Methods of predicting engraftment capability of differentiated neuronal cells
AssigneeAspen Neuroscience, Inc.
InventorsJim Mossman, Daniel Fremgen, Genevieve Beauvais
Life of this patent
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Abstract
Provided herein are methods for predicting characteristics, such as activity, function and/or differentiation state of an in vitro population of neuronal cells, for instance a population of neuronal progenitor cells, as well as methods for selecting and/or implanting an in vitro population of neuronal progenitor cells having desired characteristics. Also provided herein are computing devices for performing the provided methods as well as related compositions, articles of manufacture, and kits, including for use in methods of treating a subject having a neurodegenerative disease, for instance Parkinsons disease.
Claims38
A method of predicting whether a population of neuronal progenitor cells (NPCs) are likely to successfully engraft when implanted into a brain region, the method comprising:
- a) culturing pluripotent stem cells under conditions to neurally differentiate the pluripotent stem cells to obtain a population of NPCs;
- b) determining a gene expression level for one or more genes associated with predicted engraftment potential (G genes) in a test sample that comprises a first aliquot of the population of NPCs, wherein the one or more G genes are selected from the group consisting of:
- AC000120.3, KRT77, TTR, PRR16, MEGF10, PDE3A, GDPD2, CMTM8, APOA1, CMTM7, CDHR3, CORIN, VTN, CPNE8, EFEMP1, CD47, SPARC, JAM2, CDO1, PLXDC2, DYNLL2, ITGA3, RPS6KL1, CHRNB2, SULT4A1, PTPN3, LZTS1, RUNX1T1, TMEM145, EPHA10, CARMIL3, MANEAL, TMEM176B, MPP3, DRAXIN, ADGRB1, KIF26A, CELF5, CNTN2, ASPHD1, SVOP, ANGPT2, SLC22A15, SRRM3, GRIN2D, DACH2, CHST1, GRIN1, LHX5, and NOS2;
- and
- c) determining a predicted neuronal engraftment capability of the NPCs, wherein the determining comprises comparing the determined-gene expression level for one or more of the G genes in the test sample to a reference standard for the G gene, wherein the reference standard comprises a quantitative threshold or range of gene expression levels for the G gene, said threshold or range being empirically derived from a training set that comprises one or more reference samples of NPCs, wherein graft size produced after implantation of a second aliquot of the reference sample into a brain region has been measured, and wherein the reference standard is defined by NPC G gene expression levels that are statistically correlated with the measured graft size produced by the implanting the second aliquot of the reference sample into a subject brain;
- and
- d) formulating NPCs having a predicted engraftment capability that is above a predetermined threshold into a pharmaceutical composition.
The method of claim 0, wherein the reference standard comprises a plurality of data points and each data point is obtained by:
- a) measuring the gene expression level of the G gene in a first aliquot of a reference sample that comprises a population of neuronal progenitor cells;
- b) implanting neuronal progenitor cells from a second aliquot of the reference sample into a brain region of a test animal and measuring the size of a graft formed by the implanted neuronal progenitor cells after an incubation period;
- and
- c) statistically correlating the measured graft size produced by the implanted NPCs of the second aliquot against the expression level of the G gene in the first aliquot to generate the data point for the reference sample.
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The method of claim 0, wherein the statistical correlation is obtained by performing differential expression analysis or linear regression analysis of the plurality of data points.
The method of claim 0, wherein the reference plot is obtained by inputting gene expression levels of the one or more G genes from a plurality of reference samples into a machine learning model trained to predict engraftment potential of neuronal progenitor cells, wherein the machine learning model is trained using gene expression levels of the G genes in a plurality of reference samples of neuronal progenitor cells, and wherein the training establishes statistical relationships between gene expression levels and engraftment capability, thereby improving prediction of engraftment by the neuronal progenitor cells.
The method of claim 0, wherein the machine learning model comprises principal component analysis.
