6TSS*P1(human) SEQIDNO:50 CGGGCCCATCTGTCTTATGT COL4A6TSS*P1(human) SEQIDNO:51 CTACCGGCTGCCCAAGGTAG COL4A6TSS*P1(human) SEQIDNO:52 TTGCTTAGGTCTTAGCGTAT COL4A6TSS*P1(human) SEQIDNO:53 GGTACGCACACCAATGAGAT COL4A6TSS*P2(human) SEQIDNO:54 GAGTGACGCTCAGTTATTTG COL4A6TSS*P2(human) SEQIDNO:55 GACTGCACCGGCAACCTGCG COL4A6TSS*P2(human) SEQIDNO:56 GCCGGTGCAGTCTAAAACTG COL4A6TSS*P2(human) SEQIDNO:57 CGGGCGAGTCGCAGCGAGGA COL5A1(human) SEQIDNO:58 CCCGGGGCGGAGCGGACGTG COL5A1(human) SEQIDNO:59 CCTCGCCCGCGGCGCCCAGT COL5A1(human) SEQIDNO:60 CCCCAGGCCCGCCCGCCTAC COL5A1(human) SEQIDNO:61 CCAAGCAACGGTCTGATTGA COL5A2(human) SEQIDNO:62 CAGAGACGCGTGTTCTGATT COL5A2(human) SEQIDNO:63 AAAGTTAAAGGGTGTGTGTC COL5A2(human) SEQIDNO:64 TTAACTTTTAAGCATAGATG COL5A2(human) SEQIDNO:65 ACTGGAACCCTCGAACTCTA COL5A3(human) SEQIDNO:66 TGCACCTCCTCCTAATTCTA COL5A3(human) SEQIDNO:67 GGAGGCTGAAGTCTTGAATG COL5A3(human) SEQIDNO:68 AACAACCCAGCTCCTGGCGC COL5A3(human) SEQIDNO:69 GCGGCCGCGCCAAGATGCGC ADAMTS2(human) SEQIDNO:70 GCGAAGAGGGAAGCGGGCGG ADAMTS2(human) SEQIDNO:71 CGAGCCCGGCACCGCGGCGA ADAMTS2(human) SEQIDNO:72 GCAGAGACACCCCGAGGCGG ADAMTS2(human) SEQIDNO:73 CGACCCGCGGGGCGCTAATA ADAMTS3(human) SEQIDNO:74 CTCCGCCTAGGGCGAGAGGA ADAMTS3(human) SEQIDNO:75 CGCGCCCCCCGCACCGTGTC ADAMTS3(human) SEQIDNO:76 GTTCGGGCCCCGCATGACGT ADAMTS3(human) SEQIDNO:77 GGGGCTCTGTCCCACCAAAA ADAMTS4(human) SEQIDNO:78 AAAAAATGGGACTTGCCCAG ADAMTS4(human) SEQIDNO:79 ACAGCTGAGGGCTGATTGTG ADAMTS4(human) SEQIDNO:80 CTAGCAGCCGAATGGATAAT ADAMTS4(human) SEQIDNO:81 GTCCGCGCCTAATCAGATGG ADAMTS5(human) SEQIDNO:82 GAGGAGGGTGATCGAGGAAA ADAMTS5(human) SEQIDNO:83 GCAAAAGAGGAGGGTGATCG ADAMTS5(human) SEQIDNO:84 AGGCGCGGACTGGGAAGGGT ADAMTS5(human) SEQIDNO:85 GGGAGGTAGAGGTACAATCG ADAMTS6(human) SEQIDNO:86 AGATGTGCCTGGGCTGCGTC ADAMTS6(human) SEQIDNO:87 CCCCCTCCACGTGACGACCC ADAMTS6(human) SEQIDNO:88 GTCCTGGGTCGTCACGTGGA ADAMTS6(human) SEQIDNO:89 GCAGGCCGTGCTCGCCTCAA ADAMTS7(human) SEQIDNO:90 GCGCAGGCAGCGCCCCGCAA ADAMTS7(human) SEQIDNO:91 CACGGCCTGCGGGGCCGATG ADAMTS7(human) SEQIDNO:92 AGGGGGGCGGACATCCGTTG ADAMTS7(human) SEQIDNO:93 CACCCCGGGAAGCACCGAGT ADAMTS8(human) SEQIDNO:94 GATCCCAGGGGAGGGGAAAC ADAMTS8(human) SEQIDNO:95 GAAAGCGGTTGGGGTCTCCC ADAMTS8(human) SEQIDNO:96 TCCTCTGCGGCCAAGAGTCC ADAMTS8(human) SEQIDNO:97 GAGGAAAAAGAGACTCGGAA ADAMTS9(human) SEQIDNO:98 GCCGAGGAGGGGACATGGTC ADAMTS9(human) SEQIDNO:99 CCCCGGTGCACGCCTCTAAG ADAMTS9(human) SEQIDNO:100 GAGGCCCTGCCGGCTGCAAG ADAMTS9(human) SEQIDNO:101 GGCGGGGCGGCCCGAGTTCC ADAMTS10(human) SEQIDNO:102 GCGCCCCCGCCGCGTGGGAG ADAMTS10(human) SEQIDNO:103 AGCCAGGGACCCGGGAACTC ADAMTS10(human) SEQIDNO:104 GGGCGGGGAGGGGACGAAGC ADAMTS10(human) SEQIDNO:105 GAGGCCGCGGGGCATGCGGG ADAMTS12(human) SEQIDNO:106 GACAGTGTCCGCTTTCGGCG ADAMTS12(human) SEQIDNO:107 CTGGCCGAGCCGGCCAAATA ADAMTS12(human) SEQIDNO:108 CCGGCCAAATAGGGGAGACC ADAMTS12(human) SEQIDNO:109 GTGCACGCCCACCCCCTTAG ADAMTS13(human) SEQIDNO:110 TCTAAGATGGTTGCAGTTCA ADAMTS13(human) SEQIDNO:111 GGGAGCGGGGACCCCGGAGA ADAMTS13(human) SEQIDNO:112 GCAGGGGCCGTGGCTAGGGT ADAMTS13(human) SEQIDNO:113 CAGGCCCTGCGGCCAAGAAA ADAMTS14(human) SEQIDNO:114 GGGGATCCCAGGCGTCTGAG ADAMTS14(human) SEQIDNO:115 CCGCCCGCGCAGTGACGCTC ADAMTS14(human) SEQIDNO:116 GGCTTTGGGCCACCACTCCG ADAMTS14(human) SEQIDNO:117 GTGGCTCTCCGCTCTGGAGG ADAMTS15(human) SEQIDNO:118 GGAGAGCCACTTGGCGGGGA ADAMTS15(human) SEQIDNO:119 GCCACTCCCTCCCCGCCAAG ADAMTS15(human) SEQIDNO:120 CAGAGCGGAGAGCCACTTGG ADAMTS15(human) SEQIDNO:121 GCCCCTGTGGTCAACCTCGT ADAMTS16TSS*P1(human) SEQIDNO:122 TCGGAATTCCGAGAAGAATC ADAMTS16TSS*P1(human) SEQIDNO:123 CTGCCTACGAGGTTGACCAC ADAMTS16TSS*P1(human) SEQIDNO:124 GGGACACGTAATCGCCCCTG ADAMTS16TSS*P1(human) SEQIDNO:125 GGGAGGAGGCGAGGTCAGCG ADAMTS16TSS*P2(human) SEQIDNO:126 AGGTCAGCGGGGCGCTGAGG ADAMTS16TSS*P2(human) SEQIDNO:127 GACCGAGGAGGGGAGAGTGC ADAMTS16TSS*P2(human) SEQIDNO:128 CCGCGCGGGGACCACACAGT ADAMTS16TSS*P2(human) SEQIDNO:129 GGCGGGGCGGAGGAAAGCGA ADAMTS17(human) SEQIDNO:130 TCGACCCATCCCAACCAGTA ADAMTS17(human) SEQIDNO:131 GACAACCTAACTACGCACTG ADAMTS17(human) SEQIDNO:132 TTCCCCTCGGAAGGCGGGAG ADAMTS17(human) SEQIDNO:133 