WO2023245670A1 - Compositions and methods for treatment of neurological diseases - Google Patents
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Definitions
- compositions for regulating a primate PTBP gene expression comprising: (1) a Cas effector protein, or a nucleic acid encoding the same, (2) a guide RNA (gRNA) that targets a target sequence, or its complement, of the primate PTBP gene or a transcript thereof, or a nucleic acid encoding the same, wherein the composition is capable of suppressing the expression of the primate PTBP gene.
- the target sequence is located within a key region of the primate PTBP gene.
- the key region is SEQ ID NO: 88.
- the Cas effector protein is an RNA-targeting Cas effector protein.
- the RNA-targeting Cas effector protein is selected from the group consisting of Cas13d, CasRx, Cas13X, Cas13a, Cas13b, Cas13c, Cas13Y, and a functional domain thereof.
- the Cas effector protein is CasRx.
- the Cas effector protein is Cas13X.
- the gRNA sequence has at least 90%sequence identity to SEQ ID NOs: 1-86. In some embodiments, the gRNA sequence has at least 90%sequence identity to SEQ ID NOs: 89-112.
- the nucleic acid encoding the Cas effector protein is located on an expression vector. In some embodiments, the expression vector is a gene therapy vector. In some embodiments, the gene therapy vector is a viral gene therapy vector.
- the viral gene therapy vector is selected from the group consisting of an adeno-associated virus (AAV) vector, an adenovirus vector, a lentivirus vector, a retrovirus vector, a herpes virus, a SV40 vector, a poxvirus vector, and a combination thereof.
- AAV adeno-associated virus
- the expression vector is comprised in a nanoparticle.
- the expression vector further comprises a glial cell-specific promoter that causes expression of the Cas effector protein in a non-neuronal cell.
- the glial cell-specific promoter is selected from the group consisting of a GFAP promoter, a ALDH1L1 promoter, a EAAT1/GLAST promoter, a glutamine synthetase promoter, a S100 beta promoter and a EAAT2/GLT-1 promoter.
- the glial cell-specific promoter is a Müller glia (MG) cell-specific promoter.
- the MG cell-specific promoter is selected from the group consisting of a GFAP promoter, a ALDH1L1 promoter, a Glast (also known as Slc1a3) promoter and a Rlbp1 promoter.
- the nucleic acid encoding the gRNA is located on an expression vector.
- expression vector further comprises a promoter that causes expression of the gRNA in a non-neuronal cell.
- the promoter that causes expression of the gRNA in the non-neuronal cell is a U6 promoter.
- the primate PTBP gene is located in a non-neuronal cell.
- the non-neuronal cell is located in mature retina, striatum, substantia nigra, inner ear, spinal cord, prefrontal cortex, motor cortex, or ventral tegmental area (VTA) .
- the non-neuronal cell located in the striatum is in putamen.
- the non-neuronal cell is a glial cell. In some embodiments, the glial cell is an astrocyte.
- composition disclosed hereof further comprising one or more dopamine neuron-associated factors, or an expression vector for expression of the one or more dopamine neuron-associated factors thereof.
- the one or more dopamine neuron-associated factors are selected from the group consisting of: Lmx1a, Lmx1b, FoxA2, Nurr1, Pitx3, Gata2, Gata3, FGF8, BMP, En1, En2, PET1, a Pax family protein, SHH, a Wnt family protein, and a TGF- ⁇ family protein.
- composition disclosed hereof further comprising one or more factors selected from ⁇ -catenin, Oct4, Sox2, Klf4, Crx, Brn3a, Brn3b, Math5, Nr2e3 and Nrl, or an expression vector for expression of the one or more factors thereof.
- composition disclosed hereof further comprising an immunosuppressant.
- the immunosuppressant is selected from the group consisting of a corticosteroid, a calcineurin inhibitor, an mTOR inhibitor, an IMDH inhibitor, an immune-suppressive antibody, an interferon, a Janus kinase inhibitor and an anakinra.
- Described herein is a method of treating a disease comprising administering an effective amount of the composition disclosed thereof to a subject in need thereof.
- the composition suppresses the expression or activity of the primate PTBP protein in a non-neuronal cell, thereby allowing the non-neuronal cell to reprogram into a functional neuron.
- the disease is a neurological condition associated with degeneration of functional neurons.
- the neurological condition is a neurological condition associated with degeneration of functional neurons in the mature retina which is selected from the group consisting of: glaucoma, age-related RGC loss, optic nerve injury, retinal ischemia, LCA (Leber′s congenital amaurosis) , RP (Retinitis pigmentosa) disease and Leber′s hereditary optic neuropathy.
- the condition associated with degeneration of functional neurons is selected from the group consisting of Parkinson′s disease, Alzheimer′s disease, Huntington′s disease, Schizophrenia, depression, drug addiction, stroke, movement disorder, spinal cord injury, choreoathetosis, dyskinesias, bipolar disorder, and Autism spectrum disorder (ASD) .
- ASD Autism spectrum disorder
- the composition is administered to a glial cell or a MG cell in a mature retina to generate a functional retinal ganglion cell (RGC) neuron or a functional retinal photoreceptor.
- RRC retinal ganglion cell
- an expression level of PTBP mRNA is less than 40%compared to a corresponding control. In some embodiments, an expression level of PTBP mRNA is less than 50%compared to a corresponding control. In some embodiments, there are no co-factors expressed.
- Figures 1A-B depict a screen of efficient gRNAs.
- Figure 1A depicts downregulation of Ptbp1 mRNA with 86 gRNAs in human 293T cells, two days after transient transfection of plasmids encoding CasRx and gRNAs. Note that the gRNA 60 was used in the following experiments.
- Figure 1B depicts targeting sites of 86 gRNAs in human Ptbp1 gene, as well as the information of knockdown efficiency (red, ⁇ 0.12; green, > 0.1) .
- Key region indicates the targeting region with high knockdown efficiency. The key region corresponds to positions 951-1487 of SEQ ID NO: 87, which are position 1-536 of SEQ ID NO: 88.
- Figures 2A-B show confirmation of the key region using another RNA-targeting Cas13 protein.
- Figure 2A shows downregulation of Ptbp1 mRNA with 24 gRNAs in human 293T cells, two days after transient transfection of plasmids encoding CasRx and gRNAs. Note that gRNAs targeting the key region were highlighted as red dots.
- Figure 2B shows targeting sites of 24 gRNAs in the human Ptbp1 gene, as well as the information of knockdown efficiency (red, ⁇ 0.75; green, > 0.75) .
- Key region indicates the targeting region with high knockdown efficiency.
- the key region corresponds to positions 951-1487 of SEQ ID NO: 87, which are positions 1-536 of SEQ ID NO: 88.
- Figure 3A-C shows that gRNA 60 showed high knockdown efficiency in human 293T cells, monkey Cos7 cells, and mouse N2a cells. All values are presented as mean ⁇ SEM.; unpaired t test; *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001.
