Patent Application
20200040359
This document relates to materials and methods involved in making and using induced pluripotent stem cells.
The induced pluripotent stem cell technology allows for derivation of patient-specific pluripotent stem cells from adult somatic cells. Human induced pluripotent stem cells (iPSCs) have great potential to replace non-functioning tissues due to their unique capability of giving rise to any cell types of the body. Generation of iPSCs from the patient's own tissues allows novel autologous stem cell therapies, while circumventing immunological mismatch and ethical issues associated with the use of an embryonic cell source. iPSCs can also be used as a research tool.
This document provides materials and methods for making and using induced pluripotent stem cells (iPSCs). For example, this document provides vectors for reprogramming somatic cells into iPSCs, methods for obtaining iPSCs, and methods for using iPSCs.
As described herein, a measles virus (MV) vector having multiple reprogramming factors (RFs) can be used to reprogram safely and efficiently a somatic cell into an iPSC. Notably, the introduction of a target sequence of a microRNA (miRNA; e.g., miR375) into the MV vector can effectively increase the efficiency of reprogramming and decrease the time of reprogramming. The materials and methods provided herein increase efficiency, safety, and specificity of nuclear reprogramming, as well as minimize the risk of tumorigenicity due to sustained expression of a reprogramming factor (RF; e.g., cMYC) or vector-integration-mediated insertional mutagenesis into the host genome. Thus, the materials and methods described herein provide a new, safe way of producing pluripotent stem cells that can be differentiated into various types of cells useful for regenerative medicine approaches to treating diabetes, cardiac, respiratory, and other diseases.
In one aspect, this document features a Paramyxoviridae viral vector for reprogramming somatic cells into iPSCs, wherein the vector comprises nucleic acid encoding a plurality of reprogramming factors and a nucleic acid sequence targeted by a microRNA (miRNA) associated with pluripotency. The Paramyxoviridae viral vector can be a measles virus vector. The plurality of reprogramming factors can comprise OCT4, SOX2, and KLF4. The plurality of reprogramming factors can comprise OCT4, SOX2, KLF4, and cMYC. The OCT4, SOX2, KLF4, and cMYC can comprise human OCT4, human SOX2, human KLF4, and human cMYC. The miRNA associated with pluripotency can be a miRNA expressed in the iPSCs. The miRNA expressed can be miR375. The miR375 can be a human miR375. The nucleic acid sequence targeted by miR375 can comprise SEQ ID NO:1. The nucleic acid sequence targeted by miR375 can comprise three to five repeats of SEQ ID NO:1.
In another aspect, this document features a method for producing an induced pluripotent stem cell in vitro, the method comprising (a) introducing a Paramyxoviridae viral vector into a somatic cell, wherein the vector comprises nucleic acid encoding a plurality of reprogramming factors and a nucleic acid sequence targeted by a microRNA (miRNA) expressed in iPSCs, and (b) culturing the somatic cell under conditions to produce the induced pluripotent stem cell. The vector can be a measles virus vector. The OCT4, SOX2, KLF4, and cMYC can comprise human OCT4, human SOX2, human KLF4, and human cMYC. The somatic cell can be a human somatic cell. The miRNA can be miR375. The miR375 can be a human miR375. The nucleic acid sequence targeted by miR375 can comprise SEQ ID NO:1. The nucleic acid sequence targeted by miR375 can comprise three to five repeats of SEQ ID NO:1. The culturing the somatic cell under conditions to produce an iPSC can comprise culturing the somatic cell for about 12 to about 15 days.
In another aspect, this document features a Paramyxoviridae viral vector for reprogramming somatic cells into iPSCs. The vector comprises, or consists essentially of, nucleic acid encoding a Paramyxoviridae leader sequence, nucleic acid encoding a Paramyxoviridae virus N polypeptide, nucleic acid encoding a Paramyxoviridae virus P polypeptide, nucleic acid encoding a Paramyxoviridae virus M polypeptide, nucleic acid encoding a Paramyxoviridae virus F polypeptide, nucleic acid encoding a Paramyxoviridae virus L polypeptide, nucleic acid encoding a Paramyxoviridae trailer sequence, and nucleic acid encoding a plurality of reprogramming factors located between the nucleic acid encoding the Paramyxoviridae virus F polypeptide and the nucleic acid encoding the Paramyxoviridae virus L polypeptide. The Paramyxoviridae viral vector can be a measles virus vector, and the Paramyxoviridae virus N polypeptide, the Paramyxoviridae virus P polypeptide, the Paramyxoviridae virus M polypeptide, the Paramyxoviridae virus F polypeptide, and the Paramyxoviridae virus L polypeptides can be measles virus polypeptides, wherein the Paramyxoviridae leader sequence can be a measles virus leader sequence, and wherein the Paramyxoviridae trailer sequence can be a measles virus trailer sequence. The plurality of reprogramming factors can comprise OCT4, SOX2, and KLF4. The plurality of reprogramming factors can comprise OCT4, SOX2, KLF4, and cMYC. The OCT4 can be a human OCT4 polypeptide, the SOX2 can be a human SOX2 polypeptide, the KLF4 can be a human KLF4 polypeptide, and the cMYC can be a human cMYC polypeptide. The vector can comprise a nucleic acid sequence targeted by a microRNA (miRNA) associated with pluripotency. The miRNA associated with pluripotency can be a miRNA expressed in the iPSCs. The miRNA can be miR375. The miR375 can be a human miR375. The nucleic acid sequence targeted by miR375 can comprise SEQ ID NO:1. The nucleic acid sequence targeted by miR375 can comprise three to five repeats of SEQ ID NO:1. The nucleic acid sequence targeted by the miRNA can be located between the nucleic acid encoding the Paramyxoviridae virus F polypeptide and the nucleic acid encoding the Paramyxoviridae virus L polypeptide. The nucleic acid sequence targeted by the miRNA can be located between the nucleic acid encoding the Paramyxoviridae virus F polypeptide and the nucleic acid encoding the plurality of reprogramming factors. The vector can lack nucleic acid encoding a measles virus H polypeptide. The vector can comprise nucleic acid encoding a fluorescent polypeptide located between the nucleic acid encoding the Paramyxoviridae virus P polypeptide and the nucleic acid encoding the Paramyxoviridae virus M polypeptide. The vector can comprise a nucleic acid sequence targeted by a microRNA (miRNA) associated with pluripotency, and the nucleic acid sequence targeted by the miRNA can be located between the nucleic acid encoding the Paramyxoviridae virus P polypeptide and the nucleic acid encoding the fluorescent polypeptide. The fluorescent polypeptide can be a GFP polypeptide.
