Patent Application
20230090462
The disclosure relates to cells and methods for treatment of retinal degenerative disease.
Retinal degenerative diseases, which lead to the death of rod and cone photoreceptor cells, are the leading cause of inherited vision loss worldwide. Induced pluripotent or embryonic stem cells (iPSCs/ESCs) have been proposed as a possible source of new photoreceptors to restore vision in these conditions. Studies carried out in mouse models of retinal degeneration over the past decade have highlighted several limitations for cell replacement in the retina, such as the low efficiency of cone photoreceptor production from stem cell cultures and the poor integration of grafted cells in the host retina.
The invention provides a solution to the limitations and drawbacks associated with previous approaches in the isolation, purification and enrichment of progenitor cells. For example, the invention provides a purified or enriched population of photoreceptor precursor cells, for example, cone precursor photoreceptor cells (CPCs), methods of producing these cells and use of the cells for the treatment of ocular disorders, e.g., retinal degenerative diseases, and other diseases. The invention encompasses methods for the isolation, purification and expansion of progenitor cells using a microfluidic based cell sorting approach. The method is a scalable GMP capable protocol for production, e.g. of human cones, providing a unique ability to create universal, allogenic, cone photoreceptor cells for preserving and restoring vision.
Accordingly, in certain embodiments, a composition comprising a purified population of cells, wherein the cells are CD73+, Thyroid Hormone Receptor beta (Thrb+), CD 11b−. In this and other embodiments, the purified population of cells are derived from: embryonic retinas, embryonic retinal tissues, embryonic stem cells, mesenchymal stem cells, induced pluripotent stem cells (iPSCs), or iPSC-derived retinal organoids. In this and other embodiments, the purified population of cells are derived from embryonic retinas. In certain embodiments, the purified population of cells comprise at least 50% of a total number of cells in the composition having an expression marker profile of CD73+, Thrb+, CD11b−. In certain embodiments, the purified population of cells comprise at least 75% of a total number of cells in the composition having an expression marker profile of CD73+, Thrb+, CD11b−. In certain embodiments, the purified population of cells comprise at least 75% of a total number of cells in the composition having an expression marker profile of CD73+, Thrb+, CD11b−. In certain embodiments, the purified population of cells comprise at least 85% of a total number of cells in the composition having an expression marker profile of CD73+, Thrb+, CD11b−. In certain embodiments, the purified population of cells comprise at least 90% of a total number of cells in the composition having an expression marker profile of CD73+, Thrb+, CD11b−. In certain embodiments, the purified population of cells comprise at least 95% (or more) of a total number of cells in the composition having an expression marker profile of CD73+, Thrb+, CD11b−. CD11b−marker indicates the lack of microglia and other cells which express CD11b, e.g. monocytes, granulocytes, macrophages NK cells etc., in the purified cell population, thereby, the cells in the composition are not immunogenic. In these and other embodiments, an isolated cell having an expression marker profile of CD73+, Thrb+, CD11b−comprises a vector expressing a polypeptide, an exogenous nucleic acid sequence, polynucleotides, oligonucleotides, polypeptides, peptides or combinations thereof.
Other markers for cone photoreceptor cells, include without limitation: RXRG (retinoid X receptor (RXR) gamma), THRB (thyroid hormone receptor beta), SALL3 (Sall-like protein 3), ONECUT1 (One cut domain, family member 1), OPN1SW (Opsin 1 short wavelength), OPN 1 LW/MW (Opsin 1 long wavelength and medium wavelength), ARR3 (Arrestin -C-3), GNAT2 (G-protein subunit alpha transducing 1), CNGB3 (cyclic nucleotide-gated (CNG) channel beta subunit), PDE6H (phosphodiesterase 6H), PDE6C (phosphodiesterase 6C), GUCA1A (guanylate cyclase activator 1A).
Accordingly, in these and other embodiments, a cone photoreceptor cell comprises one or more markers comprising: RXRG, THRB, SALL3, ONECUT1, OPN1SW, OPN 1 LW/MW, ARR3, GNAT2, CNGB3, PDE6H, PDE6C, GUCA1A or combinations thereof. In certain embodiments, an early cone photoreceptor cell comprises one or more markers comprising: RXRG, THRB, SALL3, ONECUT1 or combinations thereof. The markers: RXRG, THRB, SALL3, ONECUT1 are markers for early cone photoreceptor. The markers: OPN1SW, OPN 1 LW/MW, ARR3, GNAT2, CNGB3, PDE6H, PDE6C, GUCA1A are markers for mature cone photoreceptor cells.
In certain embodiments, a method of producing progenitor photoreceptor cells, comprises culturing retinal progenitor cells; isolating CD73+cells from the cultured retinal progenitor cells; culturing the CD73+cells; subjecting the CD73+cells to a second isolation step comprising isolating CD73+Thrb+ and CD11b−cells; culturing and expanding the CD73+Thrb+CD11b−cells; to produce the progenitor photoreceptor cells. In these and other embodiments, the retinal progenitor cells are derived from embryonic retinas, embryonic stem cells, mesenchymal stem cells, induced pluripotent stem cells (iPSCs), or iPSC-derived retinal organoids. In certain embodiments, the retinal progenitor cells are derived from embryonic retinas or embryonic retinal tissues. In certain embodiments, the embryonic retinas or retinal fetal tissues are contacted with an enzyme to obtain a cell suspension. In these and other embodiments, the CD73+Thrb+CD 11 b−cells comprise at least 90% of the total cell counts. In these and other embodiments, the CD73+Thrb+CD11b−cells comprise at least 95% of the total cell counts.
In certain embodiments, a method of producing progenitor photoreceptor cells, comprises: obtaining embryonic retinas or retinal tissues and dissociating the embryonic retinas or retinal tissue with an enzyme to produce a cell suspension; culturing cells obtained from the cell suspension; isolating CD73+cells from the cell culture and further culturing CD73+cells; subjecting the CD73+cells to a second isolation step comprising isolating CD73+Thrb+ and CD 11 b−cells; culturing and expanding the CD73+Thrb+CD 11 b−cells, to produce the progenitor photoreceptor cells. In these and other embodiments, the progenitor photoreceptor cells can be cultured with one or more agents or culturing conditions to produce a desired mature cell phenotype. In these and other embodiments, the one or more agents comprise growth factors, cytokines, reprogramming factors, hormones, cells, tissues or combinations thereof. In certain embodiments, the culturing conditions comprise: culturing substrates, co-culturing environment, two- or three-dimensional culturing. In certain embodiments, progenitor photoreceptor cells are precursors of cone photoreceptor cells. In these and other embodiments, the cone photoreceptor cells identified by markers comprising: Cone Arrestin+, Red/G opsin+Rhodopsin−.
In certain embodiments, a method of producing a purified population of progenitor cells, comprises isolating cells from a biological sample; culturing and expanding the cells; isolating cells based on a first biomarker profile and further culturing of the isolated cells; subjecting the cultured isolated cells to a second isolation step based on a second biomarker profile; thereby, producing a purified population of progenitor cells. In these and other embodiments, the biological sample comprises: fetal tissues, embryonic tissues, extraembryonic, tissues, cord blood, fluids, cord tissues, bone marrow, adult tissues or combinations thereof.
In certain embodiments, a method of screening for a candidate therapeutic agent comprises contacting a cell embodied herein, with a candidate therapeutic agent; comparing genotypic and/or phenotypic characteristics and/or induction of differentiation of the cell to a baseline control in the presence or absence of the candidate therapeutic agent and correlate responses to specific genetic or phenotypic features. Typical assays known in the art can be used to identify genotypic or phenotypic changes such as, gene analysis, probes, gels, PCR, blots, enzyme assays, immunochemistry assays and the like. In these and other embodiments, the candidate agent is selected for its therapeutic properties, for example, neuroprotective, survival of cells, proliferation, does not kill the cells, induces the engraftment of the progenitors into the tissues, allows for differentiation into a desired cell-type, etc.
In certain embodiments, a method of treating an ocular or retinal disease comprises administering to a subject an effective amount of the isolated and purified population of progenitor cells embodied herein.
Other aspects are described infra.
The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, and biochemistry). It is understood that where a parameter range is provided, all integers within that range, and tenths thereof, are also provided by the invention. For example, “0.2-5 mg” is a disclosure of 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg etc. up to and including 5.0 mg.
As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof are used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising.”
