Patent Application
20120301446
Embryonic stem cells are capable of differentiating into many types of cells of the human body. The majority of somatic cells are terminally differentiated and were believed to lack the capability of changing to other types of somatic cells. Recent advances in induced pluripotent stem cell (iPSC) and transdifferentiation fields have changed this paradigm. Somatic cells can be reprogrammed to induced pluripotent stem cell (iPSC), i.e. via ectopic expression of four transcription factors, i.e. Oct4 (e.g. SEQ ID NO: 1), Sox2 (e.g. SEQ ID NO: 2), Klf4 (SEQ ID NO: 3), and cMyc (e.g. SEQ ID NO: 4) via viral transduction (Okita et al, Nature 448, 313-317 (2007); Takahashi and Yamanaka, Cell 126, 663-676 (2006)). A number of modified genetic approaches were further developed to produce iPSCs with potentially reduced risks, including using non-integrating adenoviruses to deliver reprogramming genes (Stadtfeld et al., Science 322, 945-949 (2008)), transient transfection of reprogramming plasmids (Okita et al., Science 322, 949-953 (2008)), a piggyBac transposition system and Cre-excisable viruses (Soldner et al., Cell 136, 964-977 (2009); Woltjen et al., Nature 458, 766-770 (2009)). Furthermore, strategies of exploiting endogenous gene expression in certain cell types also allowed easier reprogramming and/or with less required exogenous genes (Aasen et al., Nat Biotechnol 26, 1276-1284 (2008); Kim et al., Nature 454, 646-650 (2008); Shi et al., Cell Stem Cell 2, 525-528 (2008)). Moreover, small molecules have been identified that enhance reprogramming efficiency and replace certain reprogramming factors (Huangfu et al., Nat Biotechnol 26, 795-797 (2008); Huangfu et al., Nat Biotechnol 26, 1269-1275 (2008); Li et al., Cell Stem Cell 4, 16-19 (2009); Shi et al., Cell Stem Cell 3, 568-574 (2008); Shi et al., Cell Stem Cell 2, 525-528 (2008)). However, all of those methods to date still involve the use of genetic materials with drawbacks of introducing unknown, unwanted, or even harmful genome modifications by exogenous sequences in target cells and having inadequate control over expression levels of transgenes. To address these drawbacks, there are needs in the field to reprogram cells without relying upon or introducing exogenous genetic materials such as exogenous genes or DNA fragments or vector containing exogenous DNA or genes.
One aspect of the present disclosure relates to a transducible material comprising an effector domain. The effector domain is capable of exerting reprogramming changes of a biological sample once transduced into a biological sample. In certain embodiments, the effector domain is inherently capable of transducing into the biological sample.
In certain embodiments, the transducible material further comprises a transduction domain which is covalently or non-covalently associated with or linked to the effector domain. In certain embodiments, the transduction domain is covalently linked to the effector domain through a linker.
In certain embodiments, the transducible material is capable of selectively transducing into one or more specific biological samples or becoming transducible in a specific environment surrounding the biological sample.
Another aspect of the present disclosure relates to a composition comprising a biological sample and a transducible material, wherein the transducible material has transduced into the biological material.
Another aspect of the present disclosure relates to a method of reprogramming a biological sample by exposing the biological sample to a composition comprising a transducible material.
Another aspect of the present disclosure relates to a method of treating a disease or condition in a biological organism comprising administering a pharmaceutical composition comprising a transducible material into the biological organism.
Another aspect of the present disclosure relates to a method of developing cell-based therapies for various diseases or conditions comprising the step of reprogramming an iPSC, an embryonic stem cell, or a progenitor cell to a transplantable somatic cell or a transplantable progenitor cell using a transducible material.
Another aspect of the present disclosure relates to a method of developing disease models comprising the step of reprogramming an iPSC, an embryonic stem cell, or a progenitor cell to a transplantable somatic cell or a transplantable progenitor cell using a transducible material.
Another aspect of the present disclosure relates to a method of identifying an effector domain comprising covalently or non-covalently associating a test effector domain to a transduction domain to form a test transducible molecule, exposing the test molecule to a biological sample, and measuring a reprogramming level of the biological sample.
One aspect of the present disclosure relates to a transducible material comprising an effector domain.
In certain embodiments, a transducible material used herein refers to a material or a molecule which is not DNA or derived from DNA but is capable of crossing or transducing or being crossed through a membrane of a biological sample (e.g., a cell membrane) so that the transducible material can enter or be brought into the inside of the biological sample from the outside of the biological sample and exerts reprogramming efforts. For example, the transducible material may interact with cell-surface receptors which facilitate the entry of the material into cells through receptor mediated endocytosis.
In certain embodiments, a transducible material is a selective transducible material which is more likely to transduce into a specific type of biological samples (e.g. cancer or tumor cells) or becomes transducible in a specific microenvironment in or around a biological sample (e.g. microenvironment around cancer or tumor) than other biological samples. For example, the selective transducible material comprises a transduction domain (e.g. a cell-targeting peptide or an activatable cell penetrating peptide) that preferably delivers the selective transducible material into a specific type of biological sample or become transducible in a microenvironment around a biological sample.
Without being bounded to any theories, it is contemplated that the transducible materials may cross a cell membrane and enter into cytoplasm to reprogram cytoplasm activities such as translation, post-translation modification, signaling pathway, apoptosis pathway. It is further contemplated that the transducible material may cross the nucleus membrane and reprogram or modulate DNA or chromosomal replication, gene transcription, and RNA splicing.
An effector domain is a motif or a molecule which, once inside a biological sample, is capable of exerting reprogramming changes of the biological sample. The effector domain may interact with molecules (e.g., proteins, DNA, RNA, sugars, and lipids) in the biological sample (e.g., in cytoplasm or nuclei) and lead to changes such as proliferation, differentiation, dedifferentiation, transdifferentiation, retrodifferentiation, transdertermination, apoptosis, and morphogenesis. The effector domain can be 1) a polypeptide, or a fragment or a mimic thereof; 2) a polynucleotide which cannot be gene expressed once transduced or incorporated into the genome of the biological sample or cause genetic modification but nevertheless interacts with molecules in the biological sample (e.g., a ribozyme, an antisense molecule, a siRNA or miRNA, an oligonucleotide, and the like); and 3) a small molecule or other chemical compound (e.g. chemotherapy drugs).
In certain embodiments, an effector domain is inherently transducible, e.g. PDX1 (e.g. SEQ ID NO: 9) and NeuroD (e.g. SEQ ID NO: 7).
One example of the effector domain is a polypeptide such as a transcription factor, a chromosome remodeling protein, an antibody, or a fragment or mimic thereof. Another example of the effector domain is a small molecule which is not a polymer and binds with a biolpolymer such as protein, nucleic acid, or polysaccharide and alters the activity or function of the biopolymer. Examples of small molecules include, without limitation, acetylation inhibitors, transcription activators, signal pathway activators, signal pathway inhibitors, and methylation inhibitors.