In another aspect, this document features a method for producing an induced pluripotent stem cell in vitro. The method comprises, or consists essentially of, (a) introducing a vector into a somatic cell, and (b) culturing the somatic cell under conditions to produce the induced pluripotent stem cell. The vector is a Paramyxoviridae viral vector for reprogramming somatic cells into iPSCs. The vector comprises, or consists essentially of, nucleic acid encoding a Paramyxoviridae leader sequence, nucleic acid encoding a Paramyxoviridae virus N polypeptide, nucleic acid encoding a Paramyxoviridae virus P polypeptide, nucleic acid encoding a Paramyxoviridae virus M polypeptide, nucleic acid encoding a Paramyxoviridae virus F polypeptide, nucleic acid encoding a Paramyxoviridae virus L polypeptide, nucleic acid encoding a Paramyxoviridae trailer sequence, and nucleic acid encoding a plurality of reprogramming factors located between the nucleic acid encoding the Paramyxoviridae virus F polypeptide and the nucleic acid encoding the Paramyxoviridae virus L polypeptide. The Paramyxoviridae viral vector can be a measles virus vector, and the Paramyxoviridae virus N polypeptide, the Paramyxoviridae virus P polypeptide, the Paramyxoviridae virus M polypeptide, the Paramyxoviridae virus F polypeptide, and the Paramyxoviridae virus L polypeptides can be measles virus polypeptides, wherein the Paramyxoviridae leader sequence can be a measles virus leader sequence, and wherein the Paramyxoviridae trailer sequence can be a measles virus trailer sequence. The plurality of reprogramming factors can comprise OCT4, SOX2, and KLF4. The plurality of reprogramming factors can comprise OCT4, SOX2, KLF4, and cMYC. The OCT4 can be a human OCT4 polypeptide, the SOX2 can be a human SOX2 polypeptide, the KLF4 can be a human KLF4 polypeptide, and the cMYC can be a human cMYC polypeptide. The vector can comprise a nucleic acid sequence targeted by a microRNA (miRNA) associated with pluripotency. The miRNA associated with pluripotency can be a miRNA expressed in the iPSCs. The miRNA can be miR375. The miR375 can be a human miR375. The nucleic acid sequence targeted by miR375 can comprise SEQ ID NO:1. The nucleic acid sequence targeted by miR375 can comprise three to five repeats of SEQ ID NO:1. The nucleic acid sequence targeted by the miRNA can be located between the nucleic acid encoding the Paramyxoviridae virus F polypeptide and the nucleic acid encoding the Paramyxoviridae virus L polypeptide. The nucleic acid sequence targeted by the miRNA can be located between the nucleic acid encoding the Paramyxoviridae virus F polypeptide and the nucleic acid encoding the plurality of reprogramming factors. The vector can lack nucleic acid encoding a measles virus H polypeptide. The vector can comprise nucleic acid encoding a fluorescent polypeptide located between the nucleic acid encoding the Paramyxoviridae virus P polypeptide and the nucleic acid encoding the Paramyxoviridae virus M polypeptide. The vector can comprise a nucleic acid sequence targeted by a microRNA (miRNA) associated with pluripotency, and the nucleic acid sequence targeted by the miRNA can be located between the nucleic acid encoding the Paramyxoviridae virus P polypeptide and the nucleic acid encoding the fluorescent polypeptide. The fluorescent polypeptide can be a GFP polypeptide.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Methods and materials are described herein for use in the present disclosure; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
This document provides materials and methods for making and using iPSCs. For example, this document provides reprogramming vectors (e.g., MV based vectors) for reprogramming somatic cells into iPSCs, methods for producing iPSCs, and methods for using iPSCs. In some cases, a reprogramming vector provided herein can be introduced (e.g., transduced, microinjected, infected, transfected, or electroporated) into a somatic cell to produce iPSCs (e.g., in vitro). For example, a reprogramming vector can be introduced into a somatic cell, and the somatic cell can be cultured under conditions that reprogram the somatic cell and produce an iPSC. In some cases, an iPSC can be administered to a patient (e.g., human, non-human primate, dog, cat, or pig) in need of an autologous stem cell therapy. Examples of diseases and disorders that can be treated using autologous stem cell therapy include, for example, diabetes (e.g., type I and type II), cardiac disease, respiratory disease, and degenerative diseases (e.g., Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), macular degeneration (AMD), multiple sclerosis (MS), muscular dystrophy (MD), Parkinson's disease, hematological disorders, autoimmune disease, infectious disease (e.g. HIV, HCV infected patient) and neurological diseases).
Any appropriate somatic cell can be reprogrammed into an iPSC using the materials and methods provided herein. A somatic cell can be a prenatal cell, juvenile cell, or an adult cell. In some cases, a somatic cell that can be reprogrammed into an iPSC using the materials and methods provided herein is an adult cell. Examples of somatic cells include, without limitation, fibroblasts, white blood cells, primary tumor cells, and any primary cell. In some cases, a somatic cell that can be reprogrammed into iPSCs using the materials and methods provided herein is a fibroblast. A somatic cell can be obtained from any appropriate species. Examples of species from which somatic cells can be obtained include, without limitation, humans, non-human primates (such as monkeys), dogs, cats, horses, cows, pigs, sheep, rabbits, mice, rats, ferrets, and bats. In some cases, a somatic cell that can be reprogrammed into an iPSC using the materials and methods provided herein can be obtained from a human. A somatic cell can be obtained from a patient in need of an autologous stem cell therapy.
A reprogramming vector provided herein (e.g., a vector for reprogramming somatic cells into iPSCs) can be any appropriate vector. A reprogramming vector can be a viral vector. A viral vector can be an integrating viral vector or a non-integrating viral vector. In some cases, a viral vector is a non-integrating viral vector. A viral vector can have a DNA genome or an RNA genome. In some cases, a viral vector has an RNA genome. A viral vector can have a segmented genome or a non-segmented genome. In some cases, a viral vector has a non-segmented genome. A viral vector can have a single-stranded genome or a double stranded genome. In some cases, a viral vector has a single stranded genome. A single stranded viral vector can have a negative-, positive-, or ambi-strand genome. In some cases, a single stranded viral vector has a negative-strand genome. For example, a reprogramming vector provided herein can be a non-integrating viral vector having a non-segmented, negative-strand RNA genome (e.g., viruses in the family Paramyxoviridae). Genera within the family Paramyxoviridae include Aquaparamyxovirus, Avulavirus, Ferlavirus, Henipavirus, Morbillivirus, Respirovirus, and Rubulavirus. A reprogramming vector can be based on a species in genus Morbillivirus (e.g., canine distemper virus (CDV), cetacean morbillivirus (CeMV), feline morbillivirus (FeMV), measles virus (MV), peste-des-petits-ruminants virus (PPRV), phocine distemper virus (PDV), rinderpest virus (RPV)). In some cases, a reprogramming vector can be a MV vector. A MV vector can be based on a wildtype MV or based on an attenuated MV (e.g., a MV vaccine). Other examples of viral vectors include, without limitation, retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, Sendai viruses, and Baculoviral vectors.
A reprogramming vector provided herein (e.g., a vector for reprogramming somatic cells into iPSCs) can include one or more modifications. A reprogramming vector (e.g., a MV vector) can be deficient for the hemagglutinin (H) protein. For example, a MV vector can have the H gene inactivated or removed. A reprogramming vector (e.g., a MV vector) can be deficient for the fusion protein (F) protein. For example, a MV vector can have the F gene inactivated or removed. A reprogramming vector (e.g., a MV vector) can be deficient for the matrix (M) protein. For example, a MV vector can have the M gene inactivated or removed. A reprogramming vector (e.g., a MV vector) can include one or more mutations in the C protein. In some cases, a MV C protein can have a mutation at one or more of residues 25, 39, 44, 78, and/or 104 with respect to the sequence set forth in GenBank accession number: EU332921.1. For example, a MV C protein can have a P25L, T39S, R44G K78R, and/or M104T mutation. In some cases, a MV vector provided herein can include a R44G mutation. A reprogramming vector (e.g., a MV vector) can include one or more mutations in the V protein. In some cases, a MV V protein can have a mutation at one or more of residues 29, 46, 51, 54, 83, 97, 111, 146, 195, 219, 225, and/or 237 with respect to the sequence set forth in Genbank accession number: EU332921.1. For example, a MV V protein can have a V29I, D46E, K51R, E54K, S83P, S97P, H111Y, D146N, K195R, S219G E225G and/or G237S mutation.
A reprogramming vector provided herein (e.g., a vector for reprogramming somatic cells into iPSCs) can include one or more nucleic acid sequences encoding one or more (e.g., two, three, four, or more) RFs. ARF can be from the same species from which the somatic cell was obtained or from a different species. For example, human RFs can be used to reprogram somatic cells obtained from a human. A nucleic acid sequence encoding an RF can be present as an independent coding sequences or present in a polycistronic coding sequence. A polycistronic coding sequence is a nucleic acid sequence encoding a plurality (e.g., more than one, such as two, three, four, or more) proteins (e.g., RFs). When the RFs are present in independent coding sequences, they can be located together in the reprogramming vector or in different positions in the reprogramming vector. When RFs are present in a polycistronic coding sequence, a self-cleaving site (e.g., T2A, E2A, and F2A) can be included between the nucleic acid sequences encoding each RF. Examples of RFs that can be used to reprogramming a somatic cell into an iPSC include, without limitation, OCT3/4, KFL4, SOX2, MYC (e.g., cMYC, N-MYC or L-MYC), NANOG, GDF3, hTERT, PIN′, and LIN28. Additional examples of RFs can include those described elsewhere (see, e.g., WO 2010/017652). Examples of RF polypeptide sequences (and the nucleic acids encoding such polypeptides) can be found in the National Center for Biotechnology Information (NCBI) GenBank and in other in public databases. In some cases, a reprogramming vector provided herein can include a nucleic acid sequence encoding three RFs (e.g., OCT4, SOX2, and KLF4). In some cases, a reprogramming vector provided herein can include a nucleic acid sequence encoding four RFs (e.g., OCT4, SOX2, KLF4, and cMYC). For example, a MV vector can include a polycistronic coding sequence including human OCT4, human SOX2, human KLF4, and human cMYC, and having a 2A self-cleaving peptide between each RF.