The method of claim 0, wherein the predicted graft size is expressed as a number of neuronal cells derived from the neuronal progenitor cells within a cross section of the brain region of the test animal.
The method of claim 0, wherein the brain region is the substantia nigra and the cross section of the substantia nigra comprises approximately one sixth of the substantia nigra.
The method of claim 0, wherein a high engraftment capability is indicated if the predicted graft size is greater than 1,000 cells in the cross section of the substantia nigra.
The method of claim 0, wherein the predicted engraftment capability for the population of neuronal progenitor cells is determined by (a) measuring the gene expression level for two or more G genes in the test sample;
- (b) determining an engraftment capability prediction score for each of the two or more G genes by comparing the measured gene expression level of each G gene to the reference standard for that G gene;
- and (c) calculating an overall predicted engraftment capability score for the neuronal progenitor cells based on a statistical combination of the individual engraftment capability prediction scores for each of the two or more G genes.
The method of claim 0, wherein the combined assessment comprises determining a mean or median predicted engraftment capability.
The method of claim 0, wherein the one or more G genes are selected from the group consisting of TTR, PRR16, CMTM8, APOA1, CD47, CD01, KIR26A and CNTN2.
The method of claim 0, wherein the one or more G genes are selected from the group consisting of TTR, PRR16 and CD47.
The method of claim 0, wherein the one or more G genes are TTR, PRR16, and CD47.
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The method of claim 0, wherein the gene expression level of each of the at least one G gene is determined by RNA sequencing (RNAseq).
The method of claim 0, wherein the gene expression level of each of the at least one G gene is determined by polymerase chain reaction (PCR).
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The method of claim 231, wherein the gene expression level of the one or more G genes is determined by:
- a) obtaining an RNA sample from the test sample of neuronal progenitor cells;
- b) synthesizing complementary DNA from the RNA sample using reverse transcription;
- c) amplifying a specific nucleic acid fragment corresponding to the G gene using quantitative polymerase chain reaction (qPCR), wherein the qPCR comprises the use of a pair of primers specific to the G gene, and optionally a probe specific to the G gene;
- and
- d) determining the expression level of the G gene based on the normalized quantified amount.
The method of claim 0, wherein the gene expression level of the one or more G genes is normalized as a ratio of the relative expression level of the G gene to a housekeeping gene, optionally wherein the housekeeping gene is GAPDH.
The method of claim 0, wherein the expression levels are determined for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 of the G genes.
The method of claim 0, wherein the reference sample comprises a pooled sample of neuronal progenitor cells derived from multiple donors.
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The method of claim 281, wherein the predetermined threshold for a particular G gene is based on the expression level of the G gene in a reference sample that comprises neuronal progenitor cells that are known to exhibit high engraftment levels when implanted into a brain, and a high engraftment potential for the test sample is indicated by a gene expression level for the G gene in the test sample that is:
- a) similar to or greater than the expression level of the G gene in the reference sample if a higher expression level for the G gene is predictive of higher engraftment potential;
- or
- b) similar to or lower than the expression level for the G gene in the reference sample if a lower expression level of the G gene is predictive of higher engraftment potential.
The method of claim 0, wherein the predetermined threshold value for the particular G gene is based on a ratio of the relative expression levels in the test sample of a) the G gene, and b) a control gene.