In another embodiment, an effector domain can be at least one polypeptide that reprograms a somatic cell into a stem cell or change a cell state from one to another. For example, the effector domain can be 1) a polypeptide selected from the group consisting of Klf4 (e.g. SEQ ID NO: 3), Sox2 (e.g. SEQ ID NO: 2), Lin28 (e.g. SEQ ID NO: 5), Oct4 (e.g. SEQ ID NO: 1), cMyc (e.g. SEQ ID NO: 4), Nanog (e.g. SEQ ID NO: 6), and any combination thereof; 2) a polypeptide selected from the group consisting of Klf4, Sox2, Oct4, cMyc, and any combination thereof; 3) a polypeptide selected from the group consisting of Sox2, Oct4, Lin28, Nanog, and any combination thereof; 4) a polypeptide selected from the group consisting of Ngn3 (e.g. SEQ ID NO: 8), PDX1 (e.g. SEQ ID NO: 9), MafA (e.g. SEQ ID NO: 10), NeuroD (e.g. SEQ ID NO: 7), and any combination thereof; 5) a polypeptide comprising Foxp3 (e.g. SEQ ID NO: 11); 6) a polypeptide selected from the group consisting of Oct4, Sox2, Klf4, Lin28, Nanog, cMyc, Ngn3, PDX1, MafA, NeuroD, Foxp3, and any combination thereof; 7) a combination of polypeptides Oct4, Sox2, Klf4 and cMyc; 8) a combination of polypeptides Ngn3, PDX1 and MafA; 9) a polypeptide selected from the group consisting of pax6, ASCL1, Brn2, MYT1L, Neurod1, Neurod6, Prdm8, Npas4, Mef2c, Dlx1, Tbr1, ISL1, Foxp1, Foxp2, Nhlh2, Sox2, Brn4, Hes1, Hes5, Lhx2, Oligo2, Ngn2 (e.g. SEQ ID NO: 67), Dlx2 (e.g. SEQ ID NO: 68), Zic1, NAP1L2, Nrip3, Satb2, Chd5, Smarca1, Brm (Smarca2), Brg1 (Smarca4) and any combination thereof (the exemplary sequences are shown in Table 1); 10) a polypeptide selected from the group consisting of Neurod6, Satb2, Zic1 and any combination thereof; and 11) a polypeptide selected from the group consisting of Dlx2, Ngn2, and a combination thereof
The polypeptides provided herein encompass their homologous sequences. A “homologous sequence” as used herein, refers to a polypeptide which shares sufficient percentage of identity in amino acid sequence with a reference polypeptide to which it is homologous. In certain embodiments, a homologous sequence shares at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 93%, at least 95%, at least 97%, at least 98%, or at least 99% of identity in amino acid sequence with one of the polypeptides provided herein useful as effector domain. The polypeptides having homologous sequences have substantially the same activity as the effector domains disclosed herein.
The percentage of identity in amino acid sequence can be determined as the percentage of amino acid residues in a candidate amino acid sequence that are identical to the amino acid residues in a reference amino acid sequence, after aligning the sequences and, if necessary, introducing gaps, to achieve the maximum number of identical amino acids. Conservative substitution of the amino acid residues may be considered or may not considered as identical residues. “Conservative substitution” as used herein refers to replacing an amino acid residue with another amino acid residue that has similar physiochemical properties (e.g., hydrophobicity and molecular bulk of the side chain).
Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways known in the art. For instance, alignment of amino acid sequences may be made using publicly available tools such as BLASTp (available on the website of U.S. National Center for Biotechnology Information (NCBI): http://blast.ncbi.nlm.nih.gov/Blast.cgi, see also, Altschul S. F. et al, J. Mol. Biol., 215:403-410 (1990); Stephen F. et al, Nucleic Acids Res., 25:3389-3402 (1997)), ClustalW2 (available on the website of European Bioinformatics Institute: http://www.ebi.ac.uk/Tools/msa/clustalw2/, see also, Higgins D. G. et al, Methods in Enzymology, 266:383-402 (1996); Larkin M. A. et al, Bioinformatics (Oxford, England), 23(21): 2947-8 (2007)), and TCoffee (available on the website of Swiss Institute of Bioinformatics, see also, Poirot O. et al, Nucleic Acids Res., 31(13): 3503-6 (2003); Notredame C. et al, J. Mol. Boil., 302(1): 205-17 (2000)). Those skilled in the art may use the default parameters provided by the tool, or may customize the parameters as appropriate for the alignment, such as for example, by selecting a suitable algorithm.
The polypeptides provided herein further encompass their functional equivalents. A “functional equivalent” as used herein, refers to a polypeptide which possesses functional or structural characteristics that are substantially similar to all or part of a parent polypeptide. The polypeptides provided herein encompass their functional equivalents that can exert a substantially similar function, i.e. reprogramming a somatic cell into a stem cell or changing a cell state from one to another. A functional equivalent of the polypeptides provided herein (i.e. parent polypeptides) may be a fragment, mutant, derivative, variant, or analog of a parent polypeptide, and may contain chemical or biological modifications. The functional equivalent may have one or more amino acid substitutions, additions, deletions, insertions, truncations, modifications (e.g. phosphorylation, glycosylation, labeling, etc.), or any combination thereof, of the parent polypeptide. The functional equivalent may include naturally occurring variants of the parent polypeptide and artificial polypeptide sequences such as those obtained by recombinant methods or chemical synthesis. The functional equivalent may contain non-naturally occurring amino acid residues.
In certain embodiments, a transducible material further comprises a transduction domain. A transduction domain is a motif that is capable of facilitating the entry of the transducible material into a biological sample (e.g., a cell). The transducible domain is associated with the effector domain covalently, noncovalently or via a linker. In certain embodiments, the transduction domain is covalently linked to the effector domain through a linker. In certain embodiments, the linker is a glycine-rich linker that comprises one or more glycine residues (e.g. esggggspg (SEQ ID NO: 55)).
Examples of a transduction domain include, without limitation, polymers such as cationic lipid polymers and nanoparticles, protein transduction domains (PTD), cell penetrating peptides (CPP1), cell permeating peptides (CPP2), activatable cell penetrating peptides or conjugates (ACPP), and cell-targeting peptides (CTP).
CPP1, CPP2, and PTD are peptides known to facilitate delivery of a molecular cargo associated thereof into cells. The association between a CPP1, CPP2 or PTD and the molecular cargo can be through covalent bond or non-covalent interactions. The molecular cargo can be small chemical molecules, peptides, protein, fragment of DNA, RNA such as siRNA and miRNA, or nanosize particles. For example, CPP1 and PTD include 5 to 20 amino acid peptide motifs that are capable of penetrating cells independent of surface transporters and of cell cycle phase. CPP1 and PTD can also be capable of penetrating through blood-brain barriers. CPP1 and PTD can deliver proteins and peptides in vitro and in vivo with uniform distribution throughout the organism after parenteral administration. Cationic PTDs can act as nuclear localization signals and carry an associated molecular cargo to cell nuclei. Examples of protein transduction domains include, without limitation, TAT (e.g. YGRKKRRQRRR, SEQ ID NO: 34), poly-arginine (e.g. poly-arginine having 7-11 arginine residues such as RRRRRRR, RRRRRRRR, RRRRRRRRR, RRRRRRRRRR (SEQ ID NO: 35) and RRRRRRRRRRR (SEQ ID NO: 36)), Penetratin (Antennapedia, e.g. RQIKIWFQNRRIVIKWKK (SEQ ID NO: 38)), VP22 (e.g. DAATATRGRSAASRPTQRPRAPARSASRPRRPVQ (SEQ ID NO: 39)), Transportan (e.g. GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 40)), MAP (e.g. KLALKLALKALKAALKLA (SEQ ID NO: 41)), MTS (e.g. AAVALLPAVLLALLP (SEQ ID NO: 42)), PEP-1 (e.g. KETWWETWWTEWSQPKKKRKV (SEQ ID NO: 43)), Arg/Trp analogue (e.g. RRWRRWWRRWWRRW (SEQ ID NO: 44)), polyguanidine peptoids (e.g. polyguanidine peptoids with a 6-methylene spacer between backbone and guanidine group such as N-arg 5, 7 or 9 peptoids), HIV-1 Rev (e.g. TRQARRNRRRRWRERQR (SEQ ID NO: 60)), Flock house virus coat peptide (e.g. RRRRNRTRRNRRRVR (SEQ ID NO: 61)), DNA-binding peptides such as c-Fos (e.g. KRRIRRERNKMAAAKSRNRRRELTDT (SEQ ID NO: 62)), c-Jun (e.g. RIKAERKRMRNRIAASKSRKRKLERIAR (SEQ ID NO: 63)) and yeast GCN4 (e.g. KRARNTEAARRSRARKLQRMKQ (SEQ ID NO: 64)), and Fusogenic HA2 peptide (e.g. GLFGAIAGFIENGWEGMIDG (SEQ ID NO: 65) or GDIMGEWGNEIFGAIAGFLG (SEQ ID NO: 66)).