GAGGCTGGGTGGGAGTGATA ADAMTS18(human) SEQIDNO:134 AGGGACCGACCGCCATGAGA ADAMTS18(human) SEQIDNO:135 CCGGGCTGGTGCAAGGGCGT ADAMTS18(human) SEQIDNO:136 GTGCAAGGGCGTGGGATTCC ADAMTS18(human) SEQIDNO:137 TAATTCGCCAGGGAGCTCGA ADAMTS19(human) SEQIDNO:138 GAGGGAATGAGTAGGGAGAT ADAMTS19(human) SEQIDNO:139 GGCTGTGGGTCTGTCTTGTG ADAMTS19(human) SEQIDNO:140 GGCGCGGGAGGGAATGAGTA ADAMTS19(human) SEQIDNO:141 AGGGTACCGCAGTCCCCTTG ADAMTS20(human) SEQIDNO:142 TGCGGCTGAGTGGAGAAGGG ADAMTS20(human) SEQIDNO:143 TTGGCCGGGAACAGCCCATA ADAMTS20(human) SEQIDNO:144 GCGCGGGGAAAAGCGAGCAG ADAMTS20(human) SEQIDNO:145 GTAAGCAGGATGCAAGTGAT TLL1(human) SEQIDNO:146 TTGGCTAGGGGCCCACGGGT TLL1(human) SEQIDNO:147 GAGCAAAAGTGTGAGGATTT TLL1(human) SEQIDNO:148 GGGGAGGGCGCAGGCAAACT TLL1(human) SEQIDNO:149 CGGCACCTTCGCAACTTCGG TLL2(human) SEQIDNO:150 GCCTCGGGCGCCCGAGTGAT TLL2(human) SEQIDNO:151 TGCTCCTGAAGATCGGGACT TLL2(human) SEQIDNO:152 GCGCCCCGGGCAGGCGCCTT TLL2(human) SEQIDNO:153 CCCGCCCCCCGCTCGGTCCG BMP1(human) SEQIDNO:154 CGGAGCCGCGGACCGAGCGG BMP1(human) SEQIDNO:155 GGCCCGGCCGAGCACTGTCC BMP1(human) SEQIDNO:156 CACTGTCCCGGCCCCGAGGG BMP1(human) *TSS =Transcription Start Site Promoter 1 (P1) or P2
TABLE-US-00004 TABLE4 GenomicLocationsandPAMsofcrRNASequences GenomicLocation PAM SEQIDNO:1 hg38:17:50201809-50201828: GGG SEQIDNO:2 hg38:17:50201820-50201839: AGG SEQIDNO:3 hg38:17:50201748-50201767:+ TGG SEQIDNO:4 hg38:17:50201683-50201702:+ GGG SEQIDNO:5 hg38:7:94394807-94394826: GGG SEQIDNO:6 hg38:7:94394736-94394755: GGG SEQIDNO:7 hg38:7:94394716-94394735: GGG SEQIDNO:8 hg38:7:94394596-94394615:+ AGG SEQIDNO:9 hg38:14:75982087-75982106: AGG SEQIDNO:10 hg38:14:75982399-75982418:+ GGG SEQIDNO:11 hg38:14:75982074-75982093: TGG SEQIDNO:12 hg38:14:75982338-75982357: GGG SEQIDNO:13 hg38:19:41354142-41354161:+ CGG SEQIDNO:14 hg38:19:41354044-41354063: AGG SEQIDNO:15 hg38:19:41353785-41353804:+ CGG SEQIDNO:16 hg38:19:41354040-41354059: CGG SEQIDNO:17 hg38:1:218345204-218345223: AGG SEQIDNO:18 hg38:1:218345171-218345190: GGG SEQIDNO:19 hg38:1:218345177-218345196: GGG SEQIDNO:20 hg38:1:218345221-218345240:+ AGG SEQIDNO:21 hg38:12:48004733-48004752: GGG SEQIDNO:22 hg38:12:48004679-48004698: TGG SEQIDNO:23 hg38:12:48004682-48004701: GGG SEQIDNO:24 hg38:12:48004634-48004653:+ TGG SEQIDNO:25 hg38:2:188974289-188974308:+ GGG SEQIDNO:26 hg38:2:188974039-188974058:+ GGG SEQIDNO:27 hg38:2:188974220-188974239:+ AGG SEQIDNO:28 hg38:2:188973838-188973857:+ GGG SEQIDNO:29 hg38:13:110307345-110307364: CGG SEQIDNO:30 hg38:13:110307240-110307259:+ AGG SEQIDNO:31 hg38:13:110307269-110307288:+ TGG SEQIDNO:32 hg38:13:110307283-110307302: CGG SEQIDNO:33 hg38:13:110307127-110307146: CGG SEQIDNO:34 hg38:13:110307129-110307148:+ CGG SEQIDNO:35 hg38:13:110307080-110307099:+ AGG SEQIDNO:36 hg38:13:110307021-110307040:+ AGG SEQIDNO:37 hg38:2:227164435-227164454: GGG SEQIDNO:38 hg38:2:227164448-227164467: AGG SEQIDNO:39 hg38:2:227164449-227164468:+ GGG SEQIDNO:40 hg38:2:227164468-227164487:+ TGG SEQIDNO:41 hg38:2:227164435-227164454: GGG SEQIDNO:42 hg38:2:227164448-227164467: AGG SEQIDNO:43 hg38:2:227164329-227164348: AGG SEQIDNO:44 hg38:2:227164385-227164404: AGG SEQIDNO:45 hg38:X:108439713-108439732: GGG SEQIDNO:46 hg38:X:108439736-108439755: AGG SEQIDNO:47 hg38:X:108439581-108439600:+ TGG SEQIDNO:48 hg38:X:108439726-108439745:+ CGG SEQIDNO:49 hg38:X:108438581-108438600: GGG SEQIDNO:50 hg38:X:108438679-108438698: GGG SEQIDNO:51 hg38:X:108438493-108438512: GGG SEQIDNO:52 hg38:X:108438592-108438611: AGG SEQIDNO:53 hg38:X:108439581-108439600:+ TGG SEQIDNO:54 hg38:X:108439713-108439732: GGG SEQIDNO:55 hg38:X:108439736-108439755: AGG SEQIDNO:56 hg38:X:108439744-108439763:+ TGG SEQIDNO:57 hg38:9:134641608-134641627: AGG SEQIDNO:58 hg38:9:134641579-134641598: GGG SEQIDNO:59 hg38:9:134641640-134641659:+ GGG SEQIDNO:60 hg38:9:134641522-134641541:+ CGG SEQIDNO:61 hg38:2:189179867-189179886:+ TGG SEQIDNO:62 hg38:2:189179900-189179919: TGG SEQIDNO:63 hg38:2:189180069-189180088: TGG SEQIDNO:64 hg38:2:189179943-189179962:+ GGG SEQIDNO:65 hg38:19:10010713-10010732: GGG SEQIDNO:66 hg38:19:10010816-10010835: GGG SEQIDNO:67 hg38:19:10010786-10010805: AGG SEQIDNO:68 hg38:19:10010646-10010665: TGG SEQIDNO:69 hg38:5:179345605-179345624: CGG SEQIDNO:70 hg38:5:179345630-179345649: CGG SEQIDNO:71 hg38:5:179345657-179345676: CGG SEQIDNO:72 hg38:5:179345684-179345703:+ CGG SEQIDNO:73 hg38:4:72569368-72569387:+ AGG SEQIDNO:74 hg38:4:72569539-72569558:+ GGG SEQIDNO:75 hg38:4:72569453-72569472:+ AGG SEQIDNO:76 hg38:4:72569432-72569451: GGG SEQIDNO:77 hg38:1:161199300-161199319: AGG SEQIDNO:78 hg38:1:161199176-161199195:+ GGG SEQIDNO:79 hg38:1:161199270-161199289: GGG SEQIDNO:80 hg38:1:161199209-161199228:+ AGG SEQIDNO:81 hg38:21:26967216-26967235:+ GGG SEQIDNO:82 hg38:21:26967248-26967267:+ GGG SEQIDNO:83 hg38:21:26967242-26967261:+ AGG SEQIDNO:84 hg38:21:26967206-26967225: GGG SEQIDNO:85 hg38:5:65481993-65482012:+ GGG SEQIDNO:86 hg38:5:65481934-65481953:+ GGG SEQIDNO:87 hg38:5:65481971-65481990: AGG SEQIDNO:88 hg38:5:65481966-65481985:+ GGG SEQIDNO:89 hg38:15:78811581-78811600:+ GGG SEQIDNO:90 hg38:15:78811616-78811635:+ CGG SEQIDNO:91 hg3815:78811571-78811590: AGG SEQIDNO:92 hg38:15:78811633-78811652: CGG SEQIDNO:93 hg38:11:130428594-130428613:+ GGG SEQIDNO:94 hg38:11:130428692-130428711: GGG SEQIDNO:95 hg38:11:130428906-130428925: AGG SEQIDNO:96 hg38:11:130428926-130428945:+ AGG SEQIDNO:97 hg38:3:64688410-64688429: GGG SEQIDNO:98 hg38:3:64688158-64688177: TGG SEQIDNO:99 hg38:3:64688088-64688107:+ AGG SEQIDNO:100 hg38:3:64688202-64688221:+ AGG SEQIDNO:101 hg38:19:8610882-8610901: CGG SEQIDNO:102 hg38:19:8610910-8610929:+ GGG SEQIDNO:103 hg38:19:8610869-8610888:+ GGG SEQIDNO:104 hg38:19:8610949-8610968:+ GGG SEQIDNO:105 hg38:5:33892158-33892177: AGG SEQIDNO:106 hg38:5:33892078-33892097:+ GGG SEQIDNO:107 hg38:5:33892259-33892278:+ GGG SEQIDNO:108 hg38:5:33892268-33892287:+ CGG SEQIDNO:109 hg38:9:133422154-133422173: AGG SEQIDNO:110 hg38:9:133422219-133422238: GGG SEQIDNO:111 hg38:9:133422039-133422058:+ GGG SEQIDNO:112 hg38:9:133422279-133422298: GGG SEQIDNO:113 hg38:10:70672206-70672225:+ GGG SEQIDNO:114 hg38:10:70672269-70672288:+ GGG SEQIDNO:115 hg38:10:70672499-70672518: CGG SEQIDNO:116 hg38:10:70672250-70672269:+ GGG SEQIDNO:117 hg38:11:130448484-130448503:+ CGG SEQIDNO:118 hg38:11:130448474-130448493: GGG SEQIDNO:119 hg38:11:130448465-130448484:+ TGG SEQIDNO:120 hg38:11:130448480-130448499: CGG SEQIDNO:121 hg38:5:5146151-5146170: AGG SEQIDNO:122 hg38:5:5146075-5146094: AGG SEQIDNO:123 hg38:5:5146145-5146164:+ AGG SEQIDNO:124 hg38:5:5146164-5146183: TGG SEQIDNO:125 hg38:5:5140149-5140168: GGG SEQIDNO:126 hg38:5:5140138-5140157: CGG SEQIDNO:127 hg38:5:5140186-5140205: AGG SEQIDNO:128 hg38:5:5140010-5140029: TGG SEQIDNO:129 hg38:15:100342081-100342100: GGG SEQIDNO:130 hg38:15:100342112-100342131: GGG SEQIDNO:131 hg38:15:100342137-100342156: TGG SEQIDNO:132 hg38:15:100342049-100342068:+ GGG SEQIDNO:133 hg38:16:77435213-77435232:+ GGG SEQIDNO:134 hg38:16:77435240-77435259:+ AGG SEQIDNO:135 hg38:16:77435148-77435167:+ GGG SEQIDNO:136 hg38:16:77435156-77435175:+ CGG SEQIDNO:137 hg38:5:129460117-129460136:+ GGG SEQIDNO:138 hg38:5:129459790-129459809: GGG SEQIDNO:139 hg38:5:129460024-129460043:+ GGG SEQIDNO:140 hg38:5:129459797-129459816: GGG SEQIDNO:141 hg38:12:43552359-43552378: CGG SEQIDNO:142 hg38:12:43552341-43552360: AGG SEQIDNO:143 hg38:12:43552274-43552293: GGG SEQIDNO:144 hg38:12:43552313-43552332: GGG SEQIDNO:145 hg38:4:165873143-165873162:+ TGG SEQIDNO:146 hg38:4:165873672-165873691: GGG SEQIDNO:147 hg38:4:165873253-165873272: GGG SEQIDNO:148 hg38:4:165873615-165873634: CGG SEQIDNO:149 hg38:10:96514056-96514075:+ AGG SEQIDNO:150 hg38:10:96514019-96514038: TGG SEQIDNO:151 hg38:10:96514190-96514209:+ GGG SEQIDNO:152 hg38:10:96514142-96514161: AGG SEQIDNO:153 hg38:8:22165233-22165252: CGG SEQIDNO:154 hg38:8:22165225-22165244:+ GGG SEQIDNO:155 hg38:8:22165045-22165064:+ CGG SEQIDNO:156 hg38:8:22165057-22165076:+ GGG
[0136] The DharmaFECT Duol Transfection Reagent has been shown to be an efficient transfection reagent for transfection of small RNAs and plasmids simultaneously (Borawski, et al., 2007).