- Figure 4 shows expression levels in log2 (fragments per kilobase per million mapped reads [FPKM] + 1) values of all detected genes in RNA sequencing (RNA-seq) libraries of CasRx-Ptbp1 (y axis) compared to CasRx control (x axis) , showing that Ptbp1 was specifically downregulated.
- Cos7 cells, n 3 independent replicates for both groups.
- the gRNA 60 was used.
- Figures 5A-D shows a conversion of astrocytes into dopamine neurons in the striatum of mice.
- Figure 5A shows a schematic illustration of the AAV vectors and injection strategy.
- Vector 1 (AAV-GFAP-mCherry) encodes mCherry driven by the astrocyte-specific promoter GFAP;
- Vector 2 (AAV-GFAP-CasRx) encodes CasRx;
- Vector 3 (AAV-GFAP-CasRx-Ptbp1) encodes CasRx and gRNAs.
- the striatum was either injected with AAV-GFAP-CasRx-Ptbp1 or control vector AAV-GFAP-CasRx together with AAV-GFAP-mCherry.
- FIG. 5B shows representative images showing that mCherry (Vector 1) and CasRx (Fused with Flag, Vector 3) were specifically expressed in the mouse astrocytes. Note that GFAP is the astrocyte-specific marker.
- Figure 5C shows confocal images showing that converted mCherry+NeuN+ cells (white arrowheads) were observed in mice 2 weeks after Vector 1 + 3 injection, but not in the striatum injected with control AAVs. Note that NeuN is neuron-specific marker.
- Figure 5D shows confocal images showing that converted mCherry+DAT+ cells (white arrowheads) were observed in mice one month after Vector 1 + 3 injection, but not in the control striatum. Note that DAT is the mature dopamine neuron-specific marker.
- Figures 6A-E depict an increase of MG-to-RGC conversion efficiency by co-injecting ⁇ -catenin in middle-aged mice.
- Figure 6A shows a schematic showing MG-to-RGC conversion.
- Vector I AAV-GFAP-GFP-Cre
- Vector II AAV-GFAP-CasRx
- Vector III AAV-GFAP-beta-Catenin
- Vector IV AAV-GFAP-CasRx-Ptbp1 expresses CasRx and gRNAs.
- retinas (Ai9 mice, 4-5 months old) were injected with AAV-GFAP-CasRx-Ptbp1 or control AAV-GFAP-CasRx together with AAV-GFAP-GFP-Cre.
- Induction of RGC conversion was examined 2-3 weeks post-injection.
- ONL outer nuclear layer
- OPL outer plexiform layer
- INL inner nuclear layer
- IPL inner plexiform layer
- GCL ganglion cell layer.
- Figure 6B-E shows representative images indicating colocalization of RBPMS+tdTomato+ cells in the GCL, and tdTomato+ optic nerve for AAV-GFAP-GFP-Cre + AAV-GFAP-CasRx, AAV-GFAP-GFP-Cre + AAV-GFAP- ⁇ -catenin, AAV-GFAP-GFP-Cre + AAV-GFAP-CasRx-Ptbp1, and AAV-GFAP-GFP-Cre + AAV-GFAP-CasRx-Ptbp1 + AAV-GFAP- ⁇ -catenin groups.
- AAV-GFAP-GFP-Cre + AAV-GFAP-CasRx and AAV-GFAP-GFP-Cre + AAV-GFAP- ⁇ -catenin are control groups. Yellow arrowhead showing the colocalization of tdTomato and RBPMS in the retinas injected with AAV-GFAP-GFP-Cre, AAV-GFAP-CasRx-Ptbp1 and AAV-GFAP- ⁇ -catenin.
- RBPMS is a specific marker for RGCs.
- the gRNA 60 was used.
- Figures 7A-B depicts the conversion of astrocytes into dopamine neurons in the putamen of non-PD monkeys.
- Figure 7A shows representative images showing that the expression of mCherry+ colocalized with GFAP in the putamen injected with control AAVs.
- Figure 7B shows representative images showing a mCherry+TH+ cell in the Macaca fascicularis putamen injected with Vector 1 and 3 (yellow arrowhead) , but not in the putamen injected with control AAVs.
- the gRNA 60 was used.
- Figures 8A-C show the induction of dopaminergic neurons in a PD model monkey.
- Figure 8A shows confocal images of mCherry+ cells with (AAV-GFAP-CasRx-Ptbp1) or without (AAV-GFAP-CasRx) DAT and TH signal (white arrowheads) .
- TH is the most classical dopaminergic neuron marker
- DAT is the marker of mature dopaminergic neurons.
- Figure 8B shows confocal images of mCherry+ cells with (AAV-GFAP-CasRx-Ptbp1) or without (AAV-GFAP- CasRx) GIRK2 and TH signal (white arrowheads) .
- GIRK2 is the specific marker of substantia nigra (A9 region) dopaminergic neurons.
- Figure 8C shows confocal images of mCherry+ cells with (AAV-GFAP-CasRx-Ptbp1) or without (AAV-GFAP-CasRx) VMAT2 and TH signal (white arrowheads) . Note that the expression of VMAT2 is essential for dopamine packaging, storage, and release. For all images, the scale bars were 50 ⁇ m.
- Figure 9 shows how induced dopamine neurons alleviated the symptoms of a PD model monkey.
- the open field test was used to evaluate the behavioral recover ratio of the PD model monkeys, at 6 months and 9 months after injection of AAV-GFAP-CasRx-Ptbp1 +_AAV-GFAP-mCherry (red line) or control AAVs (AAV-GFAP-CasRx + AAV-GFAP-mCherry, blue line) .
- Figure 10 shows how induced dopaminergic neurons improved the density of PET-CT signal in putamen of a PD model monkey.
- the tracer, [18F] DOPA was used to detect the intensity of dopaminergic neurons’ signal of the PD model monkeys, at before or around one year after injection of AAV-GFAP-CasRx +AAV-GFAP-mCherry ( Figures 10A) or AAV-GFAP-CasRx-Ptbp1 + AAV-GFAP-mCherry ( Figures 10B) .
- Figures 10C shows the quantification result of PET-CT.
- Figures 10D shows the MRI images of the same monkey with the same planes shown in Figures 10B for anatomy structure validation.
- Figure 11 shows how induced dopamine neurons increased the concentration of dopamine in cerebrospinal fluid (CSF) of a PD monkey.
- the HPLC was used to evaluate the dopamine level of the PD monkeys, at before or after virus injection of AAV-GFAP-CasRx-Ptbp1 + AAV-GFAP-mCherry (red line) or control AAVs (AAV-GFAP-CasRx + AAV-GFAP-mCherry, blue line) .
- Neurodegenerative diseases are devastating diseases associated with the progressive loss of neurons in various parts of the nervous system.
- regenerative medicine has great promise for treating neurodegenerative diseases that lead to cell (e.g., neuron) loss.
- One approach employs cell replacement, while another utilizes cellular trans-differentiation.
- Trans-differentiation takes advantage of the existing cellular plasticity of endogenous cells to generate new cell types.