As used herein, the term “about” in the context of a numerical value or range means ±10% of the numerical value or range recited or claimed, unless the context requires a more limited range.
As used herein, the term “agent” or “candidate therapeutic agent” is meant to encompass any molecule, chemical entity, composition, drug, therapeutic agent, chemotherapeutic agent, or biological agent capable of preventing, ameliorating, or treating a disease or other medical condition. The term includes small molecule compounds, antisense reagents, siRNA reagents, antibodies, enzymes, peptides organic or inorganic molecules, natural or synthetic compounds and the like. An agent can be assayed in accordance with the methods of the disclosure at any stage during clinical trials, during pre-trial testing, or following FDA-approval.
In the descriptions above and in the claims, phrases such as “at least one of” or “one or more of” may occur followed by a conjunctive list of elements or features. The term “and/or” may also occur in a list of two or more elements or features. Unless otherwise implicitly or explicitly contradicted by the context in which it is used, such a phrase is intended to mean any of the listed elements or features individually or any of the recited elements or features in combination with any of the other recited elements or features. For example, the phrases “at least one of A and B;” “one or more of A and B;” and “A and/or B” are each intended to mean “A alone, B alone, or A and B together.” A similar interpretation is also intended for lists including three or more items. For example, the phrases “at least one of A, B, and C;” “one or more of A, B, and C;” and “A, B, and/or C” are each intended to mean “A alone, B alone, C alone, A and B together, A and C together, B and C together, or A and B and C together.” In addition, use of the term “based on,” above and in the claims is intended to mean, “based at least in part on,” such that an unrecited feature or element is also permissible The transitional term “comprising,” which is synonymous with “including,” “containing,” or “characterized by,” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. By contrast, the transitional phrase “consisting of” excludes any element, step, or ingredient not specified in the claim. The transitional phrase “consisting essentially of” limits the scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention.
As used herein, “effective” when referring to an amount of a therapeutic compound refers to the quantity of the compound that is sufficient to yield a desired therapeutic response without undue adverse side effects (such as toxicity, irritation, or allergic response) commensurate with a reasonable benefit/risk ratio when used in the manner of this disclosure.
As used herein, a “purified” population of cell types, e.g. progenitor cone receptor cells are substantially free of other cell populations. In certain embodiments, the cell populations are based on a biomarker profile, that is presence and/or absence of certain biomarkers, in the cell population. Purified cell populations comprise at least 50% of a total number of cells in the composition having a certain expression marker profile, for example, an expression marker profile of CD73+, Thrb+, CD11b−. Preferably, the cell population is at least 75%, at least 90%, and at least 99%, pure. For example, a purified cell population is one that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99%, or 100% (w/w) of the desired cell type. Purified also defines a degree of sterility that is safe for administration to a human subject, e.g., lacking infectious or toxic agents.
The terms “subject,” “patient,” “individual,” and the like as used herein are not intended to be limiting and can be generally interchanged. That is, an individual described as a “patient” does not necessarily have a given disease, but may be merely seeking medical advice.
The terms “therapeutically effective amount,” “treatment effective amount” and “effective amount” as used herein are synonymous unless otherwise indicated, and mean an amount of a compound, peptide or composition of the present invention that is sufficient to improve the condition, disease, or disorder being treated and/or achieved the desired benefit or goal (e.g., control of body weight). Those skilled in the art will appreciate that the therapeutic effects need not be complete or curative, as long as some benefit is provided to the subject.
As used herein, “treating” encompasses, e.g., inhibition, regression, or stasis of the progression of a disorder. Treating also encompasses the prevention or amelioration of any symptom or symptoms of the disorder. As used herein, “inhibition” of disease progression or a disease complication in a subject means preventing or reducing the disease progression and/or disease complication in the subject.
All genes, gene names, and gene products disclosed herein are intended to correspond to homologs from any species for which the compositions and methods disclosed herein are applicable. Thus, the terms include, but are not limited to genes and gene products from humans and mice. It is understood that when a gene or gene product from a particular species is disclosed, this disclosure is intended to be exemplary only, and is not to be interpreted as a limitation unless the context in which it appears clearly indicates. Thus, for example, for the genes or gene products disclosed herein, which in some embodiments relate to mammalian nucleic acid and amino acid sequences, are intended to encompass homologous and/or orthologous genes and gene products from other animals including, but not limited to other mammals, fish, amphibians, reptiles, and birds. In preferred embodiments, the genes, nucleic acid sequences, amino acid sequences, peptides, polypeptides and proteins are human.
Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.
All published foreign patents and patent applications cited herein are incorporated herein by reference. Genbank and NCBI submissions or other database references to protein sequence and/or nucleic acide sequences indicated by accession number cited herein are incorporated herein by reference. All other published references, documents, manuscripts and scientific literature cited herein are incorporated herein by reference. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
ThrB was identified, herein, as a surface marker of cone progenitor cells. This is the first time ThrB has been used as a surface marker for labeling cells that were previously impossible to identify. Thrb is a transcription factor that can be released from the membrane into the nuclei or vice versa. The studies, herein, explored the time during which Thrb can be expressed on the cell surface and use it for labeling the cone progenitor cells (ThrB is exclusively expressed only by cone progenitor cells). The technique to use a microfluidic device for isolation of cone progenitor cells also enabled the enrichment of this rare population of cells with the highest purity and viability that has been reported. The microfluidic method allowed for labeling cells with both positive (CD73/IhrB) and the deletion of the population of cells that was unwanted (Cdl lb). The release markers Cone Arrestin, R/Opsin and Blue opsin expression showed >95% positive for these markers. Furthermore, using the culture conditions described herein, these cells proliferated and allowed for obtaining large number of hCP which were transplanted into the rat eyes. This demonstrated the extremely high capacity of the hCPs to survive and engraft into the host retina. No study prior to the disclosure here, has been able to isolate, culture and transplant human cone progenitor cells and holds great promise for future clinical translation.
Cone progenitor cells were isolated, purified and expanded using a microfluidic based cell sorting approach. Isolating and purifying cone cells is important in the treatment of cone related disorders, as heretofore, there has not been a treatment to replace cones. The enriched, pure population of cone precursors meets the need for providing treatment for various cone related disorders. One example is cone dystrophy which is a degeneration of cone cells, photoreceptor responsible for both central and color vision. Stationary cone dystrophy is usually present during infancy or early childhood and symptoms remain throughout the life. Progressive cone dystrophy is associate when symptoms become worse over time. These symptoms usually develop in late childhood or early adulthood. Age-related macular degeneration, also called macular degeneration,(AMD) is a degenerative disease that causes a progressive loss of central vision. The macula is rich in cones.
Accordingly, the invention provides a unique source of high purity, viable, allogeneic human progenitor cells capable of rescuing and or replacing the functions of damaged or dead cells in many retinal diseases such as cone dystrophy and dry AMD. Key to this disclosure is the isolation, high purity and viability of the cells, manufactured in an efficient and effective system. These cells sustain and or improve vision in patients who are losing or have lost vision. In addition, these cells may be used in drug discovery and screening or other uses where retinal cells or their precursors might be needed. The inventors have been successful with two other cell types, and provides evidence that this approach has broad applicability for the isolation of other cell types throughout the body.
In addition, this is the first time pure viable progenitor cells have been isolated from human embryonic tissues using a specific set of surface markers. A novel two stage technique of sorting allows enriching cone population to greater than 95%. These cells are further cultured using growth factors and other conditions that allow further proliferation.
Accordingly, in one strategy, detailed in the examples section which follows, cone progenitor cells (CPCs) were purified, defined based on a proliferative population expressing cone arrestin and Red/Green (r/g) opsin at greater than 80% using a two multistage approach. CPCs dissociated from embryonic retinas (10-16 weeks) or ES or iPS cell retinal organoids were first sorted based on CD73 expression. After expanding this population, the cells were subjected to a second sort consisting of two different positive antibodies (CD73 and Thrb) and one negative to delete the microglia (CD11b). The positive fraction yielded the new cell line.
The cells and methods embodied herein are not limited to photoreceptor progenitor cells but are applicable to isolation and purification of all progenitor cells.
These cells can be used for cell replacement, drug discovery and screening or other uses where photoreceptors or their precursors might be needed.
This approach had broad applicability for use to isolate other cells in the eye, CNS, and throughout the body. Specifically, the inventors been successful in isolating retinal ganglion cells and retinal microglia, two very difficult targets in the eye.