[0137] An example of a dCas9 protein for use with the present technology is one having the amino acid sequence shown below (SEQ ID NO: 157 (uniprot.org/uniprot/AOA386IRG9)):
TABLE-US-00005 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNEDK NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRENASLGTYHDLLKI IKDKDELDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLEDDKVMKQ LKRRRYTGWGRLSRKLINGIRDKQSGKTILDELKSDGFANRNFMQLIHDD SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDD SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKEDNL TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI TLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVE KGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPE DNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDK PIREQAENIIHLETLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ SITGLYETRIDLSQLGGD
[0138] An example of a synthetic tracrRNA for use with the present technology is one published by Jinek, et al., (2012), which has the sequence GGAACCAUUCAAAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA AAAAGUGGCACCGAGUCGGUGCUUUUUUU (SEQ ID NO:158). The crRNA may include a region complementary to a portion of the tracrRNA. Alternatively, a linker sequence can be added between the crRNA and the tracrRNA to yield a single gRNA molecule.
[0139] VP64 is a transcriptional activator including four tandem copies of VP16 (Herpes Simplex Viral Protein 16, amino acids 437-447, connected with glycine-serine linkers). The amino acid sequence of VP64 is shown below (SEQ ID NO: 159, (parts.igem.org/Part:BBa_J176013)):
TABLE-US-00006 GACGCTTTGGACGACTTCGACTTGGACATGTTGGGTTCTGACGCTTTGGA CGACTTCGACTTGGACATGTTGGGTTCTGACGCTTTGGACGACTTCGACT TGGACATGTTGGGTTCTGACGCTTTGGACGACTTCGACTTGGACATGTTG
[0140] The transcriptional activator 65 includes four isoforms produced by alternative splicing (uniprot.org/uniprot/Q04206). Isoform 1 has the amino acid sequence shown below (SEQ ID NO: 160).
TABLE-US-00007 MDELFPLIFPAEPAQASGPYVEIIEQPKQRGMRFRYKCEGRSAGSIPGER STDTTKTHPTIKINGYTGPGTVRISLVTKDPPHRPHPHELVGKDCRDGFY EAELCPDRCIHSFQNLGIQCVKKRDLEQAISQRIQTNNNPFQVPIEEQRG DYDLNAVRLCFQVTVRDPSGRPLRLPPVLSHPIFDNRAPNTAELKICRVN RNSGSCLGGDEIFLLCDKVQKEDIEVYFTGPGWEARGSFSQADVHRQVAI VFRTPPYADPSLQAPVRVSMQLRRPSDRELSEPMEFQYLPDTDDRHRIEE KRKRTYETFKSIMKKSPFSGPTDPRPPPRRIAVPSRSSASVPKPAPQPYP FTSSLSTINYDEFPTMVFPSGQISQASALAPAPPQVLPQAPAPAPAPAMV SALAQAPAPVPVLAPGPPQAVAPPAPKPTQAGEGTLSEALLQLQFDDEDL GALLGNSTDPAVFTDLASVDNSEFQQLLNQGIPVAPHTTEPMLMEYPEAI TRLVTGAQRPPDPAPAPLGAPGLPNGLLSGDEDESSIADMDESALLSQIS S
[0141] The amino acid sequence of the transcriptional activator HSF1, SEQ ID NO: 161, is shown below (SEQ ID NO: 161 ((uniprot.org/uniprot/Q00613)).
TABLE-US-00008 MDLPVGPGAAGPSNVPAFLTKLWTLVSDPDTDALICWSPSGNSFHVEDQG QFAKEVLPKYFKHNNMASFVRQLNMYGFRKVVHIEQGGLVKPERDDTEFQ HPCFLRGQEQLLENIKRKVTSVSTLKSEDIKIRQDSVTKLLTDVQLMKGK QECMDSKLLAMKHENEALWREVASLRQKHAQQQKVVNKLIQFLISLVQSN RILGVKRKIPLMLNDSGSAHSMPKYSRQFSLEHVHGSGPYSAPSPAYSSS SLYAPDAVASSGPIISDITELAPASPMASPGGSIDERPLSSSPLVRVKEE PPSPPQSPRVEEASPGRPSSVDTLLSPTALIDSILRESEPAPASVTALTD ARGHTDTEGRPPSPPPTSTPEKCLSVACLDKNELSDHLDAMDSNLDNLQT MLSSHGFSVDTSALLDLFSPSVTVPDMSLPDLDSSLASIQELLSPQEPPR PPEAENSSPDSGKQLVHYTAQPLFLLDPGSVDTGSNDLPVLFELGEGSYF SEGDGFAEDPTISLLTGSEPPKAKDPTVS
[0142] The amino acid sequence of transcriptional activator MS2 is shown below (SEQ ID NO: 162 (uniprot.org/uniprot/P03612)).
TABLE-US-00009 MASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVR QSSAQNRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNS DCELIVKAMQGLLKDGNPIPSAIAANSGIY
[0143] The dCas9 mRNA, described above, is the limiting reagent for the protocol described below, allowing for 11 wells to be made with 5 nmol of starting material. An example protocol for plating the test plate conditions is: [0144] 1. Plate fibroblasts in a 48 well plate at a density where the wells will be 70-90 percent confluent the next day. [0145] 2. Dilute and mix COL1A1, COL1A2, and TGF-3 crRNA and tracrRNA (1:1 total crRNA to tracrRNA) to a working concentration of 2.5M according to Dharmacon's resuspension procedure [165]. This is now the gRNA solution. [0146] a. When purchased, crRNA and tracrRNA comes as 5nmol. Dilute each of these to 5M by adding 1000L of 10 mM Tris pH 7.4 to each stock of crRNA pools and tracrRNA. [0147] b. When mixed 1:1, the resulting tracrRNA:crRNA should have a working concentration of 2.5M. [0148] 3. Dilute 20pg dCas9 mRNA to a concentration of 100 ng/pL in 200L serum-free medium. [0149] 4. Add 3L of each of the three gRNA solutions, 18L of dCas9 solution, and 9 L of medium (to bring to a volume of 30 L) in a microcentrifuge tube. [0150] 5. The original protocol calls for a testing of a range of transfection reagent amounts (0.1-.8 L)to determine which amount is best for your cell type. When scaled from a 96 to 48well plate configuration and 3x mRNA amounts (3 different gRNA-Cas9 mRNA complexes, one for each gene of interest), this range is 0.9-7.2 L of transfection reagent. Generate a range of Duo Transfection reagent working solution amounts consisting of 7.2, 4.05, and 0.9 L of transfection reagent and bring each of these to a volume of 30 L with serum-free media. [0151] 6. Incubate at room temperature for 5 minutes [0152] 7. Combine 30 L of each concentration of transfection reagent with 30 L of the dCas9/gRNA solution and mix gently with a pipette. [0153] 8. Incubate at room temperature for 20 minutes [0154] 9. Add 240 L serum-free media to each combination and replace media on cells with the newly developed transfection mixtures. [0155] 10. Harvest media for collagen assay every 48 hours for 108 hours. [0156] 11. Gene activation of COL1A1, COL1A2, and TGF3 should start to occur around 24 hours and have maximal expression until about 72 hours after transfection.