In cases where the RFs are present in multiple cassettes, the RFs can be present on the same reprogramming vector or on separate reprogramming vectors. In some cases, additional RFs can be provided on one or more additional vectors. For example, OCT4 can be present on a MV vector and SOX2, KLF4 and cMYC can be present on one or more lentivirus vectors.
One or more RFs can be present at any appropriate location in the reprogramming vector provided herein (e.g., a vector for reprogramming somatic cells into iPSCs). In some cases, an RF can be located in an additional transcription unit (ATU). For example, an RF (e.g., OCT4) can be located in the ATU between P and M in a MV vector. Examples of ATUs where RFs can be localized are shown in
Expression of one or more RFs can be examined (e.g., to confirm expression and/or to evaluate expression levels) using any appropriate method. Expression of one or more RFs can be done at an mRNA level or at a protein level. Examples of methods that can be used to examine the expression of one or more RFs include, without limitation, RT-PCR (e.g., standard RT-PCR, real time RT-PCR, and/or RT-qPCR), immunostaining, flow cytometry, northern blotting, and western blotting.
The presence of one or more RFs in a reprogramming vector provided herein can be effective to reprogram a somatic cell (e.g., a human, adult somatic cell) into an iPSC. For example, a somatic cell can be reprogrammed into an iPSC about 15 days to about 25 days (e.g., about 15 days to about 19 days, about 15 days to about 18 days, about 15 days to about 17 days, about 16 days to about 20 days, about 17 days to about 20 days, about 18 days to about 20 days, or about 16 days to about 18 days) following introduction of a reprogramming vector described herein. In some cases, iPSCs can be produced about 15 to about 20 days after introduction of a MV vector having one or more nucleic acid sequence encoding OCT4, SOX2, KLF4, and cMYC.
A reprogramming vector provided herein (e.g., a vector for reprogramming somatic cells into iPSCs) can include one or more (e.g., two, three, four, or more) nucleic acid sequences that are targeted by a miRNA. For example, a nucleic acid sequence that is targeted by a miRNA associated with pluripotency (e.g., a target sequence of a miRNA specifically expressed in the iPSC, and a target sequence of a human embryonic stem cell (ESC)-enriched miRNA) can be included within a reprogramming vector provided herein. In some cases, a miRNA associated with pluripotency is not expressed in a differentiated cell (e.g., a somatic cell). Examples of miRNAs associated with pluripotency, without limitation, miR375, miRNAs from the miR-302 cluster, the miR-17 family, miR-371-373 clusters, and the chromosome 19 miRNA cluster (C19MC). Additional miRNAs associated with pluripotency include those described elsewhere (see, e.g., Razak et al., PLoS ONE. 8:e73532 (2013)); Stadler et al., Stem Cells Dev. 19:935-50 (2010); and Hinton et al., Stem Cells Dev. 19:797-807 (2010)). In some cases, a reprogramming vector provided herein (e.g., a MV vector) includes a target sequence of miR375. A nucleic acid sequence that is targeted by miR375 can include the following nucleotide sequence: aaacaagcaagccgagcgcacu (SEQ ID NO:1). See, also, miRBase database number MIMAT0000728. For example, a nucleic acid sequence that is targeted by a miRNA associated with cellular tropism of the viral vector and/or a miRNA that targets a RF (e.g., an exogenous RF expressed from a reprogramming vector) can be included within a reprogramming vector provided herein. Examples of miRNAs that can reduce or eliminate expression of a RF include, without limitation, miR302a, miR367, miR372, miR517c, miR141, and miR523.
A nucleic acid sequence that is targeted by a miRNA (e.g., a nucleic acid sequence targeted by a miRNA associated with pluripotency) can be present in one or more (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) copies in the reprogramming vector. In cases where multiple copies of a nucleic acid sequence that is targeted by a miRNA are present, the copies can be in tandem. For example, three to five tandem repeats of a target sequence of miR375 can be present in a MV vector. A nucleic acid sequence targeted by a miRNA associated with pluripotency can be present in any appropriate location in the reprogramming vector. Examples of locations in which a nucleic acid sequence that is targeted by a miRNA can be placed include, without limitation, in an untranslated region (e.g., the 3′ UTR) of N, P, or L, and any of the reprogramming factors. For example, a target sequence of miR375 can be in the 3′UTR of P in a MV vector. For example, a target sequence of miR375 can be in the 3′UTR of a RF in a MV vector.
The presence of one or more nucleic acid sequences (N, P, or L) that are targeted by a miRNA in a reprogramming vector provided herein can be effective to reduce or eliminate the reprogramming vector from an iPSC. For example, a reprogramming vector can be eliminated from an iPSC after about 3 passages to about 8 passages (e.g., about 3 passages to about 7 passages, about 3 passages to about 6 passages, about 3 passages to about 5 passages, about 3 passages to about 4 passages, about 4 passages to about 8 passages, about 5 passages to about 8 passages, or about 6 passages to about 8 passages) of the iPSC. In some cases, a reprogramming vector can be eliminated from an iPSC after about 2 to about 3 passages of the iPSC. The reduction or elimination of a reprogramming vector from an iPSC can be evaluated using any appropriate method (e.g., RT-PCR (e.g., standard RT-PCR, real time RT-PCR, and/or RT-qPCR), immunostaining, flow cytometry, northern blotting, and western blotting). For example, the reprogramming vector genome (e.g., RNA or DNA), reprogramming vector transcripts (e.g., from viral genes or from nucleic acid sequences encoding RFs), and/or reprogramming vector proteins (e.g., from viral genes or from nucleic acid sequences encoding RFs) can be detected.
The presence of one or more nucleic acid sequences that are targeted by a miRNA in a reprogramming vector provided herein can be effective to enhance the efficiency of reprogramming a somatic cell (e.g., a human, adult somatic cell) into an iPSC. For example, a somatic cell can be reprogrammed into an iPSC about 8 days to about 20 days (e.g., about 12 days to about 20 days, about 13 days to about 20 days, about 14 days to about 20 days, about 15 days to about 20 days, about 12 days to about 19 days, about 12 days to about 18 days, about 12 days to about 17 days, about 12 days to about 16 days, or about 12 days to about 15 days) following introduction of a reprogramming vector described herein. In some cases, iPSCs can be produced about 12 to about 15 days after introduction of a MV vector having one or more nucleic acid sequence encoding OCT4, SOX2, KLF4, and cMYC and having three to five tandem repeats of a target sequence of miR375.
A reprogramming vector provided herein (e.g., a vector for reprogramming somatic cells into iPSCs) can include one or more detectable labels. Examples of detectable labels include, without limitation, nucleic acids expressing fluorescent proteins (green fluorescent protein gene (GFP), blue fluorescent protein gene (BFP), cyan fluorescent protein gene (CFP), yellow fluorescent protein gene (YFP), orange fluorescent protein gene (OFP), or red fluorescent protein gene (RFP, mCherry)). In some cases, a reprogramming vector provided herein (e.g., a MV vector) can include a nucleic acid expressing GFP.
A reprogramming vector provided herein can be a universal reprogramming vector. As used herein, a “universal” reprogramming vector can be used to transduce a broad range of cell types from many different species. In some cases, a universal reprogramming vector can be a pseudotyped (e.g., vectors with modified envelopes) vector. For example, a universal reprogramming vector can include an envelope glycoprotein from Vesicular stomatitis virus (VSV-G).
A reprogramming vector provided herein can be a targeted reprogramming vector. As used herein, a “targeted” reprogramming vector can be used to transduce a cell from a specific species and/or to transduce a specific cell type. In some cases, a targeted reprogramming vector can target a cell from an animal susceptible to CDV infection (e.g., dogs or ferrets) and can include the H and/or F genes from a canine distemper virus (CDV). For example, to target a canine cell, the H and/or F genes of a MV vector can be replaced with the H and/or F genes from a CDV. In some cases, a targeted reprogramming vector can target a specific cell type (e.g., B cells or tumor cells) and can include a molecule (e.g., a single-chain variable fragment (scFv) polypeptide) that binds to a receptor expressed by the targeted cell type. Examples of receptors expressed by specific cell types include, without limitation, CD20 (expressed by B-cells), EGFR (expressed by cells from glioblastoma multiforme), HER2/neu (expressed by tumor cells), CD38 (expressed by tumor cells), CEA (expressed by carcinoma cells). For example, a reprogramming vector targeting a B-cell can include a CD20 scFv.