The method of claim 0, wherein the control gene is GAPDH and the predetermined threshold value is selected from the group consisting of:
- (a) a ratio of AC000120.3 to GAPDH expression of less than about 0.14;
- (b) a ratio of KRT77 to GAPDH expression of less than about 0.68;
- (c) a ratio of TTR to GAPDH expression of less than about 1.11;
- (d) a ratio of PRR16 to GAPDH expression of less than about 0.43;
- (e) a ratio of MEGF10 to GAPDH expression of less than about 0.79;
- (f) a ratio of PDE3A to GAPDH expression of less than about 1.00;
- (g) a ratio of GDPD2 to GAPDH expression of less than about 0.78;
- (h) a ratio of CMTM8 to GAPDH expression of less than about 1.02;
- (i) a ratio of APOA1 to GAPDH expression of less than about 0.68;
- (j) a ratio of CMTM7 to GAPDH expression of less than about 0.88;
- (k) a ratio of CDHR3 to GAPDH expression of less than about 1.09;
- (l) a ratio of CORIN to GAPDH expression of less than about 1.24;
- (m) a ratio of VTN to GAPDH expression of less than about 0.98;
- (n) a ratio of CPNE8 to GAPDH expression of less than about 0.79;
- (o) a ratio of EFEMP1 to GAPDH expression of less than about 0.83;
- (p) a ratio of CD47 to GAPDH expression of less than about 1.16;
- (q) a ratio of SPARC to GAPDH expression of less than about 1.29;
- (r) a ratio of JAM2 to GAPDH expression of less than about 0.82;
- (s) a ratio of CDO1 to GAPDH expression of less than about 1.00;
- (t) a ratio of PLXDC2 to GAPDH expression of less than about 1.00;
- (u) a ratio of DYNLL2 to GAPDH expression of greater than about 0.56;
- (v) a ratio of ITGA3 to GAPDH expression of greater than about 0.26;
- (w) a ratio of RPS6KL1 to GAPDH expression of greater than about 0.21;
- (x) a ratio of CHRNB2 to GAPDH expression of greater than about 0.23;
- (y) a ratio of SULT4A1 to GAPDH expression of greater than about 0.22;
- (z) a ratio of PTPN3 to GAPDH expression of greater than about 0.03;
- (aa) a ratio of LZTS1 to GAPDH expression of greater than about 0.19;
- (ab) a ratio of RUNX1T1 to GAPDH expression of greater than about 0.24;
- (ac) a ratio of TMEM145 to GAPDH expression of greater than about 0.05;
- (ad) a ratio of EPHA10 to GAPDH expression of greater than about 0.16;
- (ae) a ratio of CARMIL3 to GAPDH expression of greater than about 0.16;
- (af) a ratio of MANEAL to GAPDH expression of greater than about 0.24;
- (ag) a ratio of TMEM176B to GAPDH expression of greater than about 0.11;
- (ah) a ratio of MPP3 to GAPDH expression of greater than about 0.12;
- (ai) a ratio of DRAXIN to GAPDH expression of greater than about 0.27;
- (aj) a ratio of ADGRB1 to GAPDH expression of greater than about 0.07;
- (ak) a ratio of KIF26A to GAPDH expression of greater than about 0.23;
- (al) a ratio of CELF5 to GAPDH expression of greater than about 0.25;
- (am) a ratio of CNTN2 to GAPDH expression of greater than about 0.23;
- (an) a ratio of ASPHD1 to GAPDH expression of greater than about 0.08;
- (ao) a ratio of SVOP to GAPDH expression of greater than about 0.16;
- (ap) a ratio of ANGPT2 to GAPDH expression of greater than about 0.06;
- (aq) a ratio of SLC22A15 to GAPDH expression of greater than about 0.04;
- (ar) a ratio of SRRM3 to GAPDH expression of greater than about 0.17;
- (as) a ratio of GRIN2D to GAPDH expression of greater than about 0.02;
- (at) a ratio of DACH2 to GAPDH expression of greater than about 0.06;
- (au) a ratio of CHST1 to GAPDH expression of greater than about 0.04;
- (av) a ratio of GRIN1 to GAPDH expression of greater than about 0.26;
- (aw) a ratio of LHX5 to GAPDH expression of greater than about 0.06;
- and (ax) a ratio of NOS2 to GAPDH expression of greater than about 0.08.