- One challenge for this approach is to identify efficient strategies to convert certain target cells to a desired cell type (e.g., neurons) , not only in culture but more importantly in their in vivo native contexts, particularly at a desired location (e.g., a tissue or organ type) .
- polypyrimidine tract-binding protein 1 also known as Ptbp1, PTBP1, PTBP, or PTB
- Ptbp1, PTBP1, PTBP, or PTB polypyrimidine tract-binding protein 1
- Ptbp1, PTBP1, PTBP, or PTB polypyrimidine tract-binding protein 1
- the term ′′and/or′′ indicates that one or more of the stated cases may occur, alone or in combination with at least one of the stated cases, up to with all of the stated cases.
- ′′at least′′ a particular value means that particular value or more.
- ′′at least 2′′ is understood to be the same as ′′2 or more′′ i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, ..., etc.
- Astrocyte′′ generally refers to characteristic star-shaped glial cells in the brain and spinal cord that are characterized by one or more of: star shape; expression of markers like glial fibrillary acidic protein (GFAP) , aldehyde dehydrogenase 1 family member LI (ALDH1L1) , excitatory amino acid transporter 1 /glutamate aspartate transporter (EAAT1/GLAST) , glutamine synthetase, S100 beta, or excitatory amino acid transporter 1 /glutamate transporter 1 (EAAT2/GLT-1) ; participation of the blood-brain barrier together with endothelial cells; transmitter uptake and release; regulation of ionic concentration in extracellular space; reaction to neuronal injury and participation in nervous system repair; and metabolic support of surrounding neurons.
- GFAP glial fibrillary acidic protein
- ALDH1L1 aldehyde dehydrogenase 1 family member LI
- EAAT1/GLAST excitatory amino acid transporter 1
- an astrocyte refers to a non-neuronal cell in a nervous system that expresses glial fibrillary acidic protein (GFAP) , Aldehyde Dehydrogenase 1 Family Member L1 (ALDH1L1) , or both.
- GFAP glial fibrillary acidic protein
- ADH1L1 Aldehyde Dehydrogenase 1 Family Member L1
- an astrocyte refers to a non-neuronal cell in a nervous system that expresses a glial fibrillary acidic protein (GFAP) promoter-driven transgene (e.g., red fluorescent protein (RFP) , Cre recombinase) .
- GFAP glial fibrillary acidic protein
- RFP red fluorescent protein
- a Müller glia refers to a non-neuronal glial cell found in the retina and that expresses a MG-specific promoter-driven transgene (e.g., red fluorescent protein (RFP) ) .
- MG-specific promoters include promoters from GFAP, Glast (also known as Slc1a3) , and Rlbp1.
- a ′′BRN2 transcription factor′′ or ′′Brain-2 transcription factor, ′′ also called ′′POU domain, class 3, transcription factor 2′′ ( ′′POU3F2′′ ) or ′′Oct-7, ′′ can refer to a class III POU domain transcription factor, having a DNA-binding POU domain that consists of an N-terminal POU-specific domain of about 75 amino acids and a C-terminal POU-homeodomain of about 60 amino acids, which are linked via a linker comprising a short ⁇ -helical fold, and which can be predominantly expressed in the central nervous system.
- composition′′ generally refers to an agent that reprograms a differentiated non-neuronal cell to a neuronal cell through inhibiting the expression and/or function of PTB and/or nPTB.
- the composition refers to a CRISPR/Cas effector protein (which may or may not include any variants, derivatives, functional equivalents or fragments thereof) with a guide RNA (gRNA) complementary to a PTB mRNA or to a nPTB mRNA, and can knock down the expression and/or activity of PTB or nPTB, to an extent sufficient to convert a non-neuronal cell to a neuronal cell, preferably in vivo at a local microenvironment where the converted neuron is expected to be functional.
- gRNA guide RNA
- the composition may also refer to a polynucleotide encoding such CRISPR/Cas effector protein as defined above and/or the guide RNA (gRNA) .
- the polynucleotide may include a mRNA for the Cas effector, as defined above.
- the polynucleotide may also include a DNA encoding the Cas effector as defined above and/or the gRNA complementary to PTB/nPTB mRNA.
- the DNA encoding the Cas effector as defined above and/or the gRNA may be part of a vector, including a viral vector (e.g., an adeno-associated viral (AAV) vector or a lentiviral vector, or any of the other viral vectors described herein) .
- a viral vector e.g., an adeno-associated viral (AAV) vector or a lentiviral vector, or any of the other viral vectors described herein
- any AAV with tropism for glial cell or non-neuronal cell in the CNS and/or PNS can be used, including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, etc.
- any Cas effector as defined above can be used, so long as the coding sequence for the Cas effector is smaller than the packaging capacity of AAV, such as 4.7 kb, 4.5 kb, 4.0 kb, 3.5 kb, 3.0 kb, 2.5 kb, 2.0 kb, 1.5 kb or less.
- Exemplary Cas effectors that may be used include Cas13a, Cas13b, Cas13c, Cas13d, CasRx, Cas13X, Cas13Y, Cpf1, Cas9, and functional equivalents or fragments thereof.
- the term ′′composition′′ may also be used interchangeably with the Cas effector with the gRNA, or polynucleotide (e.g., DNA or vector) encoding the same.
- the Cas effector protein that can be used with the invention described herein includes CRISPR-Cas Class 2 systems utilizing a single large Cas protein to degrade target nucleic acids (e.g., mRNA) .
- Suitable Class 2 Cas effectors may include Type II Cas effectors such as Cas9 (e.g., Streptococcus pyogenes SpCas9 and S. thermophilus Cas9) .
- the suitable Cas effector may also be Class 2, Type V Cas proteins, including Cas12a (formerly known as Cpf1, such as Francisella novicida Cpf1 and Prevotella Cpf1) , C2c1 and C2c3, which lack an HNH nuclease but have RuvC nuclease activity.
- Cas12a formerly known as Cpf1, such as Francisella novicida Cpf1 and Prevotella Cpf1
- C2c1 and C2c3 which lack an HNH nuclease but have RuvC nuclease activity.
- Cas effector proteins may include Class 2, type VI Cas proteins, including Cas13 (also known as C2c2) , Cas13a, Cas13b, Cas13c, Cas13d /CasRx, Cas13X, and Cas13Y, each of which is an RNA-guided RNase (i.e., these Cas proteins use their crRNA to recognize target RNA sequences, rather than target DNA sequences as in Cas9 and Cas12a) .
- the CRISPR/Cas13 systems can achieve higher RNA digestion efficiency compared to the traditional RNAi and CRISPRi technologies, while simultaneously exhibiting much less off-target cleavage compared to RNAi.
- the composition of the invention is or encodes a Cas effector protein that, together with its canonical gRNA, targets PTB or nPTB mRNA.
- the Cas effector targets PTB or nPTB DNA.
- ′′contacting′′ cells with a composition of the disclosure refers to placing the composition (e.g., a compound, a nucleic acid, a viral vector, etc. ) in a location that will allow it to touch the cell in order to produce ′′contacted′′ cells.