Retinal degenerative diseases are generally characterized by the loss of rod and cone photoreceptors, which are the light-detecting cells of the retina (for a review see, Jones M. K. et al., Prog Retin Eye Res. 2017 May; 58:1-27; Tanna P. et al., Br J Ophthalmol. 2017 January; 101(1):25-30). Depending on the disease and genetic mutation involved, rods or cones degenerate first, causing night blindness and tunnel vision or central and daylight vision loss, respectively.
Several approaches such as gene therapy and neuroprotective factors are being explored to prevent or slow down the loss of photoreceptors, but these strategies are generally not restorative, precluding their use in advanced stages of retinal degenerative diseases. In contrast, cell-based therapies offer the potential to restore vision by replacing lost photoreceptors. Studies done in the mammalian retina have highlighted the limitations of endogenous regeneration in higher vertebrates (Ueki Y. et al., Proc Nat/Acad Sci USA. 2015 Nov 3; 112(44):13717-22. Jorstad N. L. et al., Nature. 2017 Aug 3; 548(7665):103 107), but recent studies have taken advantage of the pioneer factor activity of Ascll to trigger Muller glia reprogramming into neurons in the adult retina (Jorstad N. L. et al., 2017). However, the majority of the cells produced by reprogrammed Muller cells in these conditions were not photoreceptors and the overall efficiency of regeneration was low.
Accordingly, in certain embodiments, a method of producing progenitor photoreceptor cells, comprises culturing retinal progenitor cells isolated from a biological sample; isolating CD73+cells from the cultured retinal progenitor cells; culturing the CD73+cells; subjecting the CD73+cells to a second isolation step comprising isolating CD73++ and CD 11 b−cells; culturing and expanding the CD73+Thrb+CD 11 b−cells; to produce the progenitor photoreceptor cells. In certain embodiments, the cells are cone photoreceptor cells.
The protein encoded by the thyroid hormone receptor beta gene (RefSeq NM_000461. HGNC:HGNC:11799. Ensembl:ENSG00000151090 MIM:190160, hereby incorporated by reference) is a nuclear hormone receptor for triiodothyronine. The nucleic acid sequence for human thyroid hormone receptor beta gene can be found at GenBank Accession No.: NC_000001.11 (hereby incorporated by reference). It is one of the several receptors for thyroid hormone, and has been shown to mediate the biological activities of thyroid hormone. Knockout studies in mice suggest that the different receptors, while having certain extent of redundancy, may mediate different functions of thyroid hormone. Mutations in this gene are known to be a cause of generalized thyroid hormone resistance (GTHR), a syndrome characterized by goiter and high levels of circulating thyroid hormone (T3-T4), with normal or slightly elevated thyroid stimulating hormone (TSH). Several alternatively spliced transcript variants encoding the same protein have been observed for this gene.
An amino acid sequence for human Thrb is publically available in the UniProtKB/Swiss-Prot database under accession number P10828.2 (SEQ ID NO: 1) and is as follows:
The CD11 b gene encodes the integrin alpha M chain(HGNC: 6149 Entrez Gene: 3684 Ensembl: ENSG00000169896 OMIM: 120980) (hereby incorporated by reference). Integrins are heterodimeric integral membrane proteins composed of an alpha chain and a beta chain. This I-domain containing alpha integrin combines with the beta 2 chain (ITGB2) to form a leukocyte-specific integrin referred to as macrophage receptor 1 (‘Mac-1’), or inactivated-C3b (iC3b) receptor 3 (′CR3′). The alpha M beta 2 integrin is important in the adherence of neutrophils and monocytes to stimulated endothelium, and also in the phagocytosis of complement coated particles. Multiple transcript variants encoding different isoforms have been found for this gene.
An amino acid sequence for human CD11 b, isoform 1 is publically available in the NCBI GenBank database under accession number NP_00 11 39280.1 (hereby incorporated by reference) (SEQ ID NO: 2) and is as follows:
An amino acid sequence for human CD11 b, isoform 2 is publically available in the NCBI GenBank database under accession number NP_000623.2 (hereby incorporated by reference) (SEQ ID NO: 3) and is as follows:
Integrin ITGAM/ITGB2 is implicated in various adhesive interactions of monocytes, PGP-23,DNA,M macrophages and granulocytes as well as in mediating the uptake of complement-coated particles and pathogens (PubMed:9558116, PubMed:20008295) (hereby incorporated by reference). It is identical with CR-3, the receptor for the iC3b fragment of the third complement component. It probably recognizes the R-G-D peptide in C3b. Integrin ITGAM/ITGB2 is also a receptor for fibrinogen, factor X and ICAM1. It recognizes P1 and P2 peptides of fibrinogen gamma chain. Regulates neutrophil migration (PubMed:28807980) (hereby incorporated by reference). In association with beta subunit ITGB2/CD18, required for CD177-PRTN3-mediated activation of TNF primed neutrophils (PubMed:21193407) (hereby incorporated by reference). (UniProtKB: P11215 ITAM_HUMAN,hereby incorporated by reference).
CD73 is a cell surface enzyme which is overexpressed in the tumor microenvironment and promotes tumor growth by limiting anti-tumor immunity via the adenosine receptor pathway (HGNC:HGNC:802. Ensembl:ENSG00000135318 MIM:129190. RefSeq NG_028214.1) (hereby incorporated by reference).
A nucleotide sequence that encodes human CD73 is publically available in the NCBI GenBank database under accession number BC065937.1(hereby incorporated by reference) (SEQ ID NO: 4) and is as follows:
An amino acid sequence for human CD73 is publically available in the NCBI GenBank database under accession number AAH65937.1 (hereby incorporated by reference) (SEQ ID NO: 5) and is as follows (the signal peptide is amino acids 1-26 which are bold and underlined):
Other markers for cone photoreceptor cells, include without limitation: RXRG PGP-25,DNA (retinoid X receptor (RXR) gamma), THRB (thyroid hormone receptor beta), SALL3 (Sall-like protein 3), ONECUT1 (One cut domain, family member 1), OPN1SW (Opsin 1 short wavelength), OPN 1 LW/MW (Opsin 1 long wavelength and medium wavelength), ARR3 (Arrestin -C-3), GNAT2 (G—protein subunit alpha transducing 1), CNGB3 (cyclic nucleotide-gated (CNG) channel beta subunit), PDE6H (phosphodiesterase 6H), PDE6C (phosphodiesterase 6C), GUCA1A (guanylate cyclase activator 1A).
An amino acid sequence for human Cone Arrestin is publically available in the UniProtKB/Swiss-Prot database under accession number P36575.2(hereby incorporated by reference) (SEQ ID NO: 6) and is as follows:
An amino acid sequence for human medium wave (green) Opsin is publically PGP-26,DNA available in the NCBI GenBank database under accession number NP_001041646.1(hereby incorporated by reference) (SEQ ID NO: 7) and is as follows:
An amino acid sequence for human long wave (red) Opsin is publically available in PGP-27,DNA the NCBI GenBank database under accession number NP_064445.2 (hereby incorporated by reference) (SEQ ID NO: 8) and is as follows:
An amino acid sequence for human rhodopsin is publically available in the NCBI GenBank database under accession number NP_000530.1 (hereby incorporated by reference) (SEQ ID NO: 9) and is as follows:
Accordingly, in these and other embodiments, a cone photoreceptor cell comprises one or more markers comprising: RXRG, THRB, SALL3, ONECUT1, OPNISW, OPN 1 LW/MW, ARR3, GNAT2, CNGB3, PDE6H, PDE6C, GUCA1A or combinations thereof. In certain embodiments, an early cone photoreceptor cell comprises one or more markers comprising: RXRG, THRB, SALL3, ONECUT1 or combinations thereof. The markers: RXRG, THRB, SALL3, ONECUT1 are markers for early cone photoreceptor. The markers: OPN 1 SW, OPN 1 LW/MW, ARR3, GNAT2, CNGB3, PDE6H, PDE6C, GUCA1A are markers for mature cone photoreceptor cells.