[0157] To measure collagen production, a hydroxyproline assay can be used, optionally after concentrating the collagen solution or culture medium (see, e.g., D.D. Cissell et al., (2017). Tissue Eng Part C Methods. 2017 Apr;23(4):243-25) Alternatively, the SirCoI dye binding collagen assay can be used (www.biocolor.co.uk/product/sircol-soluble-collagen-assay). A standard curve, as shown in
[0158] Three levels of transfection reagent were compared; transfection reagents can be toxic to cells at high concentrations. The high (H), medium (M), and low (L), transfection reagent concentrations were 7.2, 4.05, and 0.9 L of transfection reagent per well, respectively. Media was harvested every 12 hours and pooled in two-day segments. Collagen amount per well was quantified every two days, and cell counts were performed after the end of media harvesting. Collagen produced per cell was estimated based on the number of cells per well at the end of media collection. A comparison of total (days 1-6) pooled collagen production (per well, not per cell) for each concentration level and the cell count is shown in
[0159] Table 5 shows mean collagen production (molecules/cell/hours) for the test groups (high, medium, low), and the group names denote the transfection level and the days at which the assay was taken (i.e., 1-2=days 1 and 2).
TABLE-US-00010 TABLE 5 Collagen Production of Test Groups. Mean Collagen Production Group (molecules/cell/hour, millions) Standard Deviation n Control 1-2 1.06 0.00 3 Control 3-4 4.86 3.91 3 Control 5-6 1.06 0.00 3 Low 1-2 1.06 0.00 3 Low 3-4 6.47 6.01 3 Low 5-6 4.49 5.94 3 Med 1-2 1.06 0.00 3 Med 3-4 6.62 5.69 3 Med 5-6 15.93 16.65 3 High 1-2 1.06 0.00 3 High 3-4 44.29 74.88 3 High 5-6 95.39 40.16 3
[0160] Quantifications that fall below the minimum detectable collagen amount are set to the minimum detectable collagen amount (1.06 million molecules/cell/hour) (Table 5). Having some quantifications fall below the minimum detectable amount is necessary to capture the upper limits of collagen production in the CRISPR conditions. Data for days 1-2 and most control samples were at or below the minimum detectable collagen amount, but every datapoint for CRISPR results at days 3-4 and 5-6 is above the minimum threshold. The fact that the results at days 1-2 matched the control wells is consistent with the expectation that CRISPR should upregulate collagen production after about two days. This increase in collagen is shown in a graphical format in
[0161] Based on the graph in
TABLE-US-00011 TABLE 6 Tukey Pairwise Comparison Chart for Table 5 and FIG. 6. Subset Group N 1 2 Control 1-2 3 1.0600 Control 5-6 3 1.0600 High 1-2 3 1.0600 Low 1-2 3 1.0600 Med 1-2 3 1.0600 Low 5-6 3 4.4900 Control 3-4 3 4.8633 Low 3-4 3 6.4667 Med 3-4 3 6.6167 Med 5-6 3 15.9333 High 3-4 3 44.2933 44.2933 High 5-6 3 95.3867
[0162] Table 6 shows the subset groupings for the experiment represented in
[0163] In order to prevent undesired crosslinking of newly synthesized collagen, BAPN is preferably added to the culture medium. Culture conditions may also be adjusted to optimize preservation of collagen structure and function. For example, since collagen is unstable if stored at 37 C., cells can be cultured below 37 C., and/or collagen can be harvested periodically (e.g., every 8-24 hours), and then stored at a lower temperature to preserve stability.
[0164] From the data in Table 5 and
[0165] As discussed above, it was found that the CRISPR system can increase collagen production about 90.29-fold in collagen production compared to the experimental control. When scaled up to a T-75 flask, this production level would yield about 554 mg collagen/week. However, the purchasing of commercially available dCas9 mRNA, gRNA (crRNA and tracrRNA) reagents for each gene of interest, and transfection reagent, would be cost prohibitive. Therefore, it is desirable to scale up using other methods of CRISPRa delivery that would cost significantly less. Suitable methods include the use of bacteria to produce dCas9 and gRNA plasmids and viral vectors produced by Human Embryonic kidney (HEK) cells to deliver and stably integrate the dCas9 and gRNA sequences into the host genome would significantly reduce the cost of scale-up and result in lower collagen production costs.
[0166] A CRISPRa process involving transduction of cells using a lentiviral vector is illustrated in
[0167] For transduction, once the virus production pipeline is established, viral plasmids are cheaply generated by bacteria, transfected into HEK cells that readily package and excrete a supply of viral particles, and added to host cell media for integration of gene of interest into host cell's genome.
[0168] The first decision to make for scaling up the collagen production process with viral delivery is deciding what kind of virus to use. Briefly, the benefits of using AAVs (adeno-associated viruses) include high titers, versatility from the availability of multiple serotypes that target different cell types, low toxicity as the virus remains in the episome or in a specific locus on chromosome 19, and low immunogenicity as there is minimal host immune response. The benefits of LVs include that they infect nearly all mammalian cell types, they can be used to deliver relatively large DNA sequences-usually about 5-6 kb in length, and they can be used to generate stable cell lines or drive stable gene expression in organs and tissues in vivo due to integration of the transgene at random locations in the genome. Because the LVs allow for quick and easy stable integration of transgenes, they are a clear choice here, especially because the same class of LV could be used for any cell type that is chosen to work with (epidermal fibroblasts, corneal fibroblasts, iPSCs, etc.). AAVs might be considered if there was a desire to develop a system to be used in a patient that targets a specific tissue type for increased collagen production. The details of the components that make up a LV and how plasmids can be used to construct LVs are further detailed herein in the example shown in
[0169] There are three main genes involved in lentivirus construction: env, gag, and pol. Env, or recombinant VSV-G, (
[0170] The last component is the transfer plasmid, which carries the vector of interest to be integrated into a host cell. This vector is usually engineered with restriction enzymes before being inserted into a bacterial cell for replication. As such, the transfer plasmid has a viral region with the vector of interest (and some other needed components) and a non-viral region with an antibiotic resistance gene. This antibiotic resistance is used for selection against bacteria without the new engineered plasmid following transformation (plasmid insertion). The viral region is set off by the aforementioned LTRs. In addition to acting as a promoter once in the host genome, these LTRs on either side of the vector of interest allow the vector to be recognized by multiple other viral proteins in the virus production and transduction process.
[0171] Once in the host genome, transcription of this vector has two main functions. The first is to select against cells that have not been transduced. In the transgene there is an antibiotic resistance gene that is transcribed by host cells (
[0172]
[0173] A lentiviral vector for expressing dCas9-VPR is commercially available (Dharmacon Edit-R CRISPa Lentiviral dCas9-VPR). This can be used together with another vector encoding the gRNA specific for the target gene, either as a single guide RNA molecule or as separate crRNA and tracrRNA molecules complexed together to form a guide RNA molecule.
[0174] Research has demonstrated that using glass as a substrate for fibroblast cells yields more collagen production than in culture. When cells are placed on a substrate that is not encountered in vivo, they produce collagen as a method of isolating themselves from the plate.
[0175] Fibroblasts also have a tendency to spread out collagen across a substrate of a higher stiffness, such as glass, rather than clumping. Following this, collagen layers would begin to stack under the cells on the plate. In most collagen producing studies, the amount of collagen produced is limited by the substrate size.
[0176]
[0177] The apparatus includes a series of stacked glass slides constrained on either fluid inlet/outlet end by jigs and watertight side walls on non-flow sides (
[0178] Following flow shearing, the culture and collagen fibrils can be removed from the glass plates and the amount of type I collagen could be assayed to determine how much was produced.