34-89. (canceled)
A method of preparing a pharmaceutical composition for treating a neurodegenerative disease, wherein the method comprises:
- a) culturing pluripotent stem cells under conditions to neurally differentiate the pluripotent stem cells to obtain a population of neuronal progenitor cells;
- b) determining a gene expression level for one or more genes associated with predicted engraftment potential (G genes) in a test sample that comprises an aliquot of the population of neuronal progenitor cells, wherein the one or more G genes are selected from the group consisting of:
- AC000120.3, KRT77, TTR, PRR16, MEGF10, PDE3A, GDPD2, CMTM8, APOA1, CMTM7, CDHR3, CORIN, VTN, CPNE8, EFEMP1, CD47, SPARC, JAM2, CDO1, PLXDC2, DYNLL2, ITGA3, RPS6KL1, CHRNB2, SULT4A1, PTPN3, LZTS1, RUNX1T1, TMEM145, EPHA10, CARMIL3, MANEAL, TMEM176B, MPP3, DRAXIN, ADGRB1, KIF26A, CELF5, CNTN2, ASPHD1, SVOP, ANGPT2, SLC22A15, SRRM3, GRIN2D, DACH2, CHST1, GRIN1, LHX5, and NOS2;
- and
- determining a predicted neuronal engraftment capability of the neuronal progenitor cells, wherein the determining comprises comparing the gene expression level for one or more of the G genes in the test sample to a reference standard for the G gene, wherein the reference standard comprises a quantitative threshold or range of gene expression levels for the G gene, said threshold or range being empirically derived from a training set that comprises one or more reference samples of neuronal progenitor cells, wherein graft size produced by neuronal progenitor cells in a first aliquot of the reference samples after implantation into a brain region has been measured, and wherein the reference standard is defined by neuronal progenitor cell G gene expression levels in a second aliquot of the reference sample that are statistically correlated with the measured graft size produced by the neuronal progenitor cells in the first aliquot of the reference sample;
- and
- formulating neuronal progenitor cells having a predicted engraftment capability that is above a predetermined threshold into a pharmaceutical composition.
A method for treating a subject having a neurodegenerative disease, the method comprising administering to a subject having a neurodegenerative disease a pharmaceutical composition that comprises neuronal progenitor cells, wherein the pharmaceutical composition is prepared by a method that comprises:
- a) culturing pluripotent stem cells under conditions to neurally differentiate the pluripotent stem cells to obtain a population of neuronal progenitor cells;
- b) determining a gene expression level for one or more genes associated with predicted engraftment potential (G genes) in a test sample that comprises an aliquot of the population of neuronal progenitor cells, wherein the one or more G genes are selected from the group consisting of:
- AC000120.3, KRT77, TTR, PRR16, MEGF10, PDE3A, GDPD2, CMTM8, APOA1, CMTM7, CDHR3, CORIN, VTN, CPNE8, EFEMP1, CD47, SPARC, JAM2, CDO1, PLXDC2, DYNLL2, ITGA3, RPS6KL1, CHRNB2, SULT4A1, PTPN3, LZTS1, RUNX1T1, TMEM145, EPHA10, CARMIL3, MANEAL, TMEM176B, MPP3, DRAXIN, ADGRB1, KIF26A, CELF5, CNTN2, ASPHD1, SVOP, ANGPT2, SLC22A15, SRRM3, GRIN2D, DACH2, CHST1, GRIN1, LHX5, and NOS2;
- and
- c) determining a predicted neuronal engraftment capability of the neuronal progenitor cells, wherein the determining comprises comparing the gene expression level for one or more of the G genes in the test sample to a reference standard for the G gene, wherein the reference standard comprises a quantitative threshold or range of gene expression levels for the G gene, said threshold or range being empirically derived from a training set that comprises one or more reference samples of neuronal progenitor cells, wherein graft size produced by NPCs in a first aliquot of the reference samples after implantation into a brain region has been measured, and wherein the reference standard is defined by NPC G gene expression levels in a second aliquot of the reference samples that are statistically correlated with the measured graft size produced by the NPCs in the first aliquot of the reference sample;
- and
- d) formulating neuronal progenitor cells having a predicted engraftment capability that is above a predetermined threshold into a pharmaceutical composition.
Dependencies are inferred from the claim text.