Cell-targeting peptides are proteins or peptides that bind to cell-surface receptors and enter cells through endocytosis. In certain embodiments, a cell-target peptide targets specific tissues or cell types, for example, GnRH peptides (e.g. SEQ ID NO: 58) target biological samples that express GnRH receptors (e.g. solid tumors and hormone-responsive cancer cell lines). More examples of cell-targeting peptides and the specific biological samples targeted are listed in Table 2.
An activatable cell penetrating peptide or conjugate (ACPP) comprises a cationic CPP1, CPP2 or PTD and a neutralizing anionic counterpart. In certain embodiments, the cationic CPP1, CPP2 or PTD and the anionic counterpart are associated via noncovalent interactions (e.g. charge-charge interaction) and/or covalent cleavable linker (e.g. matrix metalloprotease (MMP) cleavable sequence). Transduction of an ACPP into cells are inhibited until the noncovalent interactions are disrupted and/or the cleavable linker is cleaved. For example, without being bound to any theory, the anionic counterparts comprise one or more pH sensitive groups such as sulfonamide groups, which are protonated at pH 7.4 (the pH in the blood stream) and become neutral at slightly acidic pH (e.g. pH 6.8). Therefore, charge-charge interactions between cationic CPP1, CPP2 or PTD and the anionic counterpart can be interrupted in slightly acidic microenvironment (e.g. in or around tumor or cancer). MMP concentration in blood stream is lower than that in a microenvironment around tumor or cancer. Therefore, MMP cleavable sequence, which is not cleaved in the bloodstream, is cleaved in environment around tumor or cancer. The cationic CPP1, CPP2 or PTD is no longer neutralized by the anionic counterpart, and therefore is exposed to facilitate the translocation into cells (e.g. tumor or cancer cells). In certain embodiments, the CPP1, CPP2 or PTD is TAT. In certain embodiments, the anionic counterparts comprise pH-sensitive polymer (e.g. di-block copolymer) comprising pH-sensitive groups (e.g. sulfonamide groups).
For another example, an activatable cell penetrating conjugate comprises a conventional hydrophobic core made of a polymer into which an effective domain is incorporated, a peripheral hydrophilic layer composed of poly-ethylene glycol and one or more cationic CPP1s, CPP2s or PTDs, and one or more anionic counterpart that neutralize the cationic CPP12, CPP2s or PTDs through charge-charge interactions. Such charge-charge interactions are expected to shield the cationic charges during delivery until the transduction material reaches a slightly acidic microenvironment (e.g. tumor or cancer), which triggers protonation of the anionic counterparts and disrupts the charge-charge association. Subsequently the cationic CPP1s, CPP2s, or PTDs, previously quenched by the anionic counterpart, are now capable of facilitating delivery of the effector domain into the surrounding cells (e.g. tumor or cancer cells).
In certain embodiments, a selective transducible material comprises a transduction domain selected from the group consisting of cell-targeting peptides and activatable cell penetrating peptides and activatable cell penetrating conjugates.
A transduction domain is associated to an effector domain via covalent bond, non-covalent interactions or through a linker. Thus a transducible material can be made by obtaining the transduction domain and the effector domain separately and associate together through a covalent bond or non-covalent interactions (e.g., repulsive interactions, dipole interactions, hydrogen bonding interactions, dispersive interactions, charge-charge interactions, solvent, counter ion and entropic effects, and water and hydrophobic effects). In certain embodiments, the transduction material is prepared by mixing the effector domain and transducible domain. Alternatively, a transducible material can be produced by isolating the material from natural resources or recombinantly. In the case when both domains are peptides or polypeptides, the effector domain can be linked to the N-terminus or C-terminus of the transduction domain and the transducible polypeptide can be made synthetically through chemical synthesis or recombinantly through recombinant technology.
In certain embodiments, a transducible material comprises an effector domain that is inherently transducible, and a transduction domain associated with the effector domain via covalent or non-covalent interactions.
In certain embodiments, a transducible material further comprises one or more motifs that do not interrupt the function of the effector domain or the transduction domain. In certain embodiments, these motifs are linked covalently, non-covalently or through a linker to the effector domain and/or the transduction domain. In certain embodiments, these motifs facilitate the preparation and/or purification of the transducible material. One example of such motif is a polyhistidine-tag to facilitate protein purification in preparation of the transducible material. In certain embodiments, the polyhistidine-tag comprises at least six histidine residues (e.g. MGSSHHHHHHSSGLVPRGSH (“His6,” SEQ ID NO: 59)).
In certain embodiments, a transducible material includes, for example, Oct4-11R (SEQ ID NO: 12), Sox2-11R (SEQ ID NO: 13), Klf4-11R (SEQ ID NO: 14), Lin28-11R (SEQ ID NO: 16), Nanog-11R (SEQ ID NO: 17), cMyc-11R (SEQ ID NO: 15), Ngn3-11R (SEQ ID NO: 19), PDX1-11R (SEQ ID NO: 20), MafA-11R (SEQ ID NO: 21), NeuroD-11R (SEQ ID NO: 18), Foxp3-11R (SEQ ID NO: 22), pax6-11R, ASCL1-11R, Brn2-11R, MYT1L-11R, Neurod1-11R, Neurod6-11R, Prdm8-11R, Npas4-11R, Mef2c-11R, Dlx1-11R, Tbr1-11R, ISL1-11R, Foxp1-11R, Foxp2-11R, Nhlh2-11R, Brn4-11R, Hes1-11R, Hes5-11R, Lhx2-11R, Oligo2-11R, Ngn2-11R (SEQ ID NO: 69), Dlx2-11R (SEQ ID NO: 70), Zic1-11R, NAP1L2-11R, Nrip3-11R, Satb2-11R, Chd5-11R, Smarca1-11R, Brm-11R, and Brg1-11R, wherein 11R (SEQ ID NO: 37) stands for a polyarginine sequence of 11 arginine residues linking to a linker through which the polyarginine sequence is covalently linked to the effector domain. In certain embodiments, the “11R” is covalently linked to the C terminal of the effector domain. In certain embodiments, a transducible material includes, for example, His6-Oct4-11R (SEQ ID NO: 23), His6-Sox2-11R (SEQ ID NO: 24), His6-Klf4-11R (SEQ ID NO: 25), His6-Lin28-11R (SEQ ID NO: 27), His6-Nanog-11R (SEQ ID NO: 28), His6-cMyc-11R (SEQ ID NO: 26), His6-Ngn3-11R (SEQ ID NO: 30), His6-PDX1-11R (SEQ ID NO: 31), His6-MafA-11R (SEQ ID NO: 32), His6-NeuroD-11R (SEQ ID NO: 29), His6-Foxp3-11R (SEQ ID NO: 33), His6-pax6-11R, His6-ASCL1-11R, His6-Brn2-11R, His6-MYT1L-11R, His6-Neurod1-11R, His6-Neurod6-11R, His6-Prdm8-11R, His6-Npas4-11R, His6-Mef2c-11R, His6-Dlx1-11R, His6-Tbr1-11R, His6-ISL1-11R, His6-Foxp1-11R, His6-Foxp2-11R, His6-Nhlh2-11R, His6-Brn4-11R, His6-Hes1-11R, His6-Hes5-11R, His6-Lhx2-11R, His6-Oligo2-11R, His6-Ngn2-11R (SEQ ID NO: 71), His6-Dlx2-11R (SEQ ID NO: 72), His6-Zic1-11R, His6-NAP1L2-11R, His6-Nrip3-11R, His6-Satb2-11R, His6-Chd5-11R, His6-Smarca1-11R, His6-Brm-11R, and His6-Brg1-11R. In certain embodiments, the “His6” is covalently linked to the N terminal of the effector domain. Exemplary sequences of transducible materials are shown in Table 3.