It is to be understood that progenitor cells from any biological sample can be isolated and purified by the methods embodied herein. Accordingly, in certain embodiments, a method of isolating and purifying viable progenitor cells, comprises isolating cells from a biological sample; culturing and expanding the cells; isolating cells based on a first biomarker profile and further culturing of the isolated cells; subjecting the cultured isolated cells to a second isolation step based on a second biomarker profile; thereby, producing a purified population of progenitor cells. In these and other embodiments, the biological sample comprises: fetal tissues, embryonic tissues, extraembryonic, tissues, cord blood, fluids, cord tissues, bone marrow, adult tissues or combinations thereof.
Examples of markers useful to practice embodiments of the invention are as follows:
Embryonic Stem Cells: Embryonic stem cells (ES cells) are pluripotent stem cells derived from the inner cell mass of a blastocyst, an early-stage embryo. The ES cells have high potential to differentiate into a wide variety of cell types. Undifferentiated embryonic stem (ES) and induced pluripotent stem (iPS) cells can be functionally defined by their ability to differentiate into cells derived from the three germ layers the ectoderm, mesoderm, and endoderm that eventually make up to all cell types at a later time. ES cells possess two distinct properties that make them an attractive choice for cell therapy. First, as embryonic stem cells originate from early blastocysts, a very early developmental stage, retain the extraordinary plasticity to become any cell type of approximately 200 cell types that constitute the human body. Given the right combination of signals, embryonic stem cells will develop into mature cells that can function as neurons, muscles, bone, blood or other needed cell types. Another important feature of embryonic stem cells is their ability to remain in an undifferentiated state and to divide indefinitely. These so-called self-renewing cells generate unlimited well-defined, identical, genetically and genomically characterized stem cells.
Cell surface pluripotency markers. Two homeodomain transcription factors, Oct4 and Nanog, were the first proteins identified as essential for both early embryo development and pluripotency maintenance in ES cells. In addition to Oct4, Sox2, and Nanog, many other factors required for pluripotency have been identified, including Sa114, Daxl, Essrb, Tbx3, Tcll, Rifl, Nacl, and Zfp281.
TRA-1-60 and TRA-1-81. TRA-1-60 and TRA-1-81 antigens on the human embryonal carcinoma (EC) cells and human pluripotent stem cell surfaces can be used as markers in identifying and isolating ESCs. They are also routinely used to assess the pluripotency status of induced pluripotent stem (iPS) cells. Both TRA-1-60 and SSEA4 are both expressed on human embryonal carcinomas and on human embryonic stem cells. Upon differentiation, TRA-1-60 and SSEA4 expression levels decrease and SSEA1 expression increases on human embryonic stem cells over time when treated with Retinoic acid. Besides, they also express CD349/frizzled-9, stage-specific embryonic antigen (SSEA)-4, Oct-4, Nanog, and nestin. Oct-4 and Nanog, as well as several cell surface markers (SSEA-1, SSEA-4, TRA-1-60, and TRA1-81) have been used to characterize mouse and human embryonic stem cells (ESC).
Stage-specific embryonic antigens (SSEA). Conventionally, markers used for mESCs, mouse embryonic carcinomas (ECs), or human EC cells, were exploited to identify undifferentiated human embryonic stem cells (hESCs). At the pre-implantation stage, murine embryos, human germ cells, and teratocarcinoma stem cells express certain molecular receptors known as Stage-Specific Embryonic Antigens (SSEA) on their membrane surface. It is now widely recognized that SSEAs, sphingolipids, identified as a key player in identifying cells endowed with pluripotent and stem cell characteristics. During oogenesis and embryogenesis, SSEA-3 and SSEA-4 are expressed in undifferentiated primate ESC, human embryonic germ (EG) cells, human teratocarcinoma stem cells, and ESC. Currently, Stage-specific embryonic antigen-3 (SSEA-3) and SSEA-4 have been recognized to characterize undifferentiated hESCs but not on undifferentiated mESCs.
Frizzled (Fzd) and Cripto (7DGF-1). A family of Frizzled proteins is expressed in mouse and human ESC. Wnt signals execute their functions via the Fzd family receptors by bind to Fzd and the co-receptors LRP5 or LPR6, and eventually activate the Wnt/β-catenin pathway. Cripto-1 plays a crucial role for early embryonic development and has been associated with the undifferentiated status of mouse ES and human ES cells. During development, Cripto acts as a receptor for TGF-β ligands, including GDF1 and GDF3. In addition to having essential functions during embryogenesis, as an oncogene, Cripto is upregulated in tumors and promotes tumorigenesis. Frizzled (FZD) proteins comprise a family of transmembrane-spanning receptors (FZD1-10) activated by Wnt ligands. FZD9 can be activated by Wnt 2 and Wnt 8 via the canonical pathway, and by Wnt 7 via non-canonical signaling. Mouse FZD9 is present in the developing brain, in neural precursor cells in the developing neural tube, and in myotomes.
Transcription factors. The fundamental implication of transcription factors in the pluripotency maintenance was clearly exemplified by the fact that pluripotent stem cells can be derived from mouse embryonic fibroblasts by inducing transcription factors expression. In addition to extrinsic factors, the pluripotency of ES cells also depends on intrinsic determinants, such as the expression of the POU transcription factor. As such, these induced pluripotent stem (iPS) cells were developed via the overexpression of a set of specific genes Oct4, c-Myc, Sox2, and Klf4. Abundant levels of Oct4, Sox2, and Klf4 reprogram fibroblasts into the iPS cells with a pluripotent state.
A combination of either Klf4 or c-Myc with Oct4 is sufficient to generate iPS cells from NSC. Oct3/4 is specifically expressed in pluripotent stem cells. The Sox family of genes is associated with multipotent and unipotent stem cells. Sox1 induces iPS cells with similar efficiency as Sox2, and genes Sox3, Sox 15, and Sox 18 generate iPS cells, although with decreased efficiency. Klf5 has been implicated in the transcription of Oct3/4 and Nanog, and ESCs renewal and pluripotency maintenance. Klf4 and Klf2 can regulate the expression of certain transcription factors: Nanog, Tcll, Esrrb, Sa114, Tcf3, Mycn and Fbxo15. Nanog is a transcription factor and plays a crucial role in the maintenance of pluripotency and self-renewal in mouse and human ESCs and is downregulated upon ESC differentiation, which is consistent with an intimate association with pluripotent stem cell identity. Nanog has been implicated in pluripotent ES and EG cells, as well as in both mouse and human EC cells.
Induced-PS cell markers. Fully reprogrammed human iPS cells, compared to transiting or incompletely reprogrammed cells, are endowed with the important features (i) downregulation of CD 13, a fibroblast marker, (ii) upregulation of expression of SSEA-4 and TRA-1-60like pluripotent markers, (iii) silencing of viral transgenes (iv) endogenous expression of Nanog (v) a low Hoechst retaining or high Hoechst pump out potential. Similar to expression pattern found in hESC, human iPSCs also express the SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, TRA-2-49/6E, and Nanog. Gene expression and genome-wide H3K4me3 and H3K27me3 were found to be extremely similar between ES and iPS cells/. In mouse, iPSCs expressed genes expressed in undifferentiated ESCs, including Oct-3/4, Sox2, Nanog, GDF3, REX1, FGF4, ESG1, DPPA2, DPPA4, and hTERT. Similar to mESCs, mouse iPSCs do not express SSEA-3 and SSEA-4 but both cell types commonly express SSEA-1.
Germ cell markers. The precursors of primordial germ cells developed from 4-8 cells in E6.25 proximal epiblast express the transcriptional repressor Blimpl (Ohinata Y. et al., Nature. 2005; 436:207-13). Over time, these Blimpl-positive cells continuously proliferate and initiate the expression of Fragilis and Stella by E7.5 (Mise N et al., Genes Cells. 2008; 13:863-77). Reports indicate that the expression of Blimpl, Stella, Fragilis, Piwil2, Dazl, and MVH in ES cells (Mise N et al., 2008; Geijsen N. et al., Nature. 2004; 427:148-54), indicating the origin of ES cells could be germline. Depending on the stage, certain markers like Oct4, c-kit are expressed early and downregulated prior to mature Germline Specification stages. Tekt 1 and GDF9 markers are induced only at later stages. Nanos has been implicated in all development stages from blastocyst to mature sperm or oocyte.