The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
Measles vectors encoding two, three and four factors were produced.
A measles virus (MV) vector encoding OCT4 in the ATU between P and M (MV(OCT4)) and another vector that expresses SOX2 in this same position (MV(SOX2)) were produced. Two other factors, KLF4 and cMYC, were cloned in a lower position, either instead of the H gene or in the ATU after H. Eight constructs were produced and vectors (MV(S) to MV(Z)) were rescued using our rescue cell line expressing MV-H (
Using a reprogramming protocol described elsewhere (Driscoll et al., Stem Cell Research and Therapy 6:48 (2015)), an iPSC-like clone with MV(OCT4)P-ΔH(KLF4)(GFP)H or MV(T) vector in combination with 2 LV encoding SOX2 and cMYC was obtained. The clone obtained with vector MV(T) was analyzed for pluripotency markers (
Measles Vectors Expressing Three and Four Reprogramming Factors
Vectors encoding three or four reprogramming factors using a polycistronic gene were developed. Two polycistronic genes (one encoded for OCT4-SOX2-KLF4 and the other encoded for all four factors OCT4-SOX2-KLF4-cMYC) were used. Self-cleaving T2A, E2A and F2A sites were respectively added between the genes. Three (3F) and four factors (4F) vectors were recovered (
To increase transduction efficiency the spinoculation technique was established. To reduce MV-induced toxicity, the MOI of transduction was decreased. BJ cells, a neonatal foreskin human cells were purchased from the American Type Culture Collection (ATCC # CRL 2522), and were transduced with the 4F vector at a MOI of 1 instead of 6 as described elsewhere (Driscoll et al., Stem Cell Research and Therapy 6:48 (2015)). In order to induce TLR3 activation during MV reprogramming, the transduction was performed in presence of polyI:C (a TLR3 activator). Transduced cells were subjected or not to spinoculation at 1000 rpm for 1 hour at room temperature. The culture supernatants were replaced with BJ media every three days. After 9 days, optimized serum-free/feeder-free iPSC medium was added and replaced daily. The cells were monitored until reprogrammed cells form small colonies with iPSC morphology (one to two weeks after vector transduction). Pictures were taken at different stages. iPS-like clones were picked after three to four weeks, and plated at 1 colony/well in Matrigel-coated wells for further expansion.
A safety mechanism in MV that increases reprogramming efficacy was designed. A specific microRNA target sequence was inserted into the MV genome to turn off the expression of the RFs after reprogramming. A miRNA highly expressed in the iPSC cell, miR375 (Razak et al., PLoS ONE. 8:e73532 (2013)) was used.
Three repeats of the target sequence for the miR375 were cloned in the 3′ non-translated region of the P gene in the full length MV(OCT4)P genome (
The MV(OCT4)miR375 vector was recovered (
As the miRNA target sequence added in the 3′UTR of P, the transcriptional activity of P was analyzed by the expression level of the RF and the viral protein N by western blot (
293-H cells expressing MV-H were transfected with either miR375, miR control, or water and followed the propagation of the MV(OCT4)miR375 (
The ability of the MV(OCT4)mir375 to reprogram BJ cells in presence of 3LV expressing SOX2, KLF4, and cMYC was analyzed, and its efficiency was compared to MV(OCT4) and 4LV. MV(OCT4) reprogrammed BJ cells in approximately 20 days (
To verify that the MV(OCT4)mir375-derived iPSC-like clones were indeed iPSC clones, two clones derived from MV(OCT4)mir375 (one clone derived from MV(OCT4) and one clone derived from 4LV reprogramming) were tested for expression of several markers of pluripotency by immunofluorescence. All clones expressed human pluripotency-associated markers TRA-1-60, TRA-1-81, SSEA-4, OCT4, SOX2, and NANOG at passage 3 (
These results demonstrated that the introduction of three repeats of the target sequence of miR375 in the 3′ UTR of the P inhibited replication of MV(OCT4)miR375 in presence of the miR375, increased the efficiency of reprogramming compared to its parental control vector MV(OCT4), and increased the reprogramming rate.
To determine the amounts of genetic material to be inserted in the MV genome without significantly affecting the rescue and propagation of the virus, the following vectors are produced:
Single letters represent the MV genes; (xxx): represent the ATU with the corresponding RF gene clone in the ATU; and [xxx] represent H or F gene substitutions by one RF gene. The cloning strategies take advantage of the unique restriction sites as well as ATUs inserted in the genome (see, e.g.,
To determine the most efficient sequence of expression, the reprogramming gene order is rearranged using the different ATU inserted in the measles genome. Several recombinant viruses with progressively more protein expression are produced:
Uppercase letters (OSKM) represent a high level of the RF and lowercase letters (oskm) a low level of RF.
To produce a MV vector expressing the four reprogramming factor from one single genome, we use a polycistron encoding human OCT4-SOX2-KLF4 and cMYC as individual gene.
The polycitron encoding OCT4-SOX2-KLF4 separated by self cleaving 2A peptide was cloned instead of the H gene in the MV genome, and the cMYC gene in the ATU after H. The GFP was added in an ATU in front of the N gene (
The expression level of the RF and the correct processing of the polyprotein was analyzed by western blot (
Transduction efficacy of the vectors was tested at different MOI using light microscopy and flow cytometry and testing for GFP expression. BJ cells were transduced with 4F* at a MOI of 0.5 or 0.25. Transduced cells were subjected to spinoculation at 1000 rpm for 1 hour at room temperature. Early experiments from
The ability of the 4F* to reprogram BJ cells was analyzed. 4F* reprogrammed BJ cells in approximately 17 to 20 days at a MOI as low as 0.25 (
To verify that the 4F*-derived iPSC-like clones were indeed iPSC clones, two clones derived from 4F* and one clone derived from 4LV reprogramming were tested for expression of several markers of pluripotency by immunofluorescence. All clones expressed human pluripotency-associated markers TRA-1-60, TRA-1-81, SSEA-4, OCT4, SOX2, and NANOG at passage 2 (
These results demonstrated that the expression of the four reprogramming factors OCT4, SOX2, KLF4 and cMYC from one measles genome allowed efficient reprogramming of somatic cells into induce pluripotent stem cell or iPSC.
The P gene encodes for the three proteins involved in the interferon response (IFN) control (Sparrer et al., Journal of Virology 86:796-805 (2012); Takaki et al., Mol Immunol 48:497-504 (2011)). To control IFN production, a P gene from a wild type MV (MV-Pwt) was exchanged for the P gene from a MV vaccine strain in the MV(OCT4) vector.
Vectors expressing the P gene from the WT MV virus (Pwt) instead of the P gene from a MV vaccine (Pvac) were produced. An intermediate vector containing a region of the MVvac genome was modified and the Pwt gene was subcloned using Xbal and Sall restriction sites. In order to clone back the modified genome fragment into the full-length MV vector, a three-way ligation was performed to produce the final vector MV(OCT4)Pwt (
These vectors are tested for IFN induction and reprogramming efficiency. To determine if the substitution has an effect on viral transcription, the expression level of the RF and viral proteins is analyzed by western blot and immunofluorescence. The propagation of the vectors is compared to a standard MV vector on Vero-H2. Viral stability and protein expression is analyzed after 5, 10, and, 20 passages. Transduction efficacy of these vectors is analyzed and compared to the control vector MV(OCT4)Pvac.
Toxicity is assessed after transduction with different MOI using light microscope and fluorescent microscope. To determine if IFN is produced after transduction by the vectors, the activation and nuclear translocation of IRF3 is analyzed. IFN-induced IRF3 nucleus translocation is assessed by immunofluorescence and confocal microscopy after transduction. An analysis of the phosphorylation status of IRF3 is also performed as described elsewhere (McAllister et al., J Virol. 84:380-6 (2010)). Enzyme-linked immunosorbent assay of IFN-β protein and qRT-PCR analysis of IFN-β, and IFN stimulated genes ISG15, OAS and IFIT1 is performed.
The P/V/C encode three proteins; the polymerase cofactor, P; and two proteins involved in the control of the innate immune response, V and C. Another possible target for MV-induced toxicity is the matrix protein (M). To identify the protein responsible for MV-vector induced toxicity, individual Flag-tag versions of the wild type V (FVwt), C (FCwt), and/or M gene are introduced into the MV vector. The FVwt gene is introduced in the ATU after P of MV(ATU)Pvac. The FVwt is silenced for Cwt expression by introduction of stop codons as described elsewhere (Devaux et al., Journal of Virology. 82:5359-67 (2008)). FCwt is introduced at the same position. Vectors are analyzed as described herein.