- the contacting may be accomplished using any suitable method. For example, in one embodiment, contacting is by adding the compound to a culture of cells. Contacting may also be accomplished by injection or delivering the composition to a location within a body such that the composition ′′contacts′′ the targeted cell type.
- ′′differentiation, ′′ ′′differentiate, ′′ ′′converting, ′′ or ′′inducing differentiation′′ are used interchangeably to refer to changing the default cell type (genotype and/or phenotype) to a non-default cell type (genotype and/or phenotype) .
- ′′inducing differentiation in an astrocyte cell′′ can refer to inducing the cell to change its morphology from that of an astrocyte to that of a neuronal cell type (e.g., a change in gene expression as determined by genetic analysis such as a microarray) and/or phenotype (e.g., a change in protein expression) .
- an effective amount′′ can refer to the amount of an agent required to ameliorate the symptoms of a disease relative to an untreated patient.
- the effective amount (s) of active agent (s) used to practice the present invention for therapeutic treatment of a disease varies depending upon the manner of administration, age, body weight, and/or general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an ′′effective′′ amount, which may be determined as genome copies per kilogram (GC/kg) .
- a disease or condition indicates that administration in a clinically appropriate manner results in a beneficial effect for at least a statistically significant fraction of patients, such as an improvement of symptoms, a cure, a reduction in at least one disease sign or symptom, extension of life, improvement in quality of life, or other effect generally recognized as positive by medical doctors familiar with treating the particular type of disease or condition.
- the term ′′expression control sequence′′ is intended to include, at a minimum, a sequence whose presence is designed to influence expression, and can also include additional advantageous components.
- leader sequences and fusion partner sequences can be expression control sequences.
- the term can also include the design of a nucleic acid sequence such that undesirable potential initiation codons in and out of frame are removed from the sequence. It can also include the design of the nucleic acid sequence such that undesirable potential splice sites are removed. It can include sequences or polyadenylation sequences (pA) which direct the addition of a polyA tail (i.e., a string of adenine residues at the 3′-end of an mRNA, commonly referred to as polyA sequences) .
- pA polyadenylation sequences
- Expression control sequences which affect the transcription and translation stability e.g., promoters, as well as sequences which affect the translation, e.g., Kozak sequences, are known in the art per se.
- Expression control sequences can be of such nature as to modulate the nucleotide sequence to which they are operably linked such that lower expression levels or higher expression levels are achieved.
- ′′gene′′ means a DNA fragment comprising a region (transcribed region) , which is transcribed into an RNA molecule (e.g., an mRNA) in a cell, operably linked to suitable regulatory regions (e.g., a promoter) .
- a gene can comprise several operably linked fragments, such as a promoter, a 5′ leader sequence, a coding region, and/or a 3′-nontranslated sequence (3′-end) comprising a polyadenylation site.
- RNA which is biologically active, i.e., which is capable of being translated into a biologically active protein or peptide.
- glial cell′′ can generally refer to a type of supportive cell in the central nervous system (e.g., the brain and spinal cord) or the peripheral nervous system.
- glial cells do not conduct electrical impulses or exhibit action potential.
- glial cells do not transmit information with each other, or with neurons via synaptic connection or electrical signals.
- glial cells can surround neurons and provide support for and insulation between neurons.
- Non-limiting examples of glial cells include oligodendrocytes, astrocytes, Müller Glia, ependymal cells, Schwann cells, microglia, spiral ganglion glial cells, and satellite cells.
- a ′′guide sequence′′ is to be understood herein as a sequence that directs an RNA or DNA guided endonuclease to a specific site in an RNA or DNA molecule.
- a ′′guide sequence′′ is further understood herein as the section of gRNA (or crRNA) , which is required for targeting a gRNA-Cas complex to a specific site in the target RNA or DNA molecule.
- the ′′guide sequence′′ in a gRNA is complementary to a specific site in the target RNA or DNA molecule; said site is the ′′target sequence′′ .
- nucleic acid or polypeptide molecule when used to indicate the relationship between a given (recombinant) nucleic acid or polypeptide molecule and a given host organism or host cell, is understood to mean that, in nature, the nucleic acid or polypeptide molecule is produced by a host cell or organisms of the same species, preferably of the same variety or strain. If homologous to a host cell, a nucleic acid sequence encoding a polypeptide will typically (but not necessarily) be operably linked to another (heterologous) promoter sequence and, if applicable, an additional (heterologous) secretory signal sequence and/or terminator sequence than in its natural environment. It is understood that the regulatory sequences, signal sequences, terminator sequences, etc.
- ′′homologous′′ means that one single-stranded nucleic acid sequence may hybridize to a complementary single-stranded nucleic acid sequence.
- the degree of hybridization may depend on a number of factors including the amount of identity between the sequences and the hybridization conditions such as temperature and salt concentration.
- Heterologous and exogenous nucleic acids or proteins are not endogenous to the cell into which they are introduced but have been obtained from another cell or are synthetically or recombinantly produced.
- nucleic acids encode proteins, i.e., exogenous proteins, that are not normally produced by the cell in which the DNA is transcribed or expressed.
- exogenous RNA encodes for proteins not normally expressed in the cell in which the exogenous RNA is present.
- Heterologous/exogenous nucleic acids and proteins may also be referred to as foreign nucleic acids or proteins. Any nucleic acid or protein that one of skill in the art would recognize as foreign to the cell in which it is expressed is herein encompassed by the terms heterologous or exogenous nucleic acid or protein.
- heterologous and exogenous also apply to non-natural combinations of nucleic acid or amino acid sequences, e.g., combinations where at least two of the combined sequences are foreign with respect to each other.
- a ′′microRNA′′ or ′′miRNA′′ refers to a non-coding nucleic acid (RNA) sequence that binds to at least partially complementary nucleic acid sequence (mRNAs) and negatively regulates the expression of the target mRNA at the post-transcriptional level.
- RNA nucleic acid
- mRNAs at least partially complementary nucleic acid sequence
- a microRNA is typically processed from a ′′precursor′′ miRNA having a double-stranded, hairpin loop structure to a ′′mature′′ form.
- a mature microRNA sequence is about 19-25 nucleotides in length.
- miR-9′′ is a short non-coding RNA gene involved in gene regulation and highly conserved from Drosophila and mice to humans.
- the mature ⁇ 21nt miRNAs are processed from hairpin precursor sequences by the Dicer enzyme.
- miR-9 can be one of the most highly expressed microRNAs in the developing and adult vertebrate brain.
- Key transcriptional regulators such as FoxGl, Hesl or Tlx, can be direct targets of miR-9, placing it at the core of the gene network controlling the neuronal progenitor state.
- neuron can refer to an electrically excitable cell that can receive, process, and transmit information through electrical signals (e.g., membrane potential discharges) and chemical signals (e.g., synaptic transmission of neurotransmitters) .
- electrical signals e.g., membrane potential discharges
- chemical signals e.g., synaptic transmission of neurotransmitters
- the chemical signals e.g., based on release and recognition of neurotransmitters
- transduced between neurons can occur via specialized connections called synapses.