Ectoderm and endoderm markers. Ectoderm is one of the three primary germ cell layers in the very early embryo. The other two layers are the mesoderm (middle layer) and endoderm (most proximal layer), with the ectoderm as the most exterior (or distal) layer. Certain factors mark the ectoderm including Otx2, Chordin, p63/TP73L, FGF-8, Pax2. FoxJ3, Pax6, GBX2, SOX1, Nestin, beta- Tubulin, and Noggin. Endoderm formation depends on two sequential positive feedback loops mediated by Cripto and Bmp4/Wnt3 that are activated by mature or uncleaved Nodal, respectively, to sustain Nodal signaling from implantation throughout gastrulation (Ben Haim N et al., Dev Cell. 2006; 11:313-23). ENDM1 and Flkl have been used as a definitive mouse endodermal cell marker and a mesoderm cell marker, respectively (Nicetto D. et al., Science. 2019; 363:294-297).
Enzymatic and other relevant marker systems. Several additional marker systems useful in the isolation and purification of progenitor cells by the methods embodied herein, include enzymatic (alkaline phosphatase and telomerase)-based reaction, small molecules (lectins or short peptides), and quantum dots (QD) or fluorescence dyes, etc.
Hematopoietic Stem Cell Markers. Hematopoietic stern cells (HSCs) are unique, multipotent, self-renewing progenitor cells responsible for continuous supply of differentiated blood cell types of the myeloid and lymphoid lineages. These cells include lymphocytes, granulocytes, and macrophages of the immune system as well as circulating erythrocytes and platelets. HSCs are defined by two key functional abilities: (i) multipotency - the ability to form all differentiated blood cells, and (ii) long-term self-renewal —the ability to give rise to identical daughter cell that of the ancestor. I—ISC is superior with self-renewal that distinguishes HSC from multipotent progenitors and is responsible for successful secondary transplants in experimental models. A major challenge using Hematopoietic Stem Cells (HSCs) is their identification and isolation from larger pools of cells as HSCs represent a very rare, 1 in 10,000 cells of the hone marrow and 1 in 100,000 cells in the blood.
Therefore the methods embodied herein are useful in isolating and purifying large scale viable HSCs.
Examples of biomarkers that can be targeted for the isolation and enrichment of HSCs include: SLAM (Signaling Lymphocyte Activation Molecule) family of cell surface molecules which includes CD48, CD150, CD244, etc.
Lymphoid lineage markers. Hematopoietic Stem Cells (HSCs) can differentiate into cells of two primary lineages, lymphoid and myeloid. Common lymphoid progenitors can differentiate into all lymphoid lineages (Kondo M. et al., Cell. 1997; 91:661-72). During the process of lymphopoiesis, lymphoid lineage leads to the formation of B Cells, T Cells, Natural Killer (NK) Cells, and Dendritic Cells. Many of these lymphocytes are short-lived, and immune system homeostasis requires continual HSC self-renewal and differentiation. The markers, c-KittLo, Sca-1Lo, Lin and IL7R+ have shown to represent lymphoid lineage cells.
Myeloid lineage markers. Common myeloid progenitors differentiate into progenitors then can differentiate into the granulocyte/macrophage and megakaryocyte/erythroid lineages, respectively (Akashi K. et al., Nature. 2(x)( );404:193-7). During the process of myelopoiesis, the myeloid lineage develops to form Granulocytes, Monocytes, Megakaryocytes, and Dendritic cells. Circulating erythivcytes and platelets also develop from myeloid progenitor cells. Many of these myeloid cells are short-lived, and immune system homeostasis requires continual HSC self-renewal and differentiation.
The compositions embodied herein, can also be applied in the areas of drug discovery and target validation. The present disclosure comprehends the use of the progenitor cells, nucleic acid sequences and peptides, in drug discovery efforts to elucidate relationships that exist in a disease state, e.g. macular degeneration, and the regeneration or differentiation of a progenitor cell.
The screening assays of the disclosure suitably include and embody, animal models, cell-based systems and non-cell based systems. The progenitor cells embodied herein, are used for identifying agents of therapeutic interest, e.g. by screening libraries of compounds or otherwise identifying compounds of interest by any of a variety of drug screening or analysis techniques, or synthesis of novel compounds. (Moffat, J. G., et al., (2014). Phenotypic screening in cancer drug discovery —past, present and future. Nature Reviews Drug Discovery, 13(8), 588-602. doi: 10.1038/nrd4366; Swinney, D. C., & Anthony, J. (2011). How were new medicines discovered—Nature Reviews Drug Discovery, 10(7), 507-519. doi: 10.1038/nrd3480; Chackalamannil, S., et al., (2017). Comprehensive medicinal chemistry III. Amsterdam: Elsevier, E. L. Berg, et al., “Approaches to the analysis of cell signaling networks and their application in drug discovery.,” Current Opinion in Drug Discovery & Development, vol. 8, no. 1, pp. 107-114, 2005)
The assays can be of an in vitro or in vivo format. In vitro formats of interest include cell-based formats, in which contact occurs e.g., by introducing the substrate in a medium, such as an aqueous medium, in which the cell is present. In yet other embodiments, the assay may be in vivo, in which a multicellular organism that includes the cell is employed.
Multicellular organisms of interest include, but are not limited to: insects, vertebrates, such as avian species, e.g., chickens; mammals, including rodents, e.g., mice, rates; ungulates, e.g., pigs, cows, horses; dogs, cats, primates, e.g., monkeys, apes, humans; and the like. As such, the target cells of interest include, but are not limited to: insects cells, vertebrate cells, particularly avian cells, e.g., chicken cells; mammalian cells, including murine, porcine, ungulate, ovine, equine, rat, dog, cat, monkey, and human cells; and the like.
In certain embodiments, the subject methods are performed in a high throughput (HT) format. In the subject HT embodiments of the subject invention, a plurality of different cells are simultaneously assayed or tested. By simultaneously tested is meant that each of the cells in the plurality are tested at substantially the same time. In general, the number of cells that are tested simultaneously in the subject HT methods ranges from about 10 to 10,000, usually from about 100 to 10,000 and in certain embodiments from about 1000 to 5000. A variety of high throughput screening assays for determining the activity of candidate agent are known in the art and are readily adapted to the present invention.
In certain embodiments, a detectable moiety is conjugated to an agent of interest wherein the detectable moiety comprises: a luminescent moiety, a chemiluminescent moiety, a fluorescence moiety, a bioluminescent moiety, an enzyme, a natural or synthetic moiety.
Candidate Agents: The methods can be practiced with any test compounds as candidate agents. Test compounds useful in practicing the inventive method may be obtained using any of the numerous approaches in combinatorial library methods known in the art, including biological libraries, spatially-addressable parallel solid phase or solution phase libraries, synthetic library methods requiring deconvolution, the “one-bead one-compound” library method, and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, nonpeptide oligomer, or small molecule libraries of compounds (Lam, 1997, Anticancer Drug Des. 12:145).
Examples of methods for the synthesis of molecular libraries may be found in the art, for example, in: DeWitt et al., 1993, Proc. Natl. Acad. Sci. USA 90:6909-6913; Erb et al., 1994, Proc. Natl. Acad. Sci. USA 91:11422-11426; Zuckermann et al., 1994, J. Med. Chem. 37:2678-2685; Cho et al., 1992, Science 261:1303-1305; Carell et al., 1994, Angew. Chem. hit. Ed. Engl. 33:2059-2061; Carell et al., 1994, Angew. Chem. Int. Ed. Engl. 33:2061-2064; and Gallop et al., 1994, J. Med. Chem. 37:1233-1251. Libraries of compounds may be presented in solution (e.g., Houghten, 1992, Bio/Techniques 13:412-421), or on beads (Lam, 1991, Nature 354:82-84), chips (Fodor, 1993, Nature 364:555-556), bacteria (U.S. Pat. No.¶5,223,409), spores (U.S. Pat. Nos. 5,571,698; 5,403,484; and 5,223,409), plasmids (Cull et al., 1992, Proc. Natl. Acad. Sci. USA 89:1865-1869), or phage (Scott and Smith, 1990, Science 249:386-390; Devlin, 1990, Science 249:404-406; Cwirla et al., 1990, Proc. Natl. Acad. Sci. USA 87:6378-6382; and Felici, 1991, J Mol. Biol. 222:301-310).
Commercially available libraries that may be screened include, but are not limited to, the TimTec Natural Product Library (NPL), NPL-640, and TimTec NDL-3000 library. Libraries comprising compounds modeled on polyamines (i.e., polyamine analogs) may also be screened.