To identify the residue involved in the control of the IFN production by MV, site-directed mutagenesis is used to mutate residues from the vaccine to the wild type residue. An intermediate vector is used to perform the mutagenesis and sub-clone the mutated to gene into the MV vector (Devaux et al., Virology. 360:72-83 (2007); Devaux et al., J Virol. 85:348-56 (2011)). Integrity of the gene is verified by sequencing. All mutants are produced in the viral vector context to look at an effect on IFN production and toxicity in BJ cells occurring during viral transduction.
Mutation in the C protein: A sequence alignment of Cwt and Cvac was performed. There are only five residues differences: P25L, T39S, R44G, K78R and M104T, with the first residue corresponding to the wild type and the second to the vaccine. Mutation R44G is in the nuclear localization signal. G44 in the Pvac gene is mutated to R to test its effect in the control of IFN production and toxicity in MV(OCT4). Mutagenesis is performed in a way that the open reading frame of P and V remains unchanged. The vector is tested for its efficiency to block IFN production as described herein. The other four residues are also mutated and tested.
Mutation in the V protein: A sequence alignment of Vwt and Vvac was performed and 13 residues were noted having differences between both proteins: V291, D46E, K51R, E54K, S83P, S97P, H111Y, D146N, K195R, S219G, E225G and G237S, with the first residue corresponding to the Vwt and the second to the Vvac. Each residue of the Vvac is mutated to the wt residue and tested for ability to control IFN production and toxicity. Mutagenesis is performed in a way than the open reading frame of C and if possible P remains unchanged. Double and triple mutants will be produced with the most efficient mutant to increase efficacy. The vectors are rescued and assessed as described herein. The effects of the mutation on toxicity are analyzed as described herein.
The M protein in the MV vaccine has two point mutations, P64S and E89K. The Mvac is exchanged with the MwtP64S/E89K to determine if the wt version of M reduces toxicity of the MV vector.
The MV vector expressing a Pvac with one or more of the identified mutations are used for reprogramming purposes as described herein. The protocols for the characterization for the iPSC is described herein.
The functionality of iPSC-differentiation is tested in vivo. In order to test if the reprogrammed cells are functional in vivo, and can spontaneously re-differentiate into the three germ lineages in vivo, the reprogrammed cells are transplant into the kidney sub-capsule of SCID/Beige mice. All experimental mice, 6 to 8 week-old SCID/Beige mice, are purchased (e.g., from Charles Rivers). These immune-compromised mice will avoid the rejection of the engrafted cells. Mice are maintained under a 12-hour light-dark cycle. The two best MV vectors per group described herein are tested. iPSCs derived from 4LV and control MV are used as positive controls. Twenty μL of iPSC cells (2×106) are loaded into a delivery apparatus consisting of a syringe attached to a 200 uL pipette tip. Injection into a pocket under the left kidney capsule is performed. Four and eight weeks' post-injection, mice are euthanized via CO2 inhalation, and the iPSC-injected kidney is removed and tissues are embedded and flash frozen in O.C.T. compound. Seven μm cryosections are obtained and immediately fixed in 4% PFA for 30 minutes at room temperature before proceeding to hematoxylin and eosin staining. Presence of structures from the endoderm, mesoderm, and ectoderm lineages are sought using light microscope and the section are sent to the Mayo Pathology Research Core facility for high quality imaging. Three animals are used for each vector at each time point.
The proficiency of MV-derived iPSC to generate cardiac progenitor cells is analyzed in vitro using a step-wise differentiation protocol to test differentiation of iPSCs into cardiomyocyte-like cells as described elsewhere (Driscoll et al., Stem Cell Res Ther. 6:48 (2015)). After 10 days of culture, the first contracting cells are observed under light microscopy. Video of the contracting cardiomyocyte-like cell will be recorded using a Zeiss Axiovert 200M microscope and the ApoTome imaging system. Immunostaining for α-actinin, a marker of microfilaments, and troponin, a marker specific for the cardiac muscle, is use to analyzed the cells to confirm their differentiation into cardiomyocyte-like cells.
miRNA target sequences are cloned into the 3′UTR of the each RF in order to control specific expression of each RF. Each RF is silenced in a specific way and sequentially, if necessary, as the control can be independent of the viral control.
Identification of Additional miRNA to Use in the MV Vector
Level of miRNA is determined in BJ cell and MV-derived iPSC clones using HTG EdgeSeq miRNA Whole Transcriptome Assay (HTG Molecular Diagnostics, Tucson Ariz., https://www.htgmolecular.com/products/mirna-wta). miRNA highly upregulated in MV-derived iPSC clones and not expressed in BJ cells are identified and are selected to be cloned in MV vectors.
Production of MV Vectors Expressing miRNA Target Sequences
Three repeats of the target sequence in tandem for each of the selected miRNA in the 3′UTR region of OCT4 or SOX2 genes, which are expressed from an ATU are introduced in the MV vector genome. Overlapping PCR extension, restriction digestion enzymes and multiple cloning strategies are used. Two intermediate plasmids, pRS313-OCT4miRNA and pRS313-SOX2miRNA, are produced. These plasmids are used to substitute the 3′UTR region with the new modified 3′UTR-miRNA region. The final OCT4 and SOX2 genes encoding the modified 3′UTR-miRNA are reinserted in the full-length MV genome vectors (e.g., the MV(OCT4) vector). The integrity of all the constructs is verified by sequencing. OCT4 and SOX 2 genes containing a few codon differences compared to the wild type OCT4 and SOX2 gene, can be used to differentiate them from the endogenous mRNA by qRT-PCR. In order to make sure that these differences do not affect the reprogramming efficiency, these genes are cloned into the LV system, and tested for reprogramming.
The MV(OCT4)miRNA and MV(SOX2)miRNA vectors are rescued as described herein. To test the efficiency of the miRNA system, the expression level of the RF (OCT4 and SOX2) and viral protein N (control) are examined by western blot and immunofluorescence, in presence and absence of the specific or irrelevant miRNA. The propagation of the vectors is compared to a standard MV vector on Vero-H2. Viral stability and protein expression is analyzed after 5, 10, and 20 passages. Transduction efficacy of these vectors is analyzed and compared to the control vector.
Efficiency of Reprogramming of Somatic Cells, and Characterization and Functionality of the iPSC Clones
BJ cells are reprogrammed as described herein. The iPSC clones are characterized as described herein. To determine how soon the exogenous RF are cleared from the iPSC after reprogramming, qRT-PCR is performed on the OCT4 or SOX2 mRNA produced in the iPSCs. The MV-iPSC colonies are analyzed by RT-PCR for exogenous OCT4 and SOX2 mRNA, as soon as passage 2, and then passages 3, 6, and 8.
The envelope of MV vectors is modified to retarget the vector and produce a more polyvalent vector.
Vectors are produced expressing the envelope glycoprotein from Vesicular stomatitis virus (VSV-G). This protein is extensively used in other viral vector system, such as lentiviral system, to allow transduction of large varieties of cells from different species (human, mouse or other). VSV-G-pseudotyped MV is produced by introduction of a VSV-G fusion protein where the VSV-G was link to the cytoplasmic tail of the MV fusion protein (see, e.g., Spielhofer et al., Journal of Virology 72:2150-9 (1998)).
Rescue and propagation cell lines expressing a VSV-GFtail fusion protein were produced as described elsewhere (Spielhofer et al., Journal of Virology 72:2150-9 (1998)). A VSV-GFtail fusion gene was cloned into a LV vector. The integrity of the construct was verified. Rescue (293-3-46 cell from (39)) and African green Monkey Vero ((ATCC) for viral propagation) cell lines were transduced with the LV particle expressing VSV-GFtail, and new cell lines are tested for expression of VSV-GFtail at the cell surface by immuostainning and flow cytometry.
Two new MV vectors are produced, first using the MV(OCT4) vector, and then using other MV vectors described herein, one deficient in H and F, the other deficient in H, F and M. To test these vectors in other species (e.g., mouse), the mouse OCT4 (mOCT4) gene is cloned instead of the human (hOCT4) gene in the ATU in P position. The vector is rescued and propagation cell lines are produced.