- a neuron is said to be a mature neuron if it expresses one or more markers of mature neurons, e.g., microtubule-associated protein 2 (MAP2) and neuronal nuclei (NeuN) , neuron specific enolase (NSE) , 160 kDa neurofilament medium, 200 kDa neurofilament heavy, postsynaptic density protein 95 (PDS-95) , synapsin I, synaptophysin, glutamate decarboxylase 67 (GAD67) , glutamate decarboxylase 67 (GAD65) , parvalbumin, dopamine-and cAMP-regulated neuronal phosphoprotein 32 (DARPP32) , vesicular glutamate transporter 1 (vGLUT1) , vesicular glutamate transporter 2 (vGLUT2) , acetylcholine,
- MAP2 microtubule-associated protein 2
- NeuroN neuronal nuclei
- a functional neuron can refer to a neuron that is able to send or receive information through chemical or electrical signals.
- a functional neuron exhibits one or more functional properties of a mature neuron that exists in a normal nervous system, including, but not limited to: excitability (e.g., ability to exhibit action potential, or a rapid rise and subsequent fall in voltage or membrane potential across a cellular membrane) , forming synaptic connections with other neurons, pre-synaptic neurotransmitter release, or post-synaptic response (e.g., excitatory postsynaptic current or inhibitory postsynaptic current) .
- excitability e.g., ability to exhibit action potential, or a rapid rise and subsequent fall in voltage or membrane potential across a cellular membrane
- post-synaptic response e.g., excitatory postsynaptic current or inhibitory postsynaptic current
- a functional neuron is characterized in its expression of one or more markers of functional neurons, including, but not limited to, synapsin, synaptophysin, glutamate decarboxylase 67 (GAD67) , glutamate decarboxylase 67 (GAD65) , parvalbumin, dopamine-and cAMP-regulated neuronal phosphoprotein 32 (DARPP32) , vesicular glutamate transporter 1 (vGLUT1) , vesicular glutamate transporter 2 (vGLUT2) , acetylcholine, tyrosine hydroxylase (TH) , dopamine, vesicular GABA transporter (VGAT) , and gamma-aminobutyric acid (GABA) .
- markers of functional neurons including, but not limited to, synapsin, synaptophysin, glutamate decarboxylase 67 (GAD67) , glutamate decarboxylase 67 (GA
- non-neuronal cell can refer to any type of cell that is not a neuron.
- An exemplary non-neuronal cell is a cell that is of a cellular lineage other than a neuronal lineage (e.g., a hematopoietic lineage) .
- a non-neuronal cell is a cell of neuronal lineage but not a neuron, for example, a glial cell.
- a non-neuronal cell is somatic cell that is not neuron, such as, but not limited to, a glial cell, an adult primary fibroblast, an embryonic fibroblast, an epithelial cell, a melanocyte, a keratinocyte, an adipocyte, a blood cell, a bone marrow stromal cell, a Langerhans cell, a muscle cell, a rectal cell, or a chondrocyte.
- a non-neuronal cell is from a non-neuronal cell line, such as, but not limited to, a glioblastoma cell line, a Hela cell line, a NT2 cell line, an ARPE19 cell line, or a N2A cell line.
- a non-neuronal cell line such as, but not limited to, a glioblastoma cell line, a Hela cell line, a NT2 cell line, an ARPE19 cell line, or a N2A cell line.
- ′′Cell lineage′′ or ′′lineage′′ can denote the developmental history of a tissue or organ from the fertilized embryo.
- ′′Neuronal lineage′′ can refer to the developmental history from a neural stem cell to a mature neuron, including the various stages along this process (also known as neurogenesis) , such as, but not limited to, neural stem cells (neuroepithelial cells, radial glial cells) , neural progenitors (e.g., intermediate neuronal precursors) , neurons, astrocytes, oligodendrocytes, and microglia.
- neural stem cells neuroepithelial cells, radial glial cells
- neural progenitors e.g., intermediate neuronal precursors
- neurons e.g., astrocytes, oligodendrocytes, and microglia.
- the term ′′non-naturally occurring′′ when used in reference to an organism means that the organism has at least one genetic alternation that is not normally found in a naturally occurring strain of the referenced species, including wild-type strains of the referenced species.
- Genetic alterations include, for example, modifications introducing expressible nucleic acids encoding proteins or enzymes, other nucleic acid additions, nucleic acid deletions, nucleic acid substitutions, or other functional disruption of the organism′s genetic material. Such modifications include, for example, coding regions and functional fragments thereof for heterologous or homologous polypeptides for the referenced species. Additional modifications include, for example, non-coding regulatory regions in which the modifications alter expression of a gene or operon. Genetic modifications to nucleic acid molecules encoding enzymes, or functional fragments thereof, can confer a biochemical reaction capability or a metabolic pathway capability to the non-naturally occurring organism that is altered from its naturally occurring state.
- nucleic acid′′ and ′′polynucleotide, ′′ as used interchangeably herein can refer to deoxyribonucleotides or ribonucleotides and polymers thereof in either single-or double-stranded form.
- the term can encompass nucleic acids containing known nucleotide analogs or modified backbone residues or linkages which are synthetic, naturally occurring, or non-naturally occurring; which have similar binding properties as the reference nucleic acid; and which are metabolized in a manner similar to the reference nucleotides.
- Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, locked nucleic acids (LNAs) , and peptide-nucleic acids (PNAs) .
- LNAs locked nucleic acids
- PNAs peptide-nucleic acids
- a ′′nucleic acid construct′′ or ′′nucleic acid vector′′ is herein understood to mean a man-made nucleic acid molecule resulting from the use of recombinant DNA technology.
- the term ′′nucleic acid construct′′ therefore does not include naturally-occurring nucleic acid molecules, although a nucleic acid construct may comprise naturally occurring nucleic acid molecules or fragments thereof.
- a ′′vector′′ is a nucleic acid construct (typically DNA or RNA) that serves to transfer an exogenous nucleic acid sequence (i.e., DNA or RNA) into a host cell.
- a vector is preferably maintained in the host by at least one of: autonomous replication or integration into the host cell′s genome.
- the terms ′′expression vector′′ or ′′expression construct′′ refer to nucleotide sequences that are capable of affecting expression of a gene in host cells or host organisms compatible with such sequences. These expression vectors typically include at least one ′′expression cassette′′ that is the functional unit capable of affecting expression of a sequence encoding a product to be expressed, and wherein the coding sequence is operably linked to the appropriate expression control sequences which at least comprise a suitable transcription regulatory sequence and/or 3′ transcription termination signals. Additional factors necessary for or helpful in affecting expression, such as expression enhancer elements, may also be present.
- the expression vector can be introduced into a suitable host cell and can be able to affect expression of the coding sequence in an in vitro cell culture of the host cell.
- An expression vector can be suitable for expression of viral proteins and/or nucleic acids, particularly recombinant AAV proteins and/or nucleic acids.