In certain embodiments, a transducible material can be combined with one or more adjuvants such as small molecule epigenetic agents. Suitable epigenetic agents include, without limitation, histone deacetylase inhibitor and DNA methylation inhibitor. Examples of suitable adjuvants include, without limitation, trichostatin A, which is a histone deacetylase inhibitor and DNA methylation inhibitor, valproic acid, which is a histone deacetylase inhibitor and DNA methylation inhibitor, and aza-2′-deoxycytidine, which is a DNA methylation inhibitor.
Another aspect of the present disclosure relates to a composition comprising a biological sample and at least one transducible material, wherein the transducible material has transduced into the biological sample. For example, the composition includes a transducible material comprising Foxp3 (e.g. the transducible material is Foxp3, Foxp3-11R or His6-Foxp3-11R) and a T cell wherein the transducible material has transduced into the T cell; a composition includes a piPS cell and one or more transducible materials comprising a polypeptide selected from the group consisting of Oct4, Klf4, Sox2 and cMyc, and any combination thereof (e.g. the transducible material is Oct4, Klf4, Sox2, cMyc, Oct4-11R, Klf4-11R, Sox2-11R, cMyc-11R, His6-Oct4-11R, His6-Klf4-11R, His6-Sox2-11R or His6-cMyc-11R); a composition including a liver or pancreatic exocrine cell and one or more transducible materials comprising a polypeptide selected from the group consisting of Ngn3, PDX1, MafA, NeuroD, and any combination thereof (e.g. the transducible material is Ngn3, PDX1, MafA, NeuroD, Ngn3-11R, PDX1-11R, MafA-11R, His6-Ngn3-11R, His6-PDX1-11R or His6-MafA-11R) wherein the transducible materials have transduced into the liver or pancreatic exocrine cell; and a composition including a glial cell and one or more transducible materials comprising a polypeptide selected from the group consisting of pax6, ASCL1, Brn2, MYT1L, Neurod1, Neurod6, Prdm8, Npas4, Mef2c, Dlx1, Tbr1, ISL1, Foxp1, Foxp2, Nhlh2, Sox2, Brn4, Hes1, Hes5, Lhx2, Oligo2, Ngn2, Dlx2, Zic1, NAP1L2, Nrip3, Satb2, Chd5, Smarca1, Brm (Smarca2), Brg1 (Smarca4), and any combination thereof (e.g. the transducible material is pax6, ASCL1, Brn2, MYT1L, Neurod1, Neurod6, Prdm8, Npas4, Mef2c, Dlx1, Tbr1, ISL1, Foxp1, Foxp2, Nhlh2, Sox2, Brn4, Hes1, Hes5, Lhx2, Oligo2, Ngn2, Dlx2, Zic1, NAP1L2, Nrip3, Satb2, Chd5, Smarca1, Brm, Brg1, pax6-11R, ASCL1-11R, Brn2-11R, MYT1L-11R, Neurod1-11R, Neurod6-11R, Prdm8-11R, Npas4-11R, Mef2c-11R, Dlx1-11R, Tbr1-11R, ISL1-11R, Foxp1-11R, Foxp2-11R, Nhlh2-11R, Sox2-11R, Brn4-11R, Hes1-11R, Hes5-11R, Lhx2-11R, Oligo2-11R, Ngn2-11R, Dlx2-11R, Zic1-11R, NAP1L2-11R, Nrip3-11R, Satb2-11R, Chd5-11R, Smarca1-11R, Brm-11R, Brg1-11R, His6-pax6-11R, His6-ASCL1-11R, His6-Brn2-11R, His6-MYT1L-11R, His6-Neurod1-11R, His6-Neurod6-11R, His6-Prdm8-11R, His6-Npas4-11R, His6-Mef2c-11R, His6-Dlx1-11R, His6-Tbr1-11R, His6-ISL1-11R, His6-Foxp1-11R, His6-Foxp2-11R, His6-Nhlh2-11R, His6-Sox2-11R, His6-Brn4-11R, His6-Hes1-11R, His6-Hes5-11R, His6-Lhx2-11R, His6-Oligo2-11R, His6-Ngn2-11R, His6-Dlx2-11R, His6-Zic1-11R, His6-NAP1L2-11R, His6-Nrip3-11R, His6-Satb2-11R, His6-Chd5-11R, His6-Smarca1-11R, His6-Brm-11R, or His6-Brg1-11R) wherein the transducible materials have transduced into the glial cell.
Another aspect of the present disclosure relates to a method of reprogramming a biological sample by exposing the biological sample to a composition comprising a transducible material. In certain embodiments, the method preferably reprograms a specific type of biological sample (e.g. cancer or tumor cells) or biological samples in or around a specific microenvironment within a biological organism (e.g. microenvironment around cancer or tumor) than other biological samples by exposing biological samples to a composition comprising a selective transducible material.
In one embodiment, a biological sample includes a cell, a cluster of cells, a tissue, an organ, a biological body from a biological organism. The biological sample can be normal, healthy sample or abnormal, diseased sample (e.g., cancer or tumor).
A biological organism includes, for example, a microorganism (e.g., bacteria), a fungus, a plant and an animal (e.g., a human).
An organ from an animal biological organism (e.g., human) includes, for example, a circulatory organ (e.g., heart, blood and blood vessels), a digestive organ (e.g., salivary glands, esophagus, stomach, liver, gallbladder, pancreas, intestines, rectum and anus), an endocrine organ (e.g., endocrine glands such as the hypothalamus, pituitary or pituitary gland, pineal body or pineal gland, thyroid, parathyroids and adrenals, i.e., adrenal glands), an integumentary organ (e.g., skin, hair and nails), a lymphatic organ (e.g., lymph nodes and vessels, tonsils, adenoids, thymus and spleen), a muscular organ (e.g., muscles), a nervous organ (e.g., brain, spinal cord, peripheral nerves and nerves), a reproductive organ (e.g., ovaries, fallopian tubes, uterus, vagina, mammary glands, testes, vas deferens, seminal vesicles, prostate and penis), a respiratory organ (e.g., the pharynx, larynx, trachea, bronchi, lungs and diaphragm), a skeletal organ (e.g., bones, cartilage, ligaments and tendons), a urinary system (e.g., kidneys, ureters, bladder and urethra). An organ can be normal or healthy, and alternatively, abnormal or unhealthy (e.g., cancerous).
An organ from a plant biological organism includes, for example, root, stem, leaf, flower, seed and fruit.
A tissue from a biological sample (e.g. an animal) includes a connective tissue, a muscle tissue, a nervous tissue, and an epithelial tissue. A tissue can be normal or healthy, and alternatively, abnormal or unhealthy (e.g., cancerous). A tissue from a biological sample (e.g. a plant) includes an epidermis, a vascular tissue and a ground tissue.