The “universal” MV vectors are assessed. To determine the level of viral production for the new MV vectors, the propagation of the virus on Vero-VSVGFtail is analyzed. Expression of the RF and viral proteins in the MV vector-infected cells is analyzed by western blot and immunofluorescence. The transduction efficiency in human or hamster (CHO-CD46) cells is analyzed and compared with control MV system. Viral stability and protein expression are analyzed after 5, 10, and 20 passages.
Vectors are used for reprogramming experiments in human (BJ cells, ATCC) and mouse primary embryonic fibroblasts (Cell Biologics, Chicago, Ill.). Methods of reprogramming and characterizing can be adjusted as needed. All the reagents to characterize the iPSC derived from mouse fibroblasts are commercially available.
Vectors are produced expressing a chimeric envelope to specifically transduce cells from different species.
The H and F of MV and canine distemper virus (CDV) are exchanged (von Messling et al., J Virol. 75:6418-27 (2001)). MV-H is exchanged with CDV-H. This virus specifically targets cells from dogs, ferrets, and other animal susceptible to CDV infection.
Rescue and propagation cell lines expressing the H of CDV (HCDV) are produced. A LV vector expressing HCDV (LV-HCDV) similar to the LV vector expressing MV-H is produced. The rescue (293-3-46) and a modified Vero cell expressing dogSLAM receptor (Vero.dSLAM) are transduced with the LV-HCDV particle. The new cell lines are tested for expression of HCDV at the cell surface by immuostairming and flow cytometry.
Dog-targeted-MV vectors are produced, first using the MV(OCT4) vector, and then using other MV vectors described herein, using reprogramming vectors described herein. Canine fibroblasts were successfully reprogrammed either using predicted canine RF gene (Shimada et al., Mol Reprod Dev. 77:2 (2010)) or human RF (Lee et al., J Biol Chem. 286:32697-704 (2011)).
The level of viral production is analyzed in Vero-dSLAM. Expression of the RF and viral proteins in MV-CDV-pseudotyped vector transduced cells is analyzed by western blot and immunofluorescence. Transduction efficiency with the MV-HCDV-pseudotyped vector is analyzed and compared to control MV(OCT4) vector in Vero.dSLAM at 24 and 48 hours. Viral stability and protein expression are analyzed after 5, 10, and 20 passages.
Vectors are used for reprogramming experiments in canine embryonic fibroblasts (Cell Biologics, Chicago, Ill.). Reagents needed for the characterization of the canine iPSC are described elsewhere (Lee et al., J Biol Chem. 286:32697-704 (2011)).
Vectors expressing a retargeted envelope glycoprotein to a specific human receptor using a single chain antibody (scFv) are produced. MV entry is retargeted to a cell through a different receptor than it natural receptor. This is achieved by introducing CD46- and SLAM-ablating mutations at residue positions 481, 533, 548, and 549 in the H gene and the addition of an scFv in the extracellular domain of H allowing the interaction with a specific new protein (Ungerechts et al., Cancer Res. 67:10939-47 (2007)). Target receptors include (EGFR), HER2/neu, CD20, CD38, CEA as few examples (Hasegawa et al., J Virol. 81:13149-57 (2007); Nakamura et al., Nat Biotechnol. 23:209-14 (2005); Paraskevakou et al. Mol Ther. 15:677-86 (2007); Ungerechts et al., Cancer Res. 67:10939-47 (2007); Yaiw et al., Gene Ther. 18:313-7 (2011)).
Rescue and propagation cell lines are produced. For example, rescue and propagation cell lines expressing the CD20 retargeted MV-H are produced. A vector encoding for the chimeric MV-HCD46/SLAMblind(mutl) linked to CD20 scFv's (CD20HmutI) is used. The chimeric CD20HmutI is cloned into the LV vector. Rescue (293-3-46) and propagation cell lines are transduced with the LV-CD20HmutI as previously described. Both new cell lines are tested for cell surface expression of CD20HmutI.
CD20-retargeted-MV vectors are produced first using the MV(OCT4) vector, and then using other MV vectors described herein.
The CD20-retargeted-MV vectors are assessed. To determine the level of viral production for the new MV vector, the propagation of the vector in HT1080 (ATCC) and HT1080-CD20 cells is analyzed. Expression of the RF and viral proteins by the MV-CD20HmutI vector transduced cells is analyzed by western blot and immunofluorescence in HT1080-CD20 cells. Transduction efficiency with MV-CD20HmutI-pseudotyped vectors is analyzed and compared to control MV(OCT4) in HT1080-CD20 at 24 and 48 hours. Viral stability and protein expression are analyzed after 5, 10, and 20 passages.
CD20-retargeted-MV vectors are used for reprogramming experiments of human B lymphocytes. Reprogramming protocols can be adapted as needed to reprogram suspension cells. B-lymphocytes are isolated from peripheral blood samples by negative selection using non-B cell depletion beads (B cell isolation kit II from MACS, Miltenyi Biotec). The clones are characterized as described herein.
4F*, 4F(GFP)P were described in
To produce the p(+) MVvac2(GFP)PΔH(OSK)(cMYC)H (4F(GFP)P), the MV(OCT4) was used (
Measles Vectors Expressing Four Reprogramming Factors with Viral Replication Controlled by miRNA (e.g., MV(OCT4)miR375 and 4FGFP)PmiR)
MV(OCT4)miR375 from Example 2 (
To produce the p(+) MVvac2(GFP)miR375ΔH(OSK)(cMYC)H (4F(GFP)PmiR), the MV(OCT4)miR375 was used. First, OCT4 gene was replaced by GFP using AatII and MluI to get the MV(GFP)miR375. Then, the PacI-SpeI fragment from the MV(GFP)miR375 was exchanged with the PacI-SpeI fragment of the p(+)MVvac2(GFP)uNΔH(OSK)(cMYC)H to obtain p(+)MVvac2(GFP)miR375ΔH(OSK)(cMYC)H (4F(GFP)PmiR,
All Vero (ATCC #CCL81), helper 293-3-46-H2 cells, and Vero cells were maintained in DMEM medium (GE Lifesciences HyClone, SH30022.01) containing 10% fetal calf serum (FCS, Life Technologies, #10437-028) and 1% penicillin/streptomycin (P/S, Corning Mediatech, 30-002-C1) (DMEM-10). Helper 293-3-46-H2 cells were cultured with 1.2 mg/mL G418 (Cardinal Healthcare, MT61234RG) in addition to DMEM-10. Human BJ cells (neonatal foreskin human cells, ATCC #CRL 2522) were maintained in DMEM containing 10% embryonic stem cell qualified fetal calf serum (ES-FCS, Life Technologies, #16141-079) containing 0.1 mM non-essential amino acids (Corning Mediatech, 25-025-C1) and 1% P/S (media 1). iPSCs were maintained in 80% Pluriton (Stemgent, #00-0070), 20% mTeSR1 (STEMCELL Technologies, #05851), and 1% P/S (media 2). All cell lines stated above were cultured in humidified atmosphere with 5% CO2 at 37° C. under atmospheric oxygen conditions.
Recombinant MVs 3F, 4F*, F4(GFP)P, and 4F(GFP)PmiR were produced using procedures similar to those described elsewhere (Radecke et al., EMBO J. 14:5773-84 (1995)). Rescue cells (293-3-46) and propagation cells (African green monkey, Vero) were modified to express the MV-H glycoprotein (293-3-46-H2 and Vero-H2) (Driscoll et al., Stem Cell Res Ther. 6:48 (2015)). In brief, helper 293-3-46 H2 cells were transfected using calcium phosphate precipitation with two plasmids encoding for the MV genome and MV polymerase (pEMCLa). Three days after transfection, the helper cells were overlaid on Vero-H2 cells. The appearance of infectious centers was monitored by observing green fluorescent protein (GFP) expression under fluorescence microscope. Single viruses were then picked and propagated on Vero-H2 cells.
For virus stock preparation, Vero-H2 cells were infected at a multiplicity of infection (MOI) of 0.05 in OptiMEM for 2 hours at 37° C. Then, DMEM-10 medium was added on top and transferred to 32° C. until 95% of the cells expressed GFP. Cell culture media was removed, and cells were scraped in OptiMEM. Viral particles were released by two freeze-thaw cycles. Titers of virus stocks were determined by 50% end-point dilution (tissue culture infectious dose 50, or TCID50) on Vero-H2 cells using the Spearman-Karber method.