In certain embodiments, the candidate agent is a small molecule or large molecule ligand. By small molecule ligand is meant a ligand ranging in size from about 50 to about 10,000 daltons, usually from about 50 to about 5,000 daltons and more usually from about 100 to about 1000 daltons. By large molecule is meant a ligand ranging in size from about 10,000 daltons or greater in molecular weight.
The method may be practiced iteratively using different concentrations of a test candidate and/or different testing conditions, such as duration of reaction time. Test candidates that are identified by the method can be further tested by conventional methods in the art to verify specificity, dose dependency, efficacy in vivo, and the like. Test candidates may serve as lead compounds for developing additional test candidates.
A prototype compound or agent may be believed to have therapeutic activity on the basis of any information available to the artisan. For example, a prototype agent may be believed to have therapeutic activity on the basis of information contained in the Physician's Desk Reference. In addition, by way of non-limiting example, a compound may be believed to have therapeutic activity on the basis of experience of a clinician, structure of the compound, structural activity relationship data, EC50, assay data, IC50 assay data, animal or clinical studies, or any other basis, or combination of such bases.
A therapeutically-active compound or agent is an agent that has therapeutic activity, including for example, the ability of the agent to induce a specified response when administered to a subject or tested in vitro. Therapeutic activity includes treatment of a disease or condition, including both prophylactic and ameliorative treatment. Treatment of a disease or condition can include improvement of a disease or condition by any amount, including prevention, amelioration, and elimination of the disease or condition. Therapeutic activity may be conducted against any disease or condition, including in a preferred embodiment against any disease or disorder associated with damage by reactive oxygen intermediates. In order to determine therapeutic activity any method by which therapeutic activity of a compound may be evaluated can be used. For example, both in vivo and in vitro methods can be used, including for example, clinical evaluation, EC5O, and IC50 assays, and dose response curves.
Candidate compounds for use with an assay of the present disclosure or identified by assays of the present disclosure as useful pharmacological agents can be pharmacological agents already known in the art or variations thereof or can be compounds previously unknown to have any pharmacological activity. The candidate compounds can be naturally occurring or designed in the laboratory. Candidate compounds can comprise a single diastereomer, more than one diastereomer, or a single enantiomer, or more than one enantiomer.
Candidate compounds can be isolated, from microorganisms, animals or plants, for example, and can be produced recombinantly, or synthesized by chemical methods known in the art. If desired, candidate compounds of the present disclosure can be obtained using any of the numerous combinatorial library methods known in the art, including but not limited to, biological libraries, spatially addressable parallel solid phase or solution phase libraries, synthetic library methods requiring deconvolution, the “one-bead one-compound” library method, and synthetic library methods using affinity chromatography selection. The biological library approach is limited to polypeptide libraries. The other four approaches are applicable to polypeptide, non-peptide oligomers, or small molecule libraries of compounds and are preferred approaches in the present disclosure. See Lam, Anticancer Drug Des. 12: 145-167 (1997).
In an embodiment, the present disclosure provides a method of identifying a candidate compound as a suitable prodrug. A suitable prodrug includes any prodrug that may be identified by the methods of the present disclosure. Any method apparent to the artisan may be used to identify a candidate compound as a suitable prodrug.
In another aspect, the present disclosure provides methods of screening candidate compounds for suitability as therapeutic agents. Screening for suitability of therapeutic agents may include assessment of one, some or many criteria relating to the compound that may affect the ability of the compound as a therapeutic agent. Factors such as, for example, efficacy, safety, efficiency, retention, localization, tissue selectivity, degradation, or intracellular persistence may be considered. In an embodiment, a method of screening candidate compounds for suitability as therapeutic agents is provided, where the method comprises providing a candidate compound identified as a suitable prodrug, determining the therapeutic activity of the candidate compound, and determining the intracellular persistence of the candidate compound. Intracellular persistence can be measured by any technique apparent to the skilled artisan, such as for example by radioactive tracer, heavy isotope labeling, or LCMS.
In screening compounds for suitability as therapeutic agents, intracellular persistence of the candidate compound is evaluated. In an embodiment, the agents are evaluated for their ability to modulate the translation of compositions embodied herein, over a period of time in response to a candidate therapeutic agent. In other embodiments, the candidate therapeutic agent is neuroprotective, increases survivability of the cells, induces engraftment of cells etc.
In another preferred embodiment, soluble and/or membrane-bound forms of compositions embodied herein, e.g. proteins, mutants or biologically active portions thereof, can be used in the assays for screening candidate agents. When membrane-bound forms of the protein are used, it may be desirable to utilize a solubilizing agent. Examples of such solubilizing agents include non-ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, TRITON™ X-100, TRITON™ X-114, THESIT, Isotridecypoly(ethylene glycol ether)n, 3-[(3-cholamidopropyl)dimethylamminio]-1-propane sulfonate (CHAPS), 3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-1-propane sulfonate (CHAPSO), or N-dodecyl=N,N-dimethyl-3-ammonio-l-propane sulfonate.
In certain embodiments, a method of treating an ocular or retinal disease comprises administering to a subject an effective amount of the isolated and purified population of progenitor cells embodied herein.
Ocular disorders that can be treated using a method of the present disclosure include, but are not limited to, macular degeneration, choroidal neovascularization, macular edema, retinal neovascularization, proliferative vitreoretinopathy, glaucoma, and ocular inflammation.
Ocular diseases that can be treated using a method of the present disclosure include, but are not limited to, acute macular neuroretinopathy; Behcet's disease; choroidal neovascularization; diabetic uveitis; histoplasmosis; macular degeneration, such as acute macular degeneration, non-exudative age related macular degeneration and exudative age related macular degeneration; edema, such as macular edema, cystoid macular edema and diabetic macular edema; multifocal choroiditis; ocular trauma which affects a posterior ocular site or location; ocular tumors; retinal disorders, such as central retinal vein occlusion, diabetic retinopathy (including proliferative diabetic retinopathy and diabetic macular edema), proliferative vitreoretinopathy (PVR), retinal arterial occlusive disease, retinal detachment, uveitic retinal disease; sympathetic ophthalmia; Vogt Koyanagi-Harada (VKH) syndrome; uveal diffusion; a posterior ocular condition caused by or influenced by an ocular laser treatment; posterior ocular conditions caused by or influenced by a photodynamic therapy; photocoagulation, radiation retinopathy; epiretinal membrane disorders; branch retinal vein occlusion; anterior ischemic optic neuropathy; non-retinopathy diabetic retinal dysfunction; retinoschisis; retinitis pigmentosa; glaucoma; Usher syndrome, cone-rod dystrophy; Stargardt disease (fundus flavimaculatus); inherited macular degeneration; chorioretinal degeneration; Leber congenital amaurosis; congenital stationary night blindness; choroideremia; Bardet-Biedl syndrome; macular telangiectasia; Leber's hereditary optic neuropathy; retinopathy of prematurity; and disorders of color vision, including achromatopsia, protanopia, deuteranopia, and tritanopia.
In some cases, the ocular disease is glaucoma, retinitis pigmentosa, macular degeneration, retinoschisis, Leber's Congenital Amaurosis, diabetic retinopathy, achromotopsia, or color blindness.
Embodiments of the invention are also directed to treatment of any disease or disorder wherein VEGF is protective such as neurodegenerative diseases, e.g. Niemann-Pick disease, Alzheimer's disease, Parkinson's disease, Lou Gehrig's disease, schizophrenia, Gaucher disease, Fabry disease, Tay-Sachs disease, Sandhoff disease and cerebellar ataxia, but is not limited thereto.
The compositions of the present disclosure can be prepared in a variety of ways known to one of ordinary skill in the art. Regardless of their original source or the manner in which they are obtained, the compositions of the disclosure can be formulated in accordance with their use. For example, the progenitor cells, therapeutic agents etc., above can be formulated within compositions for application to cells in tissue culture or for administration to a patient or subject. Any of the pharmaceutical compositions of the disclosure can be formulated for use in the preparation of a medicament, and particular uses are indicated below in the context of treatment. These compositions can be prepared in a manner well known in the pharmaceutical art, and can be administered by a variety of routes, depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes including intranasal, vaginal and rectal delivery), pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), ocular, oral or parenteral. Methods for ocular delivery can include topical administration (eye drops), subconjunctival, periocular or intravitreal injection or introduction by balloon catheter or ophthalmic inserts surgically placed in the conjunctival sac. Parenteral administration includes intravenous, intra-arterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular administration. Parenteral administration can be in the form of a single bolus dose, or may be, for example, by a continuous perfusion pump. Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, powders, and the like. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
Human Fetus arrival: Fetal eyes arrive within 24h in 2 degree box. Dissection started directly in order to keep high viability.