- oligodendrocyte can refer to a type of glial cell that can create the myelin sheath that surrounds a neuronal axon to provide support and insulation to axons in the central nervous system.
- An oligodendrocyte can also be characterized in its expression of PDGF receptor alpha (PDGFR- ⁇ ) , SOXIO, neural/glial antigen 2 (NG2) , Olig1, Olig2, Olig3, oligodendrocyte specific protein (OSP) , Myelin basic protein (MBP) , or myelin oligodendrocyte glycoprotein (MOG) .
- PDGFR- ⁇ PDGF receptor alpha
- SOXIO neural/glial antigen 2
- NG2 neural/glial antigen 2
- Olig1, Olig2, Olig3, oligodendrocyte specific protein (OSP) Olig1, Olig2, Olig3, oligodendrocyte specific protein (OSP) , Myelin basic
- PTBP1 belongs to the subfamily of ubiquitously expressed heterogeneous nuclear ribonucleoproteins (hnRNPs) .
- the hnRNPs are RNA-binding proteins that complex with heterogeneous nuclear RNA (hnRNA) . These proteins are associated with pre-mRNAs in the nucleus and appear to influence pre-mRNA processing and other aspects of mRNA metabolism and transport.
- PTB can have four repeats of quasi-RNA recognition motif (RRM) domains that bind RNAs. Consistent with its widespread expression, PTB can contribute to the repression of a large number of alternative splicing events.
- PTB can recognize short RNA motifs, such as UCUU and UCUCU, located within a pyrimidine-rich context and is often associated with the polypyrimidine tract upstream of the 3′ splice site of both constitutive and alternative exons. In some cases, binding sites for PTB can also include exonic sequences and sequences in introns downstream of regulated exons.
- repression can be achieved through the interaction of PTB with multiple PTB binding sites surrounding the alternative exon.
- repression can involve a single PTB binding site.
- Splicing repression by PTB can occur by a direct competition between PTB and U2AF65, which, in turn, can preclude the assembly of the U2 snRNP on the branch point.
- splicing repression by PTB can involve PTB binding sites located on both sides of alternative exons and can result from cooperative interactions between PTB molecules that would loop out the RNA, thereby making the splice sites inaccessible to the splicing machinery.
- Splicing repression by PTB can also involve multimerization of PTB from a high-affinity binding site which can create a repressive wave that covers the alternative exon and prevents its recognition.
- PTB can be widely expressed in non-neuronal cells, while nPTB can be restricted to neurons.
- PTB and nPTB can undergo a programmed switch during neuronal differentiation. For example, during neuronal differentiation, PTB is gradually downregulated at the neuronal induction stage; coincidentally or consequentially, nPTB levels are gradually up-regulated to a peak level. Later, when the neuronal differentiation enters into neuronal maturation stage, nPTB level experiences reduction after its initial rise and then returns to a relatively low level as compared to its peak level during neuronal differentiation, when the cell develops into a mature neuron.
- ′′protein, ′′ ′′peptide, ′′ and ′′polypeptide′′ are used interchangeably, and can refer to an amino acid polymer or a set of two or more interacting or bound amino acid polymers, without reference to a specific mode of action, size, 3-dimensional structure, or origin.
- the term ′′promoter′′ or ′′transcription regulatory sequence′′ refers to a nucleic acid fragment that functions to control the transcription of one or more coding sequences and which is located upstream with respect to the direction of transcription of the transcription initiation site of the coding sequence.
- a promoter can be structurally identified by the presence of a binding site for a DNA-dependent RNA polymerase, transcription initiation sites, and/or any other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, enhancers, and any other sequence or sequences of nucleotides known to one of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter.
- a ′′constitutive′′ promoter is a promoter that is active in most tissues under most environmental and developmental conditions.
- An ′′inducible′′ promoter is a promoter that is environmentally or developmentally regulated, e.g. by the application of a chemical inducer or biological entity.
- the term ′′operably linked′′ refers to a linkage of polynucleotide (or polypeptide) elements in a functional relationship.
- a nucleic acid can be ′′operably linked′′ when it is placed into a functional relationship with another nucleic acid sequence.
- a transcription regulatory sequence can be operably linked to a coding sequence if it affects the transcription of the coding sequence.
- Operably linked can mean that the DNA sequences being linked are contiguous and, where necessary to join two protein encoding regions, contiguous and in reading frame.
- An expression control sequence is ′′operably linked′′ to a nucleotide sequence when the expression control sequence controls and regulates the transcription and/or the translation of the nucleotide sequence.
- an expression control sequence can include promoters, enhancers, internal ribosome entry sites (IRES) , transcription terminators, a start codon in front of a protein-encoding gene, splicing signals for introns, and stop codons.
- IRS internal ribosome entry sites
- ′′reprogramming′′ or ′′trans-differentiation′′ can refer to the generation of a cell of a certain lineage (e.g., a neuronal cell) from a different type of cell (e.g., a fibroblast cell) without an intermediate process of de-differentiating the cell into a cell exhibiting pluripotent stem cell characteristics.
- a cell of a certain lineage e.g., a neuronal cell
- a different type of cell e.g., a fibroblast cell
- ′′Pluripotent′′ can refer to the ability of a cell to form all lineages of the body or soma (i.e., the embryo proper) .
- Exemplary ′′pluripotent stem cells′′ can include embryonic stem cells and induced pluripotent stem cells.
- Sequence identity is herein defined as a relationship between two or more amino acid (polypeptide or protein) sequences and/or two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences.
- identity′′ also means the degree of sequence relatedness between amino acid or nucleic acid sequences, as the case may be, as determined by the match between strings of such sequences.
- Similarity′′ between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide.
- Identity′′ and ′′similarity′′ can be readily calculated by known methods.
- ′′Sequence identity′′ and ′′sequence similarity′′ can be determined by alignment of two peptide or two nucleotide sequences using global or local alignment algorithms, depending on the length of the two sequences. Sequences of similar lengths are preferably aligned using global alignment algorithms (e.g., Needleman Wunsch) which align the sequences optimally over the entire length, while sequences of substantially different lengths are preferably aligned using local alignment algorithms (e.g., Smith Waterman) . Sequences may then be referred to as ′′substantially identical′′ or ′′essentially similar′′ when they (when optimally aligned by, for example, the programs GAP or BESTFIT using default parameters) share at least a certain minimal percentage of sequence identity.
- global alignment algorithms e.g., Needleman Wunsch
- local alignment algorithms e.g., Smith Waterman
- GAP uses the Needleman and Wunsch global alignment algorithm to align two sequences over their entire length (full length) , maximizing the number of matches and minimizing the number of gaps. A global alignment is suitably used to determine sequence identity when the two sequences have similar lengths.
- the default scoring matrix used is nwsgapdna and for proteins the default scoring matrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919) .