A cell can be prokaryotic or eukaryotic. A prokaryotic cell includes, for example, bacteria. A eukaryotic cell includes, for example, a fungus, a plant cell, and an animal cell. The types of an animal cell (e.g., a mammalian cell or a human cell) includes, for example, a cell from circulatory/immune system or organ (e.g., a B cell, a T cell (cytotoxic T cell, natural killer T cell, regulatory T cell, T helper cell), a natural killer cell, a granulocyte (e.g., basophil granulocyte, an eosinophil granulocyte, a neutrophil granulocyte and a hypersegmented neutrophil), a monocyte or macrophage, a red blood cell (e.g., reticulocyte), a mast cell, a thrombocyte or megakaryocyte, and a dendritic cell); a cell from an endocrine system or organ (e.g., a thyroid cell (e.g., thyroid epithelial cell, parafollicular cell), a parathyroid cell (e.g., parathyroid chief cell, oxyphil cell), an adrenal cell (e.g., chromaffin cell), and a pineal cell (e.g., pinealocyte)); a cell from a nervous system or organ (e.g., a glioblast (e.g., astrocyte and oligodendrocyte), a microglia, a magnocellular neurosecretory cell, a stellate cell, a boettcher cell, and a pituitary cell (e.g., gonadotrope, corticotrope, thyrotrope, somatotrope, and lactotroph)); a cell from a respiratory system or organ (e.g., a pneumocyte (a type I pneumocyte and a type II pneumocyte), a clara cell, a goblet cell, an alveolar macrophage); a cell from circular system or organ (e.g., myocardiocyte and pericyte); a cell from digestive system or organ (e.g., a gastric chief cell, a parietal cell, a goblet cell, a paneth cell, a G cell, a D cell, an ECL cell, an I cell, a K cell, an S cell, an enteroendocrine cell, an enterochromaffin cell, an APUD cell, a liver cell (e.g., a hepatocyte and Kupffer cell)); a cell from integumentary system or organ (e.g., a bone cell (e.g., an osteoblast, an osteocyte, and an osteoclast), a teeth cell (e.g., a cementoblast, and an ameloblast), a cartilage cell (e.g., a chondroblast and a chondrocyte), a skin/hair cell (e.g., a trichocyte, a keratinocyte, and a melanocyte (Nevus cell)), a muscle cell (e.g., myocyte), an adipocyte, a fibroblast, and a tendon cell), a cell from urinary system or organ (e.g., a podocyte, a juxtaglomerular cell, an intraglomerular mesangial cell, an extraglomerular mesangial cell, a kidney proximal tubule brush border cell, and a macula densa cell), and a cell from reproductive system or organ (e.g., a spermatozoon, a sertoli cell, a leydig cell, an ovum, an oocyte). A cell can be normal, healthy cell; or a diseased or unhealthy cell (e.g., a cancer cell).
A cell further includes a mammalian stem cell which include an embryonic stem cell, a fetal stem cell, an induced pluripotent stem cell, and an adult stem cell. A stem cell is a cell that is capable of undergoing cycles of cell division while maintaining an undifferentiated state and differentiating into specialized cell types. A stem cell can be an omnipotent stem cell, a pluripotent stem cell, a multipotent stem cell, an oligopotent stem cell and an unipotent stem cell (See, Hans R. Schöler (2007). “The Potential of Stem Cells: An Inventory” in Nikolaus Knoepffler, Dagmar Schipanski, and Stefan Lorenz Sorgner. Humanbiotechnology as Social Challenge. Ashgate Publishing, Ltd. pp. 28), any of which may be induced from a somatic cell. A stem cell may also include a cancer stem cell.
In certain embodiments, the cell is a glial cell. A glial cell is a non-neuronal cell that surrounds neurons and provides support, protection and/or nutrients to neurons. A glial cell can be microglia, astrocyte, oligodendrocyte, ependymal cell, radial glia, Schwann cell, satellite cell, and enteric glia cell. In certain embodiments, the glial cell is astrocyte.
In another embodiment, “reprogramming a biological sample” used herein is exchangeable with or refers to modulating, altering, or changing the biological activities of the biological sample (e.g., cell) or modulating, altering, or changing the state or status of the biological sample from one to another. For example, by exposing a biological sample (e.g., a cell) to a transducible material, the biological activities of the cell (e.g., cell growth, cell division, cell metabolism, cell cycle, cell signaling, DNA replication, transcription, RNA splicing, protein synthesis, post-translation modification) are modulated or altered so as to lead to cell proliferation, differentiation (e.g., from progenitor cells to terminally differentiated cells), dedifferentiation (e.g., from terminally differentiated cells to pluripotent stem cells), transdifferentiation (e.g., from one type of terminally differentiated cells to another type of terminally differentiated cells), retrodifferentiation (e.g., from terminally differentiated cells to progenitor cells), transdertermination (e.g., from one type of progenitor cells to a type of terminally differentiated cells that are usually derived from another type of progenitor cells under natural conditions), apoptosis (e.g., cell death of cells or cancer cells), morphogenesis, and changes in the cell fate. For another example, the state of a biological sample can be altered or changed from abnormal or diseased state to normal or healthy state (e.g., from cancer cells to noncancer cells); from one cell type to another cell type (e.g., from undifferentiated stem cells to differentiated stem cells or specialized cells), from differentiated or specialized cells to undifferentiated cells or stem cells (e.g., an omnipotent stem cell, a pluripotent stem cell, a multipotent stem cell, an oligopotent stem cell and an unipotent stem cell) (e.g., from fibroblast cells to induced pluripotent stem cells (iPSCs)), from somatic cells to stem cells or induced stem cells, from one state of stem cells to another state of stem cells (e.g., from ominipotent stem cells to pluripotent stem cells), from one type of differentiated cells to another type of differentiated cells (e.g., T-cells to regulatory T cells, pancreatic exocrine cells to insulin-producing beta cells).
In another embodiment, a biological sample is exposed to a transducible material and reprogrammed. The biological sample can be exposed in vitro, in vivo or ex vivo. For example, the biological sample is exposed in vitro through contacting the sample with the transducible material in an environment outside of a living biological organism (e.g., in a cell culture system or a test tube). The biological sample is exposed in vivo through contacting the material with a biological organism containing the sample or introducing (e.g., through administration) the material into the organism. The transducible materials can be administered via any known administration route such as for example parenteral (e.g., subcutaneous, intraperitoneal, intravenous, including intravenous infusion, intramuscular, or intradermal injection) or non-parenteral (e.g., oral, intranasal, intraocular, sublingual, rectal, or topical) route. The biological sample is exposed ex vivo when the biological sample (e.g., a cell, a tissue or an organ) is taken outside the biological organism, contacted with the transducible material, and placed back to the same or different biological organisms. Examples of ex vivo exposures comprise removing a biological sample from the biological organism, exposing the biological sample to a transducible material, and transplanting the biological sample transduced with the transducible material back to the biological organism.
In certain embodiments, OG2-MEF cells are exposed to a composition comprising protein Oct4-11R, Sox2-11R, Klf4-11R and cMyc-11R and reprogrammed to induced pluripotent stem cells (iPSCs).
In certain embodiments, T cells are exposed to a composition comprising protein Foxp3-11R or His6-Foxp3-11R and reprogrammed to regulatory T cells (Treg cells).
In certain embodiments liver and/or pancreatic exocrine cells are exposed to a composition comprising one or more proteins selected from the group consisting of Ngn3-11R, PDX1-11R, MafA-11R, NeuroD-11R, His6-Ngn3-11R, His6-PDX1-11R, His6-MafA-11R, and His6-NeuroD-11R and reprogrammed into insulin producing cells (e.g. β cells). In certain embodiments, the composition further comprises one or more adjuvant such as Islet growth factor (e.g. betacellulin). In certain embodiments, the composition comprises His6-Ngn3-11R, His6-PDX1-11R, and His6-MafA-11R. In certain embodiments, the composition comprises His6-Ngn3-11R, His6-PDX1-11R, His6-MafA-11R and betacellulin. Without intending to be bound to a particular mechanism, it is further contemplated that such reprogramming is through transdetermination and/or transdifferentiation.