Expression and correct processing of OSK in 293T and human fibroblast was verified. All three proteins OCT4, SOX2 and KLF4 were expressed and had the same apparent molecular weight as the proteins expressed in cells infected by corresponding lentiviral vectors (LVs) (
High viral titers were documented in Vero cells infected with the replication-competent virus MV(GFP)H, but no virus was produced in cells infected with the single-cycle vectors 3F, 4F*, 4F(GFP)P, and 4F(GFP)PmiR (
Reprogramming with MV Vectors: Detailed Protocols
The ability of four factor vectors 4F(GFP)P and 4F(GFP)PmiR to reprogram human fibroblast was tested and compared to 4F* described in Example 4.
Human PBMC, T Cells.
PBMC are isolated from an Apheresis cone. The PBMC are isolated on a Ficoll gradient, and T, B, NK, CD34+ progenitor cells are isolated using magnetic beads. Cells (PBMC or isolated cells) are then activated for 1-3 days, then transduced with various MOI of the 4F vectors using a modified protocol described above. The protocol is adapted to the reprogramming of PBMC and isolated T, B, NK, CD34+ cells as described elsewhere (Ohmine et al., Stem Cell Res. Ther., 2:46 (2011)). The cells are transferred on a matrigel-coated plate to allow iPSC to adhere. At day 3 and 5 days post-transduction, 1 mL of optimized serum-free/feeder-free iPSC medium is added, and then starting at day 7, it is replaced daily. The REproTeSR™ reprogramming media (STEMCELL Technologies), which is specifically design for the reprogramming of blood-derived cells under feeder free conditions, is used. The cells are monitored until cells form small colonies with iPSC morphology (1 to 2 weeks after transduction). Pictures are taken at different stages. iPS-like clones should appear around days 15 to 25, and are picked after 3 to 4 weeks, and are plated at 1 clone/well in Matrigel-coated wells for further expansion.
Human fibroblast cells.
Human fibroblast cells (7×104/well) were seeded on matrigel-coated plates in fibroblast media. Cells were transduced with at different multiplicity of infection (MOI, 0.5 or 0.25) in 500 μL OptiMEM. Cells transduced with 3F were co-transduced with LV-cMYC (50 μL). Cell transduced with 4F*, 4F(GFP)P, or 4F(GFP)PmiR were transduced with the Measles vector alone. Cell and virus were subjected to spinoculation at 1100 rpm for one hour at room temperature (25° C.), then the inoculum was left overnight on top of the cells. The day after, cells were washed once with PBS, and fibroblast media added and was changed every two days until day 7 for 3F and day 8 for 4F*, 4F(GFP)P or 4F(GFP)PmiR. Feeding fibroblast media was changed to iPSC media and was changed every day thereafter. iPSCs clones were picked on the basis of size and morphology, around day 25-30 and transferred individually on a matrigel-coated 12 well and cultured in iPSC media for further expansion and study (
For reprogramming with small molecules, SB431542 (Stemgent, 04-0010-05, 5 μM), PD0325901 (Stemgent, 04-0006-02, 0.2 μM), and Thiazovivin (Stemgent, 04-0017, 0.5 μM) small molecules were added to iPSC media from day 7 to 14.
Reprogramming with the 3F, 4F*, 4(GFP)P or 4F(GFP)PmiR Leads to iPSC-Like Clones
The ability of 3F, 4F*, 4F(GFP)P or 4F(GFP)PmiR to reprogram human fibroblasts was documented, and the effect of supplementation of small molecules (sm, 2sm), SB431542 (5 μM), PD0325901 (0.2 μM) and Thiazovivin (0.5 μM) was tested to determine their impact reprogramming efficiency. Although reprogramming at MOI 0.5 with 4F*, 4F(GFP)P or 4F(GFP)P miR, with or without small molecules, led to noticeable toxicity over the first week, GFP expressing cells remained visible over 30 days. Around 2 weeks (12 to 17 days) post transduction, GFP positive, sharp-edge, flat, and tightly packed iPSC-like clones emerged in the fibroblast monolayer (
Elimination of the Vector from the iPSC-Like Clones
The elimination of the 4F* vector from the established iPSC was tested. The two most abundant MV mRNAs, nucleoprotein (N) and phosphoprotein (P), were detected in iPSC clones produced without small molecules up to passages P3 to P5 (
Verification that the iPSC-Like Clones are Indeed iPSCs
To verify that the 4F-derived iPSC-like clones were indeed iPSC clones, the 3F, 4F*, 4F(GFP)P-derived clones were verified to express human pluripotency-associated markers, SSEA-4, TRA-1-60, TRA-1-81, OCT4, SOX2 and NANOG, at passage 3-5 and after prolonged culture (at passage 20-25), indicating stability of the derived clones (
To confirm that the 3F, 4F*, 4F(GFP)P-derived iPSC clones were pluripotent, their differentiation propensity into the three germ layers; endoderm, ectoderm and mesoderm was assessed. First, the multi-lineages propensity of 3F, 4F*, 4F(GFP)P-derived clones was confirmed by formation of embryoid bodies (EBs) and spontaneous differentiation into mesoderm (CD31) endoderm (FOXA2), or ectoderm (β-III tubulin). All 3F, 4F*, 4F(GFP)P-derived iPSCs could form EBs and differentiate spontaneously into ectoderm, endoderm or mesoderm and maintained their differentiation propensity over 20 passages, indicating stability of the pluripotency state (
In addition, the global gene expression profiles of MV four factor vector-derived iPSC clones were determined and compared against global gene expression profiles of parental fibroblast cells and H9 human ES cells (GSM551202; GEO DataSets, Boston, Mass.). Microarray analysis was conducted according to manufacturer's instructions for the Affymetrix 3′ IVT Plus kit (Thermofisher Scientific, 902416). Briefly, RNA quality was assessed by Agilent Bioanalyzer (Agilent Technologies). Reverse transcription to second strand cDNA was generated from 100 ng of high quality total RNA. Subsequently, the products were in vitro transcribed to generate biotin-labeled cRNA. The IVT products were then bead-purified (Affymetrix), fragmented, and hybridized onto Affymetrix U133Plus 2 GeneChips® at 45° C. for 16 hours. Subsequent to hybridization, the arrays were washed and stained with streptavidinphycoerythrin and scanned in an Affymetrix GeneChip® Scanner 3000 (Santa Clara, Calif.). Control parameters were confirmed to be within normal ranges before normalization, and data reduction was initiated using the GeneChip™ Command Console™ Software. The .cel files were processed using Partek Genomics Suite software, version 6.6 (Partek Inc., St. Louis, Mo., USA). The files were normalized using quantile normalization with a log probes using base 2. The differential expression was performed using the ANOVA method in Partek. The scatter plots were generate using R script version 3.1.1. The heat map was generated using Excel software.
Scatter plot analysis demonstrated that the transcriptome of MV four factor vector-derived iPSC clones showed higher similarity to those of ES than parental fibroblast cells. Heat map analysis of differentially expressed genes further confirmed that gene expression patterns of derived iPSC clones were similar to those of human ESCs, but highly divergent from control human fibroblast cells. The transcriptome of three independent MV four factor vector-derived iPSC clones exhibited striking similarity to each other. Thus, global gene expression profiles support a high degree of similarity in transcriptome between MV four factor vector-derived iPSCs and human ES cells.
Construct having the structure shown in
Control of OCT4, SOX2 and KLF4: Single, Double, Triple Combinations Vector without miRNA Control
Two PCR fragments encoding RF1 or RF2-RF3 are cloned instead of the H gene using the EcoRV and SmaI restriction sites. A new measles virus intergenic region containing a stop and start of transcription is inserted between RF1 and RF2-RF3. Cloning is performed in accord with the “rule of six” using an intermediate vector pCGΔH(GFP)
(GFP)H. Unique restriction site, such as Swal and Fsel, are introduced upstream and downstream RF2-RF3 to facilitate interchanging the factors for the following constructs and the introduction of the miRNA. The resulting plasmid pCGΔH(RF1)(RF2-RF3)(GFP)H is then modified by the introduction of cMyc (M) in place of the GFP, using restriction digestion, leading to the pCGΔH(RF1)(RF2-RF3)(M)H. Then, the PacI-SpeI fragment from the MV(GFP)P is exchanged with the PacI-SpeI fragment of the pCGΔH(RF1)(RF2-RF3)(M)H to obtain p(+)MVvac2(GFP)PΔH(RF1)(RF2-RF3)(M)H (MV4FP(RF1)(RF2-RF3)(M)).