- Sequence alignments and scores for percentage sequence identity may be determined using computer programs, such as the GCG Wisconsin Package, Version 10.3, available from Accelrys Inc., 9685 Scranton Road, San Diego, CA 92121-3752 USA, or using open source software, such as the program ′′needle′′ (using the global Needleman Wunsch algorithm) or ′′water′′ (using the local Smith Waterman algorithm) in EmbossWIN version 2.10.0, using the same parameters as for GAP above, or using the default settings (both for ′needle′ and for ′water′ and both for protein and for DNA alignments, the default Gap opening penalty is 10.0 and the default gap extension penalty is 0.5; default scoring matrices are Blossum62 for proteins and DNAFull for DNA) .
- GCG Wisconsin Package Version 10.3, available from Accelrys Inc., 9685 Scranton Road, San Diego, CA 92121-3752 USA
- open source software such as the program ′′needle′′ (using the global Needleman Wunsch algorithm) or ′′water
- nucleic acid and protein sequences of the present invention can be further used as a ′′query sequence′′ to perform a search against public databases to, for example, identify other family members or related sequences.
- search can be performed using the BLASTn and BLASTx programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215: 403-10.
- Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25 (17) : 3389-3402.
- the default parameters of the respective programs e.g., BLASTx and BLASTn
- ′′subject′′ and ′′patient′′ as used interchangeably can refer to, except where indicated, mammals such as humans and non-human primates, as well as rabbits, rats, mice, goats, pigs, and other mammalian species.
- mammals such as humans and non-human primates, as well as rabbits, rats, mice, goats, pigs, and other mammalian species.
- the term does not necessarily indicate that the subject has been diagnosed with a particular disease, but instead can refer to an individual under medical supervision.
- a ′′target sequence′′ denotes an order of nucleotides within a nucleic acid that is to be targeted (e.g., wherein an alteration is to be introduced or to be detected) .
- a ′′target sequence′′ is further to be understood herein as the section within the RNA or DNA molecule that is to be targeted by the gRNA-CAS complex by its complementarity to the ′′guide sequence′′ in the gRNA.
- an antisense oligonucleotide or miRNA is targeted by its complementarity to the ′′target sequence′′ within the RNA or DNA molecule that is to be targeted.
- the target sequence can be an order of nucleotides comprised by a first strand of a DNA duplex.
- mammalian species that can benefit from the disclosed methods and composition can include, but are not limited to, primates, such as apes, chimpanzees, orangutans, and humans.
- a ′′vector′′ is a nucleic acid that can be capable of transporting another nucleic acid into a cell.
- a vector can be capable of directing expression of a protein or proteins encoded by one or more genes, or a microRNA encoded by a polynucleotide, carried by the vector when it is present in the appropriate environment.
- a substance as a medicament as described in this document can also be interpreted as the use of said substance in the manufacture of a medicament.
- a substance is used for treatment or as a medicament, it can also be used for the manufacture of a medicament for treatment.
- Products for use as a medicament described herein can be used in methods of treatments, wherein such methods of treatment comprise the administration of the product for use.
- a ′′viral vector′′ is a viral-derived nucleic acid that can be capable of transporting another nucleic acid into a cell.
- a viral vector can be capable of directing expression of a protein or proteins encoded by one or more genes, or a microRNA encoded by a polynucleotide, carried by the vector when it is present in the appropriate environment.
- examples of viral vectors include, but are not limited to, retroviral, adenoviral, lentiviral and adeno-associated viral vectors.
- compositions for regulating a primate PTBP gene expression comprising: a Cas effector protein, or a nucleic acid encoding the same, and a guide RNA (gRNA) that targets a target sequence, or its complement, of the primate PTBP gene or a transcript thereof, or a nucleic acid encoding the same.
- the composition described herein is capable of suppressing the expression of the primate PTBP gene.
- a composition described herein can comprise a nucleic acid molecule that targets a target sequence or its complement in a primate PTB gene, transcript, or mRNA in order to suppress the expression or activity of PTB in the non-neuronal cell. This suppression allows the non-neural cell to reprogram into a functional neuron.
- the nucleic acid molecule in the composition comprising the composition can be a nucleic acid molecule that targets a target sequence in a primate PTB gene, transcript, or mRNA; or in case of a double-stranded PT, DNA, or RNA sequence, the nucleic acid molecule can target either one of the two complementary strands.
- Targeting of the target sequence (or its complement) in a primate PTB gene, transcript, or mRNA by the nucleic acid molecule is understood to mean that at least a part of the nucleic acid molecule is substantially complementary to the target sequence in the primate PTB nucleic acid, such that it can base pair with the primate PTB nucleic acid, under, for example, physiological conditions, so as to exerts its biological effect, i.e. to suppress the expression or activity of PTB in the non-neuronal cell.
- Target sequences can be identified within a primate or human PTB gene, transcript, or mRNA efficiency target sequence, the targeting of which by the nucleic acid molecules of the invention ensures high efficiency suppression of the expression or activity of PTB in the non-neuronal cell.
- the composition comprises: i) a CRISPR/Cas effector protein and a gRNA complementary to a Polypyrimidine Tract Binding protein (PTB) mRNA or ii) at least one expression vector encoding a CRISPR/Cas effector protein and a guide RNA (gRNA) complementary to a PTB mRNA.
- PTB Polypyrimidine Tract Binding protein
- gRNA guide RNA
- a single composition that suppresses the expression and/or activity of PTB/nPTB in a non-human primate or human non-neuronal cell (e.g., MG cell in mature retina or astrocyte in striatum) can directly convert the non-neuronal cell into a mature neuron (e.g., a retinal ganglion cell (RGC) neuron, a retinal photoreceptor, or dopamine neuron, respectively) .
- a mature neuron e.g., a retinal ganglion cell (RGC) neuron, a retinal photoreceptor, or dopamine neuron, respectively
- the direct conversion of a non-neuronal cell into a neuron by a single composition can mean that the conversion of the non-neuronal cell into the neuron requires no other intervention than contacting with the single composition.
- PTB reduction can induce a number of key neuronal differentiation factors.
- PTB and nPTB can be involved in two separate but intertwined loops, that can be important in neuronal differentiation.
- PTB can suppress a neuronal induction loop in which the microRNA miR-124 can inhibit the transcriptional repressor RE1-Silencing Transcription factor (REST) , which in turn can block the induction of miR-124 and many neuronal-specific genes (loop I) .
- REST transcriptional repressor RE1-Silencing Transcription factor
- nPTB is part of a second loop for neuronal maturation that includes the transcription activator Brn2 and miR-9 (loop II) .
- loop II nPTB can inhibit Brn2 and consequentially can inhibit miR-9, and miR-9 in turn can inhibit nPTB.
- the expression level of miR-9 or Brn2 in a non-neuronal cell can affect the conversion of the non-neuronal cell into a mature neuron by a composition that suppresses the expression or activity of PTB in the non-neuronal cell.
- a human adult fibroblast cell can have a low expression level of miR-9 and Brn2.
- a single agent that suppresses the expression or activity of PTB in a human adult fibroblast cell can induce the human adult fibroblast cell to differentiate into a neuron-like cell, e.g., expression of Tuj1 protein, but not into a mature neuron, e.g., expression of NeuN protein or other markers of a mature neuron.
- the subject method and composition in some embodiments are particularly effective in creating a reinforcing feedback loop in molecular changes that direct the conversion of a non-neuronal cell into a neuron.
- PTB expression or activity is initially downregulated by an exogenous anti-PTB agent, which can in turn lead to upregulation of miR-124 level.
- the upregulated miR-124 can thus reinforce the inhibition of PTB in the cell; such a positive reinforcing effect can be long-lasting, even though in some cases, the anti-PTB agent, e.g., an antisense oligonucleotide against PTB, may be present and active merely temporarily in the cell.
- the anti-PTB agent e.g., an antisense oligonucleotide against PTB
- a single composition e.g., a Cas with PTB/nPTB-targeting gRNA or a polynucleotide encoding the same
- a single composition that suppresses the expression or activity of PTB/nPTB in a human non-neuronal cell can directly convert the non-neuronal cell into a mature neuron, optionally when the human non-neuronal cell expresses miR-9 or Brn2 at a level that is higher than that expressed in a human adult fibroblast.
- An exemplary human non-neuronal cell that can be used in the method provided herein expresses miR-9 or Brn2 at a level that is at least two times higher than that expressed in a human adult fibroblast.
- the human non-neuronal cell expresses miR-9 or Brn2 at a level that is at least about 1.2, 1.5, 2, 5, 10, 15, 20 or 50 times higher than that expressed in a human adult fibroblast.
- a single composition that suppresses the expression or activity of PTB/nPTB (e.g., Cas with PTB/nPTB-targeting gRNA) in a human non-neuronal cell can directly convert the non-neuronal cell into a mature neuron, when the human non-neuronal cell expresses both miR-9 and Brn2 at a level that is higher than that expressed in a human adult fibroblast.
- PTB/nPTB e.g., Cas with PTB/nPTB-targeting gRNA
- An exemplary human non-neuronal cell that can be used in the method as provided herein express both miR-9 and Brn2 at a level that is at least two times higher than that expressed in a human adult fibroblast.
- the human non-neuronal cell expresses both miR-9 and Brn2 at a level that is at least about 1.2, 1.5, 2, 5, 10, 15, 20 or 50 times higher than that expressed in a human adult fibroblast.
- a single composition e.g., Cas with PTB/nPTB-targeting gRNA
- a single composition that suppresses the expression or activity of PTB/nPTB in a human non-neuronal cell can directly convert the non-neuronal cell into a mature neuron, when the human non-neuronal cell expresses endogenous miR-9 or endogenous Brn2 at a level that is higher than that expressed in a human adult fibroblast.
- no exogenous miR-9 is introduced into the human non-neuronal cell.
- no exogenous Brn2 is introduced into the human non-neuronal cell.
- the expression level of miR-9 or Brn 2 in a non-neuronal cell can be assessed by any technique one skilled in the art would appreciate.
- the expression level of miR-9 in a cell can be measured by reverse transcription (RT) -polymerase chain reaction (PCR) , miRNA array, RNA sequencing (RNA-seq) , and multiplex miRNA assays.
- RT reverse transcription
- RNA-seq RNA sequencing
- multiplex miRNA assays multiplex miRNA assays.
- Expression levels of miR-9 can also be assayed by in situ methods like in situ hybridization.
- Expression levels of Brn2 as a protein can be assayed by conventional techniques, like Western blot, enzyme-linked immunosorbent assay (ELISA) , and immunostaining, or by other techniques, such as, but not limited to, protein microarray and spectrometry methods (e.g., high performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC/MS) ) .
- protein microarray and spectrometry methods e.g., high performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC/MS)
- information on the expression level of miR-9 in a cell or a certain type of tissue/cells can be obtained by referring to publicly available databases for microRNAs, such as, but not limited to, Human MiRNA Expression Database (HMED) , miRGator 3.0, miRmine, and PhenomiR.
- HMED Human MiRNA Expression Database
- miRmine miRmine
- PhenomiR Phenom
- information on the expression level of miR-9 in a cell or a certain type of tissue/cells can be obtained by referring to publicly available databases for protein expression, including, but not limited to, The Human Protein Atlas, GeMDBJ Proteomics, Human Proteinpedia, and Kahn Dynamic Proteomics Database.
- a single composition e.g., Cas with PTB-targeting gRNA
- a single composition that suppresses the expression or activity of PTB in the human astrocyte leads to immediate decrease in expression or activity of PTB, an initial increase in expression level of nPTB, and a subsequent decrease in expression level of nPTB.
- a single composition e.g., Cas with PTB-targeting gRNA
- a single composition that suppresses the expression or activity of PTB directly converts a human astrocyte to a mature neuron.
- the expression level of miR-9 or Brn2 in the non-neuronal cell can be correlated with whether or not nPTB expression level in the non-neuronal cell decreases after the initial increase following PTB expression or activity is suppressed by a composition.
- nPTB expression level in the non-neuronal cells decreases after the initial increase following PTB expression or activity is suppressed by a composition, while in human adult fibroblast, as described above, in some cases, the subsequent decrease in nPTB expression level may not happen.
- an exemplary non-neuronal cell that can be reprogrammed into a mature neuron herein can include a glial cell, such as, but not limited, an astrocyte, an oligodendrocyte, an ependymal cell, a microglia, a Muller glia, a spiral ganglion glial cell, a Schwan cell, a NG2 cell, and a satellite cell.
- a glial cell can be a primate glial cell, for instance, a human glial cell or a non-human primate glial cell.
- the glial cell is a primate astrocyte, for instance, a human astrocyte or a non-human primate astrocyte.
- a glial cell that can be used herein is a glial cell isolated from a brain.
- a glial cell is a glial cell in a cell culture, for instance, divided from a parental glial cell.
- a glial cell as provided herein is a glial cell differentiated from a different type of cell under external induction, for instance, differentiated in vitro from a neuronal stem cell in a culture medium containing differentiation factors, or differentiated from an induced pluripotent stem cell.
- a glial cell is a glial cell in a nervous system, for example, a MG cell in the mature retina, or an astrocyte residing in a brain region, such as in the striatum.
- an astrocyte that can be herein is a glial cell that is of a star-shape in brain or spinal cord.
- an astrocyte expresses one or more of well-recognized astrocyte markers, including, but not limited to, glial fibrillary acidic protein (GFAP) and aldehyde dehydrogenase 1 family member LI (ALDH1L1) , excitatory amino acid transporter 1 /glutamate aspartate transporter (EAAT1/GLAST) , glutamine synthetase, S100 beta, or excitatory amino acid transporter 1 /glutamate transporter 1 (EAAT2/GLT-1) .
- GFAP glial fibrillary acidic protein
- ALDH1L1 aldehyde dehydrogenase 1 family member LI
- EAAT1/GLAST excitatory amino acid transporter 1 /glutamate aspartate transporter
- glutamine synthetase glutamine synthetase
- an astrocyte expresses glial fibrillary acidic protein (GFAP) , Aldehyde Dehydrogenase 1 Family Member LI (