In certain embodiments, glial cells (e.g. astrocytes) are exposed to a composition comprising one or more proteins selected from the group consisting of pax6-11R, ASCL1-11R, Brn2-11R, MYT1L-11R, Neurod1-11R, Neurod6-11R, Prdm8-11R, Npas4-11R, Mef2c-11R, Dlx1-11R, Tbr1-11R, ISL1-11R, Foxp1-11R, Foxp2-11R, Nhlh2-11R, Sox2-11R, Brn4-11R, Hes1-11R, Hes5-11R, Lhx2-11R, Oligo2-11R, Ngn2-11R, Dlx2-11R, Zic1-11R, NAP1L2-11R, Nrip3-11R, Satb2-11R, Chd5-11R, Smarca1-11R, Brm-11R, Brg1-11R, His6-pax6-11R, His6-ASCL1-11R, His6-Brn2-11R, His6-MYT1L-11R, His6-Neurod1-11R, His6-Neurod6-11R, His6-Prdm8-11R, His6-Npas4-11R, His6-Mef2c-11R, His6-Dlx1-11R, His6-Tbr1-11R, His6-ISL1-11R, His6-Foxp1-11R, His6-Foxp2-11R, His6-Nhlh2-11R, His6-Sox2-11R, His6-Brn4-11R, His6-Hes1-11R, His6-Hes5-11R, His6-Lhx2-11R, His6-Oligo2-11R, His6-Ngn2-11R, His6-Dlx2-11R, His6-Zic1-11R, His6-NAP1L2-11R, His6-Nrip3-11R, His6-Satb2-11R, His6-Chd5-11R, His6-Smarca1-11R, His6-Brm-11R, and His6-Brg1-11R, and reprogrammed into neurons. In certain embodiments, the composition further comprises one or more adjuvant such as epigenetic agents (e.g. trichostatin A, valproic acid, aza-2′-deoxycytidine). In certain embodiments, the composition comprises Ngn2-11R, Dlx2-11R, Neurod6-11R, Satb2-11R, ASCL1-11R, Brn2-11R, Zic1-11R, Npas4-11R, His6-Ngn2-11R, His6-Dlx2-11R, His6-Neurod6-11R, His6-Satb2-11R, His6-ASCL1-11R, His6-Brn2-11R, His6-Zic1-11R, His6-Npas4-11R, or any combination thereof. Without intending to be bound to a particular mechanism, it is further contemplated that such reprogramming is through transdetermination and/or transdifferentiation.
Another aspect of the present disclosure relates to a method of treating, preventing or reducing a disease or condition in a biological organism by administering a composition comprising a transducible material into the organism. In certain embodiments, the composition is a pharmaceutical composition comprising a transducible material. In certain embodiments, the composition comprises a selective transducible material. The treatment, prevention or reduction of a disease or condition is associated with the change or reprogramming of a biological sample (e.g., a cell, a tissue or an organ) in the organism.
The present disclosure also provides use of a transducible material in manufacturing a medicament for treating preventing or reducing a disease or condition in a biological organism. In certain embodiments, the transducible material is a selective transducible material. The treatment, prevention or reduction of a disease or condition is associated with the change or reprogramming of a biological sample (e.g., a cell, a tissue or an organ) in the organism.
In certain embodiments, the disease or condition treatable by the method or treatable by the medicament include, without limitations, tumor, cancer, metabolic diseases or conditions (e.g. type I and type II diabetes and obesity), inflammatory conditions, cardiac diseases, neurogenerative diseases (e.g. anemia, amyotrophic lateral sclerosis, spinal cord injury, burns, or arthritis), autoimmune diseases or conditions (e.g. acute disseminated encephalomyelitis (ADEM), Addison's disease, alopecia greata, ankylosing spondylitis, antiphospholipid antibody syndrome (APS), anemia (e.g. autoimmune hemolytic anemia and pernicious anaemia), arthritis, psoriatic arthritis, rheumatoid arthritis, diabetes mellitus type 1, autoimmune hepatitis, autoimmune inner ear disease, bullous pemphigoid, coeliac disease, Chagas disease, chronic obstructive pulmonary disease, Crohns disease, dermatomyositis, endometriosis, Goodpasture's syndrome, Graves' disease, Guillain-Barre syndrome (GBS), Hashimoto's disease, hidradenitis suppurativa, Kawasaki disease, IgA nephropathy, idiopathic thrombocytopenic purpura, interstitial cyctitis, lupus erythematosus, mixed connective tissue disease, morphea, multiple sclerosis (MS), myasthenia gravis, narcolepsy, neuromyotonia, pemphigus vulgaris, psoriasis, polymyositis, primary billiary cirrhosis, schizophrenia, scleroderma, Sjogren's syndrome, stiff person syndrome, temporal arteritis (“Giant cell arteritis”), ulcerative colitis, vasculitis, vitiligo, and Wegener's granulomatosis).
For example, it is contemplated that a transducible material, or a medicament manufactured from a transducible material can be administered to a biological organism having a tumor to activate the apoptosis of the tumor cells or make tumor cells more sensitive to chemotherapy, radiotherapy, or cancer drugs.
In certain embodiments, a transducible material, or a medicament manufactured from a transducible material can be administered to a biological organism to enhance or attenuate immune system and thus treat or prevent immune-related diseases or inflammatory diseases. For example, protein Foxp3-11R or His6-Foxp3-11R is transduced to T cells and programs them to Treg cells, which suppress the overactive immune system and thus is a treatment for auto-immune diseases
In certain embodiments, a transducible material, or a medicament manufactured from a transducible material can be administered to a biological organism to treat neurological diseases or conditions such asischemic and hemorrhagic stroke, spinal cord injury, brain injury, Huntington's disease, Alzheimer's disease, Parkinson's disease, Schizophrenia, Autism, Ataxia, Amyotrophic Lateral Sclerosis, Lou Gehrig's Disease, Lyme Disease, Meningitis, Migraine, Motor Neuron Diseases, Neuropathry, pain, brain damage (e.g. frontal lobe damage, parietal lobe damage, temporal lobe damage, and occipital lobe damage), brain dysfunction (e.g. aphasia, dysarthria, apraxia, agnosia, amnesia), spinal cord disorders (e.g. spinal injury, spinal inflammation, spinal pathology), peripheral nervous system disorders, cranial nerve disorders, autonomic nervous system disorders, seizure disorders such as epilepsy, movement disorders such as Parkinson's disease, sleep disorders, headaches (including migraine), lower back and neck pain, neuropathic pain, delirium and dementia such as Alzheimer's disease, dizziness and vertigo, stupor and coma, head injury, stroke (e.g. cerebrovascular attack), tumors of the nervous system (e.g. glioma), multiple sclerosis (MS) and other demyelinating diseases, infections of the brain or spinal cord (e.g. meningitis), and prion diseases (e.g. mad cow disease).