Vector with miRNA Control of the Reprogramming Factors
The pCGΔH(RF1)(RF2-RF3)(M)H is used to introduce the miRNA target sequence in the 3′UTR of RF1. Three repeats of the miRNA target sequence is inserted in the 3′ UTR of the RF1 gene, and one NcoI restriction site (or any other appropriate restriction site) is introduced between the first and second repeat to facilitate cloning. Two PCR fragments are generated; one covering the RF1 gene, the other covering the RF2-RF3 gene. Both fragments are linked using the NcoI site, and the resulting fragment is cloned back in the intermediate vector and then in the full-length MV vector using unique restriction sites to produce MV4FP(RF1)miR(RF2-RF3)(M).
The pCGΔH(RF1)(RF2-RF3)(M)H is used to introduce the miRNA target sequence in the 3′UTR of RF2-RF3. Three repeats of the miRNA target sequence are inserted in the 3′ UTR of the bicistron RF2-RF3, and one NcoI restriction site (or any other appropriate restriction site) is introduced between the first and second repeat to facilitate cloning. Two PCR fragments are generated; one covering the RF2-RF3 gene, the other covering the cMYC gene. Both fragments are linked using the NcoI site, and the resulting fragment is cloned back in the intermediate vector and then in the full-length MV vector using unique restriction sites to produce MV4FP(RF1)(RF2-RF3)miR(M).
The pCGΔH(OSK)(M)H is used to introduce the miRNA target sequence in the 3′UTR of OSK. Three repeats of the miRNA target sequence are inserted in the 3′ UTR of the tricistron OSK, and one NcoI restriction site (or any other appropriate restriction site) is introduced between the first and second repeat to facilitate cloning. Two PCR fragments are generated; one covering the KLF4 gene, the other covering the cMYC gene. Both fragments are linked using the NcoI site, and the resulting fragment is cloned back in the pCGΔH(OSK)(M)H and then in the full-length MV vector using unique restriction sites to produce MV4FP(OSK)miR(M).
cMYC Control: Single, Double, Triple Combinations
The pCGΔH(OSK)(cMYC)H is used to introduce the miRNA target sequence. Three repeats of the miRNA target sequence are inserted in the 3′ UTR of the cMyc gene, and one NcoI restriction site (or any other appropriate restriction site) is introduced between the first and second repeat to facilitate cloning. Two PCR fragments are generated; one covering the cMYC gene and the start of the ATU encoding cMYC including one of the unique restriction site (such as MluI) that is used for the cloning of the modified cMYC, the other covering the SpeI and a part of the L gene. Both fragments are linked using the NcoI site, and the resulting fragment is cloned back in the intermediate vector and then in the full-length MV vector using unique restriction sites to produce MV4FP(RF1)(RF2-RF3)(M)miR.
The vector MV4FP(RF1)miR(RF2-RF3)(M)miR is obtained by sub-cloning the MluI-SpeI fragment of MV4FP(OSK)(M)miR in place of the MluI-SpeI fragment of MV4FP(RF1)miR(RF2-RF3)(M).
The vector MV4FP(RF1)(RF2-RF3)miR(M)miR is obtained by sub-cloning the MluI-SpeI fragment of MV4FP(OSK)(M)miR in place of the MluI-SpeI fragment of MV4FP(RF1)(RF2-RF3)miR(M).
The proficiency of MV vector to derive naïve iPSC is analyzed in vitro after reprogramming somatic cells in presence of compound identified to maintain the naïve state of human embryonic stem cells (hESC). Human cells (PBMCs or Fibroblasts) are transduced with MV4F as described in Example 10. Starting from day 3 to 4, compounds identified to maintain the naïve state of the hESC are added in the media and changed every other day. Compounds can be a cocktail containing a mix of some of the following inhibitors: MEKi, GSK3i, JNKi, P38i, PKC, ROCKi, BMPi, BRAFi, SRCi; and growth factors: bFGF, TGFb, hLIF, Activin (Gafni et al., Nature, 504:282-280 (2013); Chan et al., Cell Stem Cell, 13:663-675, (2013); Valamehr et al., Stem Cell Report, 2:366-381, (2014); Ware et al., Proc Natl Acad Sci USA, 111:4484-4489, (2014); and Theunissen et al., Cell Stem Cell, 15:471-487, (2014)). This list is not restrictive. The NutriSTEM is used as reprogramming media, this media is the equivalent of the Pluriton media that was used previously to establish the MV reprogramming system. As the bFGF (20 ng/mL for primed iPSC reprogramming) for primed iPSC reprogramming is added to the media, the amount of bFGF can be tittered down as needed to induce maintenance of the iPSC to a naïve state. Expression of naïve markers in MV4F-derived iPSC clones is confirmed by expression of markers such as OCT4, NANOG, STELLA, DPPAS, DNMT3L, REX1, KLF4, KLF5, KLF2, DPPA2, UTF1, OTX1, ZIC2, or ESSRB.
The proficiency of MV-derived iPSC (Naïve or primed) to generate pancreatic progenitor cells is analyzed in vitro using a step-wise differentiation protocol to test differentiation of iPSCs into beta cells (or insulin producing cells). The iPSCs are subjected to a stepwise differentiation process, which is a combination of several protocols (Rezania et al., Nat. Biotechnol., 32:1121-1133 (2014); and Pagliuca et al., Cell, 159:428-439 (2014)). Briefly, the iPSC are first differentiated into definitive endoderm with Activin A and CHIR-99021 for 3 days (Stage 1); then into primitive gut tube with ascorbic acid and FGF7 for 2 days (Stage 2); then into posterior foregut with ascorbic acid, FGF7, SANT-1, retinoic acid, LDN193189 and PKC activator for 2 days (Stage 3); then into PDX1-positive pancreatic endoderm cells with SANT-1 and LDN198189 for 3 days (Stage 4); then into PDX1/NEUROD1-positive pancreatic endocrine precursors with SANT-1, LDN1938189, T3, ALKS inhibitor and zinc sulfate for 3 days (Stage 5); then into NKX6.1/insulin-positive beta-cells with T3, ALKS inhibitor, zinc sulfate, heparin and gamma secretase inhibitor for 15 days (Stage 6); and finally into NKX6.1/MAFA/insulin-positive beta-cells with T3, ALKS inhibitor, AXL inhibitor R428, betacellulin for 7 days (Stage 7).
The functionality of the beta cell are tested in vivo. They are used to reverse STZ-induced diabetes in mice. Briefly, SCID/Beige mice, immune-compromised mice are used. To induce diabetes, mice are given 5 consecutive intraperitoneal injections of Streptozotocin. Once diabetes is established, by obtaining 2 consecutive blood glucose readings of >300 mg/dL, mice are given the beta-like cells graft. The following experimental group receives beta-like cells generated with 4F vectors. Twenty μL of beta-like cells are injected into a pocket under the left kidney capsule. One-day post-injection, blood sugars are recorded, and then every 3 days for the next 2 weeks, then every week for 2 months until beta-like cells function is observed. Human C-peptide secretion is analyzed 1 week after injection, then every 2 weeks. If an effect is observed in the first few days, mice are kept for an observation phase (60 days), to verify the long-term survival and functionality of the cells. At the end of the observation period, a left nephrectomy is performed to remove the beta-like cell grafts. The mice are observed for 7 days for reoccurrence of the diabetes to confirm that the effect is due to the engrafted beta-like cells. Mice are then euthanized via CO2 inhalation. The mice overall health is followed and is assessed for tumor and metastasis formation. If any tumor is detected, they are analyzed after H&E staining for malignancy pattern.
If the beta-like cells are not responsive to glucose in vitro, they might need a phase of maturation in the animal to become fully mature. If this is the case, the cells are implanted and are followed to determine if they are able to continue their differentiation into mature and functional beta-like cells, in vivo. The mice are kept for 120 days. Every month, a glucose challenge test is performed to determine if the cells have matured and are able secrete insulin in response to the excessive glucose intake. At the end of the observation period, the mice are euthanized via CO2 inhalation. If the renal sub capsular site is unsuitable for delivery of the beta-like cell, a small capsule (TheraCyteR System from TheraCyte, Inc) is used to deliver the cells. The small capsule is implanted on the flank of the mice.
It is to be understood that while the disclosure has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the disclosure, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
This application claims the benefit of the filing date of U.S. Provisional Application No. 62/402,310, filed on Sep. 30, 2016. The contents of U.S. Application No. 62/402,310 are incorporated herein by reference in their entirety.
This invention was made with government support under AI105233 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2017/054268 | 9/29/2017 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62402310 | Sep 2016 | US |