[0179] An alternative method of collagen growth using glass as a substrate is to utilize glass beads. An example device is depicted in
Examples
Example 1. CRISPR Using Pooled Transfection
[0180] Three levels of transfection reagent were compared for a CRISPR design. The transfection reagent was DharmaFECT 1 Transfection Reagent as shown in Table 2. In all cell culture media, the media should be concentrated at least 10 times using a dialysis (50 Daltons molecular weight cutoff) against PBS to promote propeptide cleavage. The high, medium, and low, transfection reagent concentrations were 7.2, 4.05, and 0.9 L of transfection reagent per well, respectively. Media was harvested every 12 hours and pooled in two-day segments. Collagen amount per well was quantified every two days, and cell counts were performed after the end of media harvesting. Collagen produced per cell was estimated based on the number of cells per well at the end of media collection. A comparison of total (days 1-6) pooled collagen production (per well, not per cell) for each concentration level and the cell count is shown in
[0181] Quantifications that fell below the minimum detectable collagen amount were set to the minimum detectable collagen amount (1.06 million molecules/cell/hour). Having some quantifications fall below the minimum detectable amount was necessary to capture the upper limits of collagen production in the CRISPR conditions. In other words, we could have not diluted the samples before quantifying collagen, but then the upper limits of CRISPR collagen production would have been above the upper threshold for collagen quantification and not have been detectable. Note that days 1-2 data and most control samples were at or below the minimum detectable collagen amount, but every datapoint for CRISPR results for days 3-4 and 5-6 were above the minimum threshold. This is important for when fold change and statistical significance was calculated, as the actual collagen production for the control wells is likely much lower than what we are saying it is. Lastly, early timepoints (days 1-2) matching the control wells makes sense as the CRISPR was expected to upregulate collagen production after about two days. Based on the graph in
[0182] An important feature of
[0183] The peak of collagen production seen using CRISPR showed about a 90.29-fold increase (42.1% std) in collagen production compared to the experimental control.
[0184] Cells that have been genetically modified with CRISPR can be cultured in media containing the top performing chemical conditions found in Example 2, including a control with CRISPR cells in traditional media and a second control.
[0185] In addition to the scale up methods depicted in
TABLE-US-00012 TABLE 20 Reagents Needed for a Pooled Transduction. Catalog Number Item Name (Dharmacon) CRISPRa Human COL1A1 GSGH11890- Set of 4 EG1277 [71] Lentiviral sgRNA CRISPRa Human COL1A2 GSGH11890- Set of 4 EG1278 [250] Lentiviral sgRNA CRISPRa Human TGF-3 GSGH11890- Set of 4 Lentiviral sgRNA EG7040 [153] QIAprep Spin Miniprep U-002005-05-000 Kit Trans-Lentiviral shRNA TLP5912 [155] Packaging Kit with Calcium Phosphate CRISPRa Lentiviral T-2001-01 [251] BlastdCas9-VPR Particles 10 mM Tris-HCl Buffer pH B-006000-100 7.4
Example 2. Influence of Additives on Collagen Production
[0186] A variety of chemical stimulants were studied to be potentially used to increase the collagen production of fibroblast cells. Acetaldehyde, also known as ethanal, is a derivative of ethanol that has been shown to increase the levels of collagen produced in baboon liver myofibroblasts and human dermal fibroblasts when added to the culture media in concentrations up to 300 M.
[0187] Ascorbic acid is known to be beneficial to the production of collagen in cell types including bovine, mouse, and human. It was hypothesized that the addition of caffeine to media could be beneficial to any lentiviral based CRISPR design solutions because of its demonstrated effect on increasing the activity of lentivirus in the gene therapy space. However, caffeine was shown to have a negative effect on collagen production in concentrations in media as little as 1-5 mM. This is most likely due to the inhibition of the accumulation of several growth factors, including interleukin-8.
[0188] While in high concentrations the addition of ethanol to cell culture media is commonly known to be extremely negative, a number of studies have been performed at concentrations of 50 mM in order to hypothesize the cause of alcoholic liver fibrosis. While fibrosis is commonly synonymous with an increase in the amount of collagen present in tissue, studies performed at these concentrations in myofibroblasts and fibroblasts report no direct effect on the level of collagen production.
[0189] Glutamate has not been studied extensively for its impact on collagen production despite its close relationship to glutamine, which has been known to be a very positive influence on collagen transcription rates. One study showed a 400% increase from the control in glutamate-supplemented media used to culture human dermal fibroblasts.
[0190] Many studies have been conducted in order to observe the response of human dermal fibroblasts in the presence of glutamine in media. Concentrations seen in these studies range up to 10 mM, demonstrating a maximum benefit for the production of collagen of nearly 300% of the control at 250 M. This effect has been thought to increase the level of collagen gene transcription through its conversion into pyrroline-5-carboxylate.
[0191] Hyaluronic acid showed a neutral effect on collagen production in human dermal fibroblasts plated on an undisclosed surface at 500 pg/ml in media. It increased the rate of cell division and general fibril production when present at 150 pg/ml in media for human dermal fibroblast cultures plated on collagen, however researchers did not specifically quantify the production of collagen. When it was incorporated into the culture surface at a ratio of 1:19 hyaluronic acid to collagen by weight, it increased the amount of collagen produced by embryonic chick fibroblasts up to 230% of control. Based on the information presented in these studies and the well-known tendency of cells to stop producing collagen once enveloped in it, the group hypothesized that it interferes with a feedback process, fooling cells into making more collagen than they actually require for a suitable microenvironment.
[0192] There has been a demonstrated positive correlation between the level of Insulin-like Growth Factor 1 (IGF-1) in media and the production of collagen by manipulation of rat serum applied to media used to culture human dermal fibroblasts. Other studies quantify these values in human lung fibroblasts at a maximum of a 300% increase from control at a concentration of 100 ng/mL. Macroscopically, this effect can be seen in diabeteic individuals who are slow to heal wounds or suffer from accelerated atherosclerosis.
[0193] Interleukins (IL) encompass a wide range of glycoproteins associated with immune response. Researchers have looked into types 1p, 4, 6, 8, 10, and 13 for their specific effect on collagen production. IL-4 has demonstrated a maximum positive effect of a 250% increase from control. The lowest concentration of any of these types that is needed for an observable, positive effect is IL-1p at 2.5 M in human chondrocytes. Out of the six types listed above, only IL-10 was found to reduce the level of collagen production.
[0194] Lactate is commonly found in high concentrations in the body after alcohol consumption, especially in individuals suffering from alcoholic liver fibrosis. Therefore, its addition into media could lead to an increase in collagen production even outside of the whole organ system. One study seems to agree with this logical argument by demonstrating a statistically significant increase in collagen production upon addition of 5 mM to media used to culture baboon liver myofibroblasts. However, in human dermal fibroblasts a concentration of 40 mM was shown to decrease collagen production.
[0195] Lathyrogens have been used to inhibit the formation of collagen crosslinks without cytotoxic effects. The most popular lathrogen used in cell culture is -aminopropionitrile (BAPN) which operates by irreversibly blocking lysyl oxidase. Other cellular effects include prevented development of adhesive strength and a buildup of GuHCI-extractable collagen crosslink precursors. No research with any cell type has shown adverse effects on cell viability, collagen synthesis, or non collagen protein synthesis. However, one research study demonstrated inhibited fibroblast migration in a dose-dependent fashion at 0.25 and 0.5 mM BAPN. Previous research has used BAPN successfully at concentrations of 0.1 mM-0.5 mM.