Dissection and dissociation: Dissection of the retina was performed on all eyes. Retinas were then dissociated in papain for 30 min in the incubator. Cell suspension was then centrifuged and seeded in a T25 or 175 flask (depending on the number of cells).
Culture for 1-2 weeks: Cells were cultured in a 2D layer for 1-2 weeks using FDA approved media (no animal products). Upon reaching confluence (about 10 million cells) cells were then passaged and centrifuged to start sorting.
First sort with CD73 double positive (PE and APC): Cells were stained with CD73 (PE and APC) for 30 min in 2 degrees. After washing and preparation of the cell sorter, cells are sorted by gating about 3-20% of the positive population. Presort and Postsort analysis was performed.
Culture back for 2 weeks: Positive sorted cells were then cultured in a 175 or T175 for 1-2 weeks until reaching confluence (10 million cells.)
Second sort with CD73/Thrb positive and CDllb negative: The second sort consisted of two different positive antibodies (CD73 and Thrb) and one negative to delete the microglia (CD 11 b). The positive fraction is the new cell line.
Culture to reach high yield: The final cell line was cultured with the same media and flasks to reach the number of cells needed for experiments.
The first sorting or enrichment step (CD73/CD11b−) yielded about 30-40% precursor photoreceptor cells. The second sorting (CD73+/THR-beta+/CD11b−) for purification of precursor photoreceptor cells yielded a viable 98% pure population of photoreceptor cells with a Ki-67 index of about 20% (proliferation). Accordingly, the method herein has proved to be successful in isolating retinal ganglion cells and retinal microglia, two very difficult targets in the eye.
These cells can be used for cell replacement, drug discovery and screening or other uses where photoreceptors or their precursors might be needed.
This approach has broad applicability for use to isolate other cells in the eye, central nervous system (CNS) and throughout the body.
Cell Culture
Media 1: Ultraculture media (Lonza)-500 ml; 100x 1-glutamax (Gibco)-5 ml; rhEGF (Peprotech)- 1000 μl(10 μg/l ml); rhFGF (peprotech)- 5000 (1014/m1); Primocin (Invivogen)-1000 μl; Filter and store at 4° C.
Media 2: DMEM/F12- 500 ml; 100x 1-Glutamax 5.5.m1;100x non-essential amino acid- 5.5 ml; Sodium Pyruvate- 5 ml; β-mercaptoethanol- 40; bFGF- 1000u1 Anti-Anti- 5 ml Knock serum replacement (Gibco)- 65m.
For hCPs mix media 1 and 2 in equal quantity just before feeding cells.
Step 1. Prepare papain solution (10 ml per 5 plates)
Step 2. Eyecup collection (perform during papain activation)
Single sort (2 weeks
)
.23
.7
2
.55
7.22
4
67
6.77
2
Db sort Positive (2 weeks
)
.14
05
4.55
1
52
5
78.
3
.2
4
.5
.00
.47
.88
.2
8.02
.77
.7
7
sort Negative (2 weeks
)
55
.27
66
2.59
.41
7
.2
5
2.4
7
5
.27
sort Positive (2 weeks
)
.25
48
.42
7
.28
.52
.23
.79
7.22
.41
7
.02
04
4.
.8
.0
.02
Positive (
weeks
)
.26
7.7
2.74
.35
.19
.19
.27
.0
2
.27
4
.20
.87
Db Unsorted
.2
2
.01
.5
2
.27
2.
.73
.06
5
.55
6
8.
2
.21
Db Unsorted
.72
.74
.35
.81
.83
.28
.01
1.
.06
0.8
.25
8
88
.40
.2
8
2
6.1
7
.23
.32
9
Db sort Positive
27
.2
.02
.2
.1
.23
.35
9
2
7
7
.04
.82
.83
4.8
indicates data missing or illegible when filed
Sort
Cells were analyzed with a flow cytometer prior to sorting which are set up as the control for running the sort. Markers of choice (depending on the sort), CD73, Thrb and CD11b are analyzed with the flow cytometer by tuning the voltages in order to put the desired population in the highest quadrant of the intensity. After this first gating strategy was performed, the the microfluidic flow is started.
Cells were first pushed through the negative chambers in order to stabilize the pressure (around 150 mPa) but also the flow of cells in front of the lasers (40 ms between two cells). In order for the machine to know its velocity, a cell must be stained in at least two different fluorochromes, this way the machine can calculate the time it takes the cell to go from the first laser to the second one, hence measuring its velocity. As soon as both these variables are stable, lasers can be started and fluorescent data starts to appear showing an approximate of 5000-7000 cells that are being pushed in front of the lasers. Each cell passes in front of the three lasers and its fluorescent intensity is measured and reported on the graph.
The same gating strategy that was used on the control on the cell sorter and reported on the sorting machine and the sort was started. However, in order to capture properly each cell, the valve speed and delay of action must be properly set up. The arrival window was open and was then fitted to the exact population of cells flowing through the microfluidic device (this depends on cell size, shape, granularity, intensity, stiffness, concentration).
During the entire sorting process (usually 2 hours) the percent of sorted, gated and positive cells was measured and reported in function of time. At the end of the sort, the cartridge was freed from the machine and cells can be taken back to the cell culture for next steps.
Flow Cytometry
hCPs [(5×105/mL in media) were trypsinized and cell pellet collected was processed for the phenotype then analyzed using a Flow Cytometry assay.
Flow Cytometry was performed using MACSQuant flow cytometer (Miltenyi, San Diego) (100). Cells were collected and fixed with Perm/Fix buffer (BD Biosciences) at 4° C. for 15 min. Cells were then washed in wash buffer (BD Biosciences) and incubated, at room temperature, in block buffer (Pharmingen staining buffer with 2% goat serum) for 30 min. Blocked cells were seeded onto a flat bottom 96-well plate (treated, sterile, polystyrene, Thomas Scientific) and stained with conjugated primary antibodies (DAPI-Vioblue, Cone Arrestin-FITC, S-opsin-FITC, R/G opsin-FITC, Blue opsin- Rhodopsin-FITC, Recoverin-APC, Calbidin- FITC, RBPMS-APC, PKCa-FITC and Brna3a-FITC, KI67-APC) overnight at room temperature. Primary antibodies were diluted in 200 μL of antibody buffer (TBS, 0.3% Triton X-100 and 1% goat serum). After cells were washed three times for 15 min, secondary antibodies were goat-derived anti-rabbit and anti-mouse and diluted 1:200 in antibody buffer (Jackson Immunoresearch Laboratory). Secondary antibodies were applied and left at room temperature for 3 h. light scatter and fluorescence signals from each well were measured using the MACSQuant flow cytometer (2×105 events were recorded). The results were analyzed using the MACSQuantify software (Miltenyi Biotec). For each primary antibody DAPI-positive single cell population was gated. The ratio of positive cells in the gated population was estimated in comparison with blank and species-specific isotype control.
Immunohistochemistry (IHC)
Live hCPs grown in chamber slides and cryosection from Long Evans left eye were fixed with 4% paraformaldehyde in 0.1 M PBS (Irvine Scientific) at room temperature for 20 min. These fixed cells and sections were blocked and permeabilized with a blocking solution [(iris-buffered saline (TBS), 0.3% Triton X-100 and 3% goat serum (Jackson Immunoresearch Laboratories, West Grove, Pa.) for 15 min. Samples were then rinsed twice with 0.1 M TBS buffer for 15 min each time, mounted on polysine microscope slides and incubated with primary antibodies overnight at 4° C. (DAPI-Vioblue, Cone Anrestin-CY3, R/G opsin-Cy3, TRA-1-85- FITC, STEM 121-FITC) at concentrations determined in laboratory. The next day, samples were rinsed three times with TBS for 15 min. Secondary antibodies (goat-derived anti-mouse and anti-rabbit) were applied for lh at room temperature. Samples were then washed one last time with TBS before being mounted on polysine microscope slide with lox viscosity slide mounting medium. Digital images were obtained with an Epifluroscent microscope using 20x objective. Electronic image files were managed using Matlab software.