For example, to treat a neurological disorder or to treat damaged neurons, a polypeptide or a composition comprising such polynucleotide is transduced to glial cells and reprograms them to neurons, which polypeptide is selected from the group consisting of pax6-11R, ASCL1-11R, Brn2-11R, MYT1L-11R, Neurod1-11R, Neurod6-11R, Prdm8-11R, Npas4-11R, Mef2c-11R, Dlx1-11R, Tbr1-11R, ISL1-11R, Foxp1-11R, Foxp2-11R, Nhlh2-11R, Sox2-11R, Brn4-11R, Hes1-11R, Hes5-11R, Lhx2-11R, Oligo2-11R, Ngn2-11R, Dlx2-11R, Zic1-11R, NAP1L2-11R, Nrip3-11R, Satb2-11R, Chd5-11R, Smarca1-11R, Brm-11R, Brg1-11R, His6-pax6-11R, His6-ASCL1-11R, His6-Brn2-11R, His6-MYT1L-11R, His6-Neurod1-11R, His6-Neurod6-11R, His6-Prdm8-11R, His6-Npas4-11R, His6-Mef2c-11R, His6-Dlx1-11R, His6-Tbr1-11R, His6-ISL1-11R, His6-Foxp1-11R, His6-Foxp2-11R, His6-Nhlh2-11R, His6-Sox2-11R, His6-Brn4-11R, His6-Hes1-11R, His6-Hes5-11R, His6-Lhx2-11R, His6-Oligo2-11R, His6-Ngn2-11R, His6-Dlx2-11R, His6-Zic1-11R, His6-NAP1L2-11R, His6-Nrip3-11R, His6-Satb2-11R, His6-Chd5-11R, His6-Smarca1-11R, His6-Brm-11R, and His6-Brg1-11R, and any combination thereof. Without intending to be bound to a particular mechanism, it is further contemplated that such reprogramming is through transdetermination and/or transdifferentiation. In certain embodiments, a composition comprising a polypeptide selected from the group consisting of Ngn2-11R, Dlx2-11R, Neurod6-11R, Satb2-11R, ASCL1-11R, Brn2-11R, Zic1-11R, Npas4-11R, His6-Ngn2-11R, His6-Dlx2-11R, His6-Neurod6-11R, His6-Satb2-11R, His6-ASCL1-11R, His6-Brn2-11R, His6-Zic1-11R, His6-Npas4-11R, or any combination thereof is transduced into astrocytes and reprograms them to neurons.
In certain embodiments, one or more adjuvant such as epigenetic agents (e.g. trichostatin A, valproic acid, aza-2′-deoxycytidine) is/are also administered to the biological organism.
Another aspect of the present disclosure relates to a method of reprogramming iPSCs, embryonic stem cells, or other types of stem or progenitor cells to certain types of somatic cells or progenitor cells, which can be developed as cell-based therapies for various diseases or conditions, including neurological disorders, anemia, neurodegenerative diseases, cancer, amyotrophic lateral sclerosis, spinal cord injury, burns, heart diseases, diabetes, and arthritis. The stem cells or progenitor cells may be patient-specific or non-patient-specific, repaired to rid of molecular defects or not, before they are exposed to transducible materials for controlled differentiation or reprogramming. The reprogrammed cells may be enriched, purified, or manipulated before transplanted back to patients.
Another aspect of the present disclosure relates to a method of reprogramming iPSCs, embryonic stem cells, or other types of stem or progenitor cells to certain types of somatic cells or progenitor cells, which can be used as disease models for drug screening, mechanism study, toxicity assay, or other research and drug discovery and development tools. For example, the method comprises exposing an iPSC, an embryonic stem cell, or a progenitor cell to a composition comprising a transducible material to reprogram the iPSC, embryonic stem cell, or progenitor cell to a transplantable somatic cell or a transplantable progenitor cell; transplanting the transplantable somatic cell or transplantable progenitor cell into a biological sample or a biological organism; developing the biological sample or biological organism to become a disease model. For another example, the method comprises reprogramming patient-specific cells to iPSCs using a transducible materials; further generating different type of cells from patient specific iPSCs with or without transducible materials; and developing a disease model using patient-specific iPSCs or iPSC-derived cells. For another example, the method of developing drug screening or toxicity models comprises reprogramming somatic cells, progenitor cells, or multipotent cells to iPSCs using a transducible material; further generating different type of cells from iPSCs with or without exposing to transducible materials; and using iPSCs and/or iPSC-derived cells to screen the effects and/or toxicities of different compounds.
Another aspect of the present disclosure relates to a method of developing cell-based therapies for various diseases or conditions comprising the step of reprogramming an iPSC, an embryonic stem cell, or a progenitor cell to a transplantable somatic or progenitor cell using a transducible material; transplanting the transplantable somatic or progenitor cell into a biological sample or biological organism; assessing the therapeutic effect of the transplantable somatic or progenitor cell.
Another aspect of the present disclosure relates to a method of identifying a effector domain, wherein the method comprises the steps of covalently linking a test effector domain to a know transduction domain to form a test transducible molecule; exposing the test molecule to a biological sample, and measuring the reprogramming of the biological sample to indicate whether the test effector domain can exerts a change in the biological sample. It is also contemplated that another aspect of the present disclosure relates to a method of identifying a transducible domain, wherein the method comprises the steps of covalently linking a known effector domain to a test transduction domain to form a test transducible molecule; exposing the test molecule to a biological sample, and measuring the location of the test molecule in or the reprogramming effect of the biological sample to indicate whether the test transduction domain can transduce the effector domain into the biological sample.
The following examples are provided to better illustrate the claimed invention and are not to be interpreted in any way as limiting the scope of the invention. All specific compositions, materials, and methods described below, in whole or in part, fall within the scope of the invention. These specific compositions, materials, and methods are not intended to limit the invention, but merely to illustrate specific embodiments falling within the scope of the invention. One skilled in the art may develop equivalent compositions, materials, and methods without the exercise of inventive capacity and without departing from the scope of the invention. It will be understood that many variations can be made in the procedures herein described while still remaining within the bounds of the invention. It is the intention of the inventors that such variations are included within the scope of the invention.
1.a. Preparation of Transducible Material Oct4-11R, Sox2-11R, Klf4-11R, and cMyc-11R.
A poly-arginine protein transduction domain was fused to the C-terminal of each reprogramming proteins Oct4, Sox2, Klf4 and cMyc through a linker SEQ ID NO. 55 to form a fused protein Oct4-11R, Sox2-11R, Klf4-11R and cMyc-11R respectively (
1.b. Cell Permeability and Stability of Transducible Material Oct4-11R, Sox2-11R, Klf4-11R, and cMyc-11R
A transducible material (Oct4-11R, Sox2-11R, Klf4-11R, or cMyc-11R) was added to mouse embryonic fibroblast (MEF) cells at various concentrations for 6-72 hours. Cell morphology and protein presence were examined by immunocytochemistry. The transducible materials were found to enter cells at concentrations of 0.5-8 μg/ml within 6 hours, and translocated into nucleus (
1.c. Reprogramming OG2/Oct4-GFP Reporter MEF Cells.