[0196] Proline stabilizes collagen during post translational modifications. One study found a range of concentrations from 5-10 mM in media applied to human dermal fibroblasts that resulted in a maximum increase of 200% in collagen production as compared to the control value.
[0197] The effects of pyrroline-5-carboxylate on collagen production have been documented multiple times in human dermal fibroblasts. Information presented in these studies suggests an optimal concentration of 1 mM for a maximum increase of 260% of the control value. Interestingly, this effect can be seen in as little as 6 hours. It is thought to have such a potent effect because it enables IGF-1. Additionally, it can be converted to proline.
[0198] The subfamilies within the TGF- family all have been shown to have a positive impact on collagen production. Generally, it has a positive effect on the number of ribosomes in the cell, the organelle responsible for the translation of all proteins, including collagen. TGF-1 applied to rat liver M cells at a concentration in media as low as 1 ng/ml demonstrated an unquantified increase in collagen production from control. It has been shown that types of collagen produced by TGF- vary, with collagen type I being especially associated with TGF-3. Sources seem to agree on a concentration of 12.5 ng/ml for maximum efficacy in human dermal fibroblasts.
[0199] Based on a weighted scoring (Table 13), the seven best additives were selected and were added to the fibroblast media separately in a Phase 1 screening study using concentrations presented in Table 14. Table 14 shows the additives and concentrations to be tested. A common concentration cited in literature should be a standard condition, plus one concentration at 50% of that value, and another concentration at 150% that value (Table 14). Positive effects of these additives have been shown in other cell lines, but its main effects on corneal fibroblasts need to be explored independently before combinations of additives can be tested. This screening of factors phase is common in designed experiments in bioengineering applications. Specifically when using a factorial design for media composition it is recommended to perform an screening experiment of the unknown domain before applying advanced designs that allow optimization. Screening studies can be done in a number of ways. The simplest screening experimental design is each variable at two levels, however this assumes a linear relationship between the input and output. For this study a three-level design was chosen in order to determine a maxima. This phase of the experiment determined which additives have a statistically significant effect on collagen production and what concentration of each additive has the highest positive impact. This data was fed into Phase 2 of the experiment where the top performing additives were used within their optimal range of concentrations.
[0200] The seven additives also allowed for minimizing the number of plates and maximizing the number of used wells. This resulted in 3 plates, each with a standard media control group. In phase 1 of testing 7 media additives were tested at 3 concentrations with a sample size of 4 for each condition. Additives and concentrations used were determined by the prior research described above. Collagen amount per well was quantified, but cell counts were not performed. Collagen produced per cell was estimated based on the number of cells expected per well. The results are shown in Table 15. Some groups fell below the SirCol standard curve range of 1-50 ug. These values were represented as 1.00 ug/100 uL and were assigned the lowest rating for efficacy in the resulting trade study.
[0201] Three different top performing additives were identified (BAP, ACE, and ASC). Based on single additive performance, with only three media additives resulting in higher collagen production than the control, the three different additives went on to the DOE. For the three additives that advanced the highest ranking of each additive was used as the DOE centerpoint. Due to a lack of available stock, ACE low was substituted for ACE medium. As a result of these modifications, the final selections from Phase 1 were BAP low, acetaldehyde low, and ascorbate low for the centerpoint and all three at medium concentration for the high point (100%).
[0202] In phase 2 the top performing media additives were fed into a full factorial DOE. The concentrations used were determined by a variety of factors including a trade study, remaining laboratory supplies, and finally the lower cost associated with a lower concentration in a near-tie situation. A near-tie situation was defined by the group as concentrations of the same chemical that scored within 1% total score. For BAP, the concentration in the centerpoint was determined to be the low concentration from phase 1 at 0.25 mM due to it scoring the highest in the trade study. For ascorbate, a near-tie situation was observed so the low concentration from phase 1 at 0.5 mM was chosen. For acetaldehyde, due to a stock issue the low concentration at 0.2 mM was chosen. For the 100% concentration limit needed to complete construction of the full-factorial DOE plan, each of these values was doubled, which corresponds to the medium conditions from phase 1.
[0203] As shown in
REFERENCES
[0204] Wu, F., Nerlich, M., & Docheva, D. (2017). Tendon injuries: Basic science and new repair proposals. EFORT open reviews, 2(7), 332-342.; doi.org/10.1302/2058-5241.2.160075. [0205] (nhs.uk/conditions/hand-tendon-repair/recovery/) Recovery Hand Tendon Repair. (2018, June 14). Retrieved Feb. 13, 2020, from nhs.uk/conditions/hand-tendon-repair/recovery/. [0206] (orthocarolina.com/media/how-does-a-patch-repair-a-rotator-cuff-tear) Rotator Cuff Surgery. (2018, May 21). Retrieved Feb. 16, 2020, from orthocarolina.com/media/how-does-a-patch-repair-a-rotator-cuff-tear. [0207] Vig K, Chaudhari A, Tripathi S, Dixit S, Sahu R, Pillai S, Dennis V.A., Singh S.R. Advances in Skin Regeneration Using Tissue Engineering 18, 789, 2017. [0208] Yao, J. Q., Blanchard, C. R., & Bloor, S. (2005). Zimmer Collagen Repair Patch for Rotator [0209] Cuff Tendon Repair. Zimmer, Inc.