Quantitative Real-Time RT-PCR (aRT2-PCR)
RNA extraction was performed using the Qiagen kit. Cells were treated with trypsin and pellet collected was disrupted using Buffer RLT (1 ml of RLT buffer add l00 of β-mercaptoethanol) and the tube was flicked to disrupt the cells. One volume of 70% ethanol (not provided in the kit) was added and mixed well by pipetting. The suspension was transferred into the RNeasy spin column (provided in the kit). Close lid and gently centrifuged at 8000g for 15 seconds. The flow through was discarded and about 7000 of RW1 buffer was added and then spun for 15 sec at 8000 x g. The flow through was discarded and 5000 of RPE buffer (both buffers provided in the kit; RPE buffer add ethanol before using) was added and then spun for 15 sec at 8000 x g. This was followed by second wash with RPE buffer for 2 min at 8000 x g. The flow through was discarded and the spin column was placed in a new 1.5 ml collection tube and RNA was eluted by gently adding RNAase free water directly to the spin column. The lid was closed and centrifuged for 1 in at 8000 x g. RNA was quantified using nanodrop and 1 μg of total RNA was used for cDNA synthesis using iScript cDNA advance transcription kit (Bio-rad).
Reaction protocol used: Priming 5 min at 25° C. followed by reverse transcription step for 20 mins at 46° C. and finally RT inactivation for 1 min at 95° C. Synthesized cDNA was used for gene expression analysis.
The volume was calculated for 96 well plates and data was quantified using Biorad software.
In-Vivo Transplantation
Twenty-three male domestic rats of the Long Evans breed (age 12 weeks, approximate weight 200g) were used as recipients in the experiment. Prior to surgery, all animals were pre-anesthetized with GAS. Rats were anesthetized with 2%-3% isoflurane (Abbott, Solna, Sweden) in combination with oxygen. Transplantation was performed on non-immuno-suppressant rats. Rats were sedated using 2-4% isoflurane by placing the rats in the inhalation chamber, followed by intraperitoneal injection of ketamine (40-80 mg/kg) and xyalzine (10 mg/kg) for anesthesia. Eyes were first anesthetized using topical ophthalmic proparacacine (0.5%) followed by Genteal to keep the lens moist during the surgery.
Recipient rats were injected in sub-retinal space with hCP single-cells injections. A conjunctival incision and a small sclerotomy were performed using a fine disposal scalpel. Cells were injected into the subretinal space using a glass pipette (internal diameter, 150 urn) attached to a 50-μL Hamilton syringe via a polyethylene tubing. The hCPs were injected into the retina bleb as a single-cell suspension in PBS. All samples contained approximately 1×105 cells and the injection volume were 2 μL for all replicates. Using a glass coverslip applied on the eye checked bleb presence. Subretinal space injection was considered successful is a shining bleb was seen under the dissection surgical microscope. Triple antibiotic (Bac/Neo/Poly) was given locally at the end of the surgery to prevent further infection. The rats were then placed in their cages for a 14 days study.
The research protocol was reviewed and approved by the Schepens Eye Research Institute Animal Facility and was in accordance with the Association for Research in Vision Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research.
Tissue Processing
Three Days post transplantation rats were sacrificed by CO2 suffocation for 2 min. Eyes were enucleated and placed in 4% paraformaldehyde for 24 hrs. Tissues were subsequently saturated with increase concentration of sucrose (5%, 10%, 20%) containing Sorensen phosphate buffer. Eyes were left in 30% sucrose overnight or till dissection. The tissues were embedded in cryo-section gelatin medium overnight and sectioned at 15 μm thickness on a cryostat. During the sectioning process, every 5th section was stained and examined by epi-fluorescence for hCPs presence with TRA-1-85 and STEM 121-FITC (human cells marker), R/G opsin and Cone Anrestin-APC (host photoreceptor marker) and DAPI-Vioblue (cell nuclei).
Micro-Electrode Array Assay
Using a 1 mL pipette tip, a 2 mL cell suspension containing around 500,000 hCP was placed on the MaxWell MaxOne Multielectrode Array and aligned to the electrode. 16,000 electrodes on 4 mm2 was used. A 5-mm coverslip was placed between the microscope objective and the cell suspension to maintain an optically aberration-free transition zone from the air to the liquid. The HD-MEA chip was plugged into the interfacing circuit board. The cells were allowed to acclimate to the MEA under a 50% contrast background for 20 min.
The data recorded on the MEA was sampled at 20 kHz, and filtered on-chip, approximately between 0.5 Hz and 12 kHz. Data was then filtered with a 280 Hz high-pass filter and 7 kHz low-pass filter to reduce offset effect and high frequency noises.
Light stimuli were programmed and sent to a LED projector. The projected image was centered on the selected electrode region, and light stimuli were run sequentially. Prior to each stimulus, a 50% contrast background was projected onto the cell suspension for 5 min so that the cells could adapt to the mean projected photopic level.
Frozen formulation delivered to clinical site (alternatively cells delivered in culture vessels at 37 degrees C.).
Direct injection of 50,000-10×106 cells into subretinal under macula.
3 port pars plana vitrectomy approach, or delivered with device aimed at subretinal space.
No systemic immunosupression (local anti-inflammatory treatment) anticipated. Cone dystrophy, an orphan indication with no available treatments:
Phase 1/2a in patients <20/200 without significant cones in macula but with preserved outer retinal structures (RPEBM/CC) first for safety.
Progressing through subsequent phases into patients with better function.
Outcomes and assessment: Visual acuity (VA) for functional and ophthalmic coherence tomography (OCT) for structural evaluation.
Other more novel outcome measures (e.g. ellipsoid zone (EZ) on OCT) expected to be available by proposed trial date.
Followed by dry AMD, other indication where cone structure and function is impaired (e.g. Retinal Detachment).
Stargardt's and Cone dystrophies are retinal diseases caused by a gene mutation in cone photoreceptors, which leads to death of these cells. In early stages of these diseases, gene therapy is useful for treatment; however, no treatment is available for later stages of these diseases. The methods and cells described herein provide a solution to a longstanding clinical problem in treating theses pathologies. Rare cone progenitors cells (hCP) were isolated and enriched from human fetal embryonic retinal tissue. For stem cell-based therapy it is crucial to obtain a purified highly enriched, e.g., at least 85% pure (e.g., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100% pure) or a homogenous cell population. For example, 95% pure cone progenitor cells were achieved using a 2 step sorting technique. Fetal retina was first dissociated and cultured to obtain the desired cell number of 10M. The first sorting step involved tagging the cells with CD73 surface antigen, which is exclusively expressed in the photoreceptor precursor population. The positive cells were further cultured in hypoxia conditions (10% oxygen) and sorted with CD73+Thrb+ and CD 11 b- cells using Miltenyi Tyto cell sorter. Double positive staining of cells ensured higher purity with CD11b- acting as negative channel that allowed us to delete specific cell population (i.e. microglia). These cells could be cultured and expanded in hypoxia conditions. The phenotypical expression of these cells was confirmed using a MacsQuant cell analyzer and identified these cells as human cone progenitor cells after testing for cone arrestin, s-opsin, m-opsin and blue-opsin expression (95%). These cells were found to have low PKCa expression (4%), and no rhodopsin, Brn3a, NeuN, and RBPMS indicating the purity of these cells with few contaminating cells of non-cone lineage. This strategy to isolate, enrich and culture was used to obtain large number of hCPs which were transplanted into rat eyes showing extremely high capacity to survive and engraftment into the host inner retina. The cells are also evaluated in photoreceptor degeneration rat models. The data demonstrated the ability of hCP to integrate, survive and engraft in a xenograft model indicating that the purified cell population is useful to treat retinal diseases such as cone dystrophies and/or photoreceptor degenerative conditions.
While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference. Genbank and NCBI submissions indicated by accession number cited herein are hereby incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are hereby incorporated by reference.
While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.
This Application is a national stage application under 35 U.S.C. § 371 of International Patent Application No. PCT/US2020/022929, filed Mar. 16, 2020, which claims the benefit of and priority under 35 U.S.C. § 119(e) to U.S. Provisional Application 62/819,160 filed on Mar. 15, 2019, the entire contents of which are hereby expressly incorporated by reference herein. The content of the text file named “036770-581001WO_SL.TXT”, which was created on Apr. 29, 2020, and is 46,671 bytes in size, is hereby incorporated by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2020/022929 | 3/16/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62819160 | Mar 2019 | US |