The protein transduction condition described in paragraph 0047 was used to reprogram OG2/Oct4-GFP reporter MEF cells. Cells were treated in 4 cycles. In each cycle the fibroblasts (initially seeded at the density of 5×104 cells/well in a six-well plate) were first treated with transducible materials Oct4-11R, Sox2-11R, Klf4-11R and cMyc-11R at 8 μg/ml in the mESC growth media supplemented with or without 1 mM valproic acid (VPA, a inhibitor of the enzyme histone deacetylase 1 (HDAC1)) for overnight, followed by changing to the same media without the transducible material and VPA, and culturing for additional 36 hours before the next cycle of the treatment. After completing repeated protein transduction of a transducible material, the treated cells were transferred onto irradiated MEF feeder cells and kept in mESC growth media until colonies emerged around day 30-35 (
The generated murine piPS cells have been stably expanded for over twenty passages, and were morphologically indistinguishable to classic mES cells, forming compact domed small colonies (
To examine the developmental potential of piPS cells, standard in vitro differentiation using embryoid bodies (EB) or monolayer chemically defined step-wise differentiation, as well as in vivo chimerism assays were performed. piPS cells efficiently formed EB in suspension, and differentiated into cells in the three primary germ layers, including primitive endoderm (AFP, Sox17), foregut endoderm (FoxA2), pancreatic cells endoderm (PDX1, Pax6), mesoderm (Brachyury), and neural (Sox1) and neuronal cells (βIII-tubulin)-ectoderm (
A poly-arginine protein transduction domain was fused respectively to the C-terminal of each reprogramming protein (Ngn3, PDX1 and MafA) through a linker (SEQ ID NO: 55) to form His6-Ngn3-11R, His6-PDX1-11R and His6-MafA-11R respectively (
Six CD-1 mice (Charles River Laboratory) were divided into two groups: the treatment group and the control group. Transducible material His6-Ngn3-11R (1 mg/kg), His6-PDX1-11R (1 mg/kg), and His6-MafA-11R (1 mg/kg) were injected into each mouse by intraperitoneal (IP) in treatment group (Mouse-4, Mouse-5 and Mouse-6) and BSA (1 mg/kg) was injected into each mouse in the control group (Mouse-1, Mouse-2 and Mouse-3). There was no Greenish-brown or Yellow aspirate when needle penetrated into each mouse peritonea. Injections were repeated every day for 7 days. Mice of both treatment and control group were sacrificed on the 3rd day after the completion of all injections. The mouse liver and pancreas were washed with 1×PBS and fixed by 4% paraformaldehyde for overnight. Then the liver and pancreatic tissues were processed by standard Paraffin Embedding protocol. The Tissue sections, 5-micro in thickness, were prepared routinely with histology microtomes and mounted on standard histology glass slides. The wax in tissues was dissolved by xylene during processing of tissue sections. Tissue sectioning and histologic and immunohistochemical staining were performed using routine methods. For indirect fluorescent-antibody (IFA) assay, the slides were blocked with 0.05% Tween-20 (TBST) and 3% BSA for 1 hour at RT and were incubated with mouse anti-insulin antibody (Invitrogen) at 4° C. overnight. The slides were washed three times with PBS for 15 minutes at RT and incubated with fluorescein isothiocyanate (FITC) conjugated swine anti-mouse antibody (KPL) for 2 hours at RT. Same concentration of Mouse IgG was used as isotype control. Anti-DAPI antibody was added to slides as a nuclear marker. The slides were washed as before and mounted with aqueous mounting media (Biomeda, Foster City, Calif.). Endothelial markers were identified under the microscope (Olympus BX51, San Diego, Calif.) and merged cells were analyzed by Microsuite Biological Suite program (Olympus BX51, San Diego, Calif.) (
A poly-arginine protein transduction domain was fused to the C-terminal of each reprogramming protein Foxp3 through a linker (SEQ ID NO: 55) to form His6-Foxp3-11R (
100 ml of healthy human blood was collected from a donor and the peripheral blood mononuclear cells (PBMCs) were isolated by density-gradient centrifugation using Histopaque-1077 (Sigma-Aldrich, St Louis, Mo.). CD14+ monocytes were removed by magnetic bead selection (Miltenyi Biotec, Auburn, Calif.). Briefly, 108 PBMCs were incubated with 200 μL anti-CD14 microbeads (Miltenyi Biotec) in ice for 30 minutes. The cells were washed with cold 1×PBS with 2% FCS and centrifuged at 300 g for 10 minutes and then resuspended in 1×PBS with 2% FCS. The cell suspension was applied to the magnetic column and unbinding cells were passed through by washing 3 times with 1×PBS with 2% FCS. The PBMC/mono- were harvested by centrifuged at 300 g for 10 minutes.
The PBMC/mono- were cultured in 6-well plates (Becton Dickinson, Gaithersburg, Md.) supplemented with 10% FBS, nonessential amino acids, 2 mM glutamine, 1 mM sodium pyruvate, 25 mM HEPES, 200 units/ml penicillin, and streptomycin at 37° C. and 5% CO2. After 1 hour of culture, His6-Foxp3-11R (10 μg/ml, 20 μg/ml, or 50 μg/ml) was added to the cells. BSA (100 μg/ml) was added to another well as control. Same concentration of His6-Foxp3-11R or BSA was added after cultured for two days. After 5 days of culture, the cells were washed with PBS twice. The cells were re-suspended in 100 μL diluted and added rabbit anti-human CD25 for 90 minutes. The cells were washed three time with cold 1×PBS supplied 2% FBS and then the conjugated-PE mouse anti-human CD4 as well as conjugated-FITC goat anti-rabbit IgG were added to the cells for 60 minutes in ice. Conjugated-PE mouse IgG and rabbit IgG were incubated with another group cells as Isotype control. The cells were washed with PBS for flow cytometric analysis using a Beckman Coulter FC500 cytometer with Cytomics CXP software (Beckman Coulter, Fullerton, Calif.) (
100 ml of healthy human blood was collected from a donor and the peripheral blood mononuclear cells (PBMCs) were isolated by density-gradient centrifugation using Histopaque-1077 (Sigma-Aldrich, St Louis, Mo.). The PBMC/mono− were cultured in E-well plates (Becton Dickinson, Gaithersburg, Md.) supplemented with 10% FBS, nonessential amino acids, 2 mM glutamine, 1 mM sodium pyruvate, 25 mM HEPES, 200 units/ml penicillin, and streptomycin at 37° C. and 5% CO2. After 1 hour of culture, Foxp3 (10 μg/ml, 50 μg/ml, 100 μg/ml) were added to the cells. BSA (100 μg/ml) was added to another well as control. Same concentration of the Foxp3 or BSA was added after cultured two days. Following 5 days of culture, the cells were washed with PBS twice. The cells were re-suspended in 100 μL diluted and added rabbit anti-human CD25 for 90 minutes. The cells were washed three time with cold 1×PBS supplied 2% FBS and then the conjugated-PE mouse anti-human CD4 as well as conjugated-FITC goat anti-rabbit IgG were added to the cells for 60 minutes in ice. Conjugated-PE mouse IgG and rabbit IgG were incubated with another group cells as Isotype control. The cells were washed with PBS for flow cytometric analysis using a Beckman Coulter FC500 cytometer with Cytomics CXP software (Beckman Coulter, Fullerton, Calif.) (
A poly-arginine protein transduction domain was fused respectively to the C-terminal of each reprogramming protein (Ngn2 and Dlx2) through a linker (SEQ ID NO: 55) to form His6-Ngn2-11R (SEQ ID NO: 71), and His6-Dlx2-11R (SEQ ID NO: 72) respectively (
Mouse astrocytes were plated on 24-well plates and cultured in neural reprogramming medium with the transducible proteins (His-Ngn2-11R, His-Dlx2-11R, or both) at concentrations of 1 μg/ml for 10 consecutive days. Culture medium were changed daily. On day 11, the cells were switched to the neuronal differentiation medium. On day 18, the cells were fixed and analyzed by immunostaining.
The cells were stained with Tuj1 antibody and DAPI. Tuj1 (class III β-tubulin) is a marker of neurons, and DAPI is a chemical that binds to DNA and therefore a marker for nuclei. As shown in
The present application claims the benefit of U.S. Provisional Patent Application No. 61/337,522, filed on Feb. 4, 2010, and U.S. Provisional Patent Application No. 61/360,841, filed on Jul. 1, 2010, both of which are incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US11/23259 | 2/1/2011 | WO | 00 | 7/31/2012 |
| Number | Date | Country | |
|---|---|---|---|
| 61337522 | Feb 2010 | US | |
| 61360841 | Jul 2010 | US |