[0210] OKeefe, R. J., Chu, C. R., Jacobs, J. J., & Einhorn, T. A. (2013). Orthopaedic basic science: foundations of clinical practice (4th ed.). Rosemont, IL: American Academy of Orthopaedic Surgeons. [0211] Packer, H. (2016, February 04), reprinted with permission from CRISPR guide RNA format affects genome editing outcomes; retrieved from (idtdna.com/pages/education/decoded/article/crispr-guide-rna-format-affects-genome-editing-). [0212] Biolabs: (neb.com, 2019), reprinted with permission from New England Biolabs from neb.com. [0213] Biolabs, 2019: Biolabs, N. E. CRISPR/Cas9 & Targeted Genome Editing: New Era in Molecular Biology. Retrieved from (neb.com/tools-and-resources/feature-articles/crispr-cas9-and-targeted-genome-editing-a-new-era-in-molecular-biology). [0214] Lodish H, Berk A, Zipursky SL, et al., Molecular Cell Biology. 4th edition. New York: W. H. Freeman; 2000. Section 22.3, Collagen: The Fibrous Proteins of the Matrix; available from: (ncbi.nlm.nih.gov/books/NBK21582/). [0215] Schleip, R. (2012). Fascia: the tensional network of the human body the science and clinical applications in manual and movement therapy. Edinburgh: Churchill Livingstone/Elsevier. [0216] NIH, COL1A1 and COL1A2 genesGenetics Home Reference(2019, May 28). Retrieved from (ghr.nlm.nih.gov/gene/COLlAl) and retrieved from (ghr.nlm.nih.gov/gene/COLlA2). [0217] Gelse, K., Poschl, E., & Aigner, T. (2003). Collagens-structure, function, and biosynthesis. Advanced Drug Delivery Reviews. Available from: (core.ac.uk/download/pdf/2756387.pdf). [0218] Wu M, Crane JS. Biochemistry, Collagen Synthesis. [Updated 2019 Apr 21]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2019 Jan. Available from: (ncbi.nlm.nih.gov/books/NBK507709/). [0219] Stuart, J. M., Townes, A. S., & Kang, A. H. (1982). Nature and specificity of the immune response to collagen in type II collagen-induced arthritis in mice. The Journal of clinical investigation, 69(3), 673-683. doi:10.1172/jci110495. [0220] Lynn, A. K., Yannas, I. V., & Bonfield, W. (2004). Antigenicity and immunogenicity of collagen. Journal of Biomedical Materials Research, 71B(2), 343-354. doi:10.1002/jbm.b.30096. [0221] Tuteja, R. (2005). Type I signal peptidase: An overview. Archives of Biochemistry and Biophysics. https://doi.org/10.1016/j.abb.2005.07.013. [0222] Bonfanti, L., Mironov, A. A., Martinez-Menarguez, J. A., Martella, O., Fusella, A., Baldassarre, M., . . . Luini, A. (1998). Procollagen traverses the Golgi stack without leaving the lumen of cisternae: Evidence for cisternal maturation. Cell. https://doi.org/10.1016/S0092-8674(00)81723-7. [0223] Uchio, Y., Ochi, M., Matsusaki, M., Kurioka, H., & Katsube, K. (2000). Human chondrocyte proliferation and matrix synthesis cultured in Atelocollagen gel. Journal of Biomedical Materials Research, 50(2), 138-143. doi:10.1002/(sici)1097-4636(200005)50:2<138::aid-jbm7>3.0.co;2-k. [0224] CRISPR Guide. (n.d.). Retrieved from (addgene.org/crispr/guide/). [0225] CRISPR Guide Design Software. (n.d.). Retrieved from (benchling.com/crispr/) [0226] COL1A1 crRNA Pool: horizondiscovery.com/en/crisprmod/crispra/products/crispra-crrna?nodeid=entrezgene-1277 [0227] COL1A2 crRNA Pool: [0228] horizondiscovery.com/en/crisprmod/crispra/products/crispra-crrna?nodeid=entrezgene-1278 [0229] TGF-3 crRNA Pool: [0230] horizondiscovery.com/en/crisprmod/crispra/products/crispra-crrna?nodeid=entrezgene-7043 [0231] Borawski, J., et al., Optimization Procedure for small interfering RNA Transfection in a 384-well format. J. Bimolecular Screening. 12, 546-559 (2007). [0232] Pellegrini, A. (2016, February 1). How to Design gRNAs to Target Your Favorite Gene [Web log post]. Retrieved from (blog.benchling.com/2016/02/01/how-to-design-grnas-to-target-your-favorite-gene/). [0233] Wyatt, D., & Ramsden, D. (2015, April 16). CRISPR 101: Non-Homologous End Joining [Web log post]. Retrieved from (blog.addgene.org/crispr-101-non-homologous-end-joining). [0234] Mohr, S. E., Hu, Y., Ewen-Campen, B., Housden, B. E., Viswanatha, R., & Perrimon, N. (2016). CRISPR guide RNA design for research applications. The FEBS journal, 283(17), 3232-3238. https://doi.org/10.1111/febs.13777. [0235] Zhang, Feng; Wen, Yan; Guo, Xiong; CRISPR/Cas9 for genome editing: progress, implications and challenges, Human Molecular Genetics, Volume 23, Issue R1,15 Sep. 2014, Pages R40-R46, (doi.org/10.1093/hmg/ddul25). [0236] COL1A1 gene, 2019 Genetics Home ReferenceNIH. (2019, May 28). Retrieved from (ghr.nlm.nih.gov/gene/COL1A1). [0237] COL1A2 gene, 2019 Genetics Home ReferenceNIH. (2019, May 28). Retrieved from (ghr.nlm.nih.gov/gene/COL1A2). [0238] Introduction to Transfection. no date (n.d.). Retrieved from (thermofisher.com/us/en/home/references/gibco-cell-culture-basics/transfection-basics/introduction-to-transfection.html). [0239] Human COL1A1 gene (1277). (n.d.). Retrieved from (dharmacon.horizondiscovery.com/biology-overview-entrezgene-1277-col1a1/). [0240] MeSH Browser: Transduction, Genetic. (n.d.). Retrieved from (meshb.nlm.nih.gov/record/ui?name=Transduction, Genetic). [0241] Edit-R CRISPRa crRNA. (n.d.). Retrieved from (dharmacon.horizondiscovery.com/crispr-activation/crispra-guide-rna/edit-r-crispra-crrna/?term=&p [73]005D982AF75C&sourceld=entrezgene/1277). [0242] Multiplex CRISPR/Cas9-based genome engineering from a single lentiviral vector. (n.d.). Retrieved from (addgene.org/browse/article/8975/). [0243] Kabadi, Ami M.; David G. Ousterout, Isaac B. Hilton, Charles A. Gersbach, Multiplex CRISPR/Cas9-based genome engineering from a single lentiviral vector, Nucleic Acids Research, Volume 42, Issue 19,29 October [145] 2014, Page e147, (doi.org/10.1093/nar/gku749). [0244] Inspired Cell Solutions, 2018: Dharmacon Edit-R CRISPRa transcriptional activation system with synthetic guide RNA. (2018). Cambridge, MA: Horizon [150] Discovery Group Company. [0245] Electroporation, 2015: Electroporation of Dharmacon Edit-R dCas9-VPR mRNA and synthetic guide RNA for gene modulation.. (2015, October). Retrieved from (dharmacon.horizondiscovery.com/uploadedFiles/Resources/dcas9-vpr-mrna-electroporation-protocol). [0246] Transfection Protocol. (2016, September). Retrieved from (dharmacon.horizondiscovery.com/uploadedFiles/Resources/dcas9-vpr-mrna-quick-protocol.pdf). [0247] Human TGF1 gene (7040). (n.d.). Retrieved from (dharmacon.horizondiscovery.com/biology-overview-entrezgene-7040-tgfb1/). [0248] Transcriptional Activation System, 2018: Dharmacon Edit-R transcriptional activation system with expressed sgRNA. (2018). Retrieved from dharmacon.horizondiscovery.com/uploadedFiles/Resources/edit-r-crispra-lentiviral-guide-rna-manu). [0249] Trans-Lentiviral shRNA Packaging System, 2016: Trans-Lentiviral shRNA Packaging System. (n.d.). Retrieved from (dharmacon.horizondiscovery.com/viral-packaging/trans-Ientiviral-shrna-packaging-system/). [0250] Mandenius, C.-F. and Brundin, A. (2008), Bioprocess optimization using design-of-experiments methodology. Biotechnol Progress, 24: 1191-1203. doi:10.1002/btpr.67. [0251] Wang Y.-H,Yang B,Ren J,Dong M.-L,Liang D,Xu A.-L. Optimization of medium composition for the production of clavulanic acid by Streptomyces clavuligerus. Process Biochem. 2004; 40:1161-1166. [0252] Jinek, M., et al. A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity. Science. 337(6096), 816-821 (2012). [0253] Tanenbaum, M. E., et al. A Protein Tagging System for Signal Amplification in Gene Expression and Fluorescence Imaging. Cell 159(3):635-646 (2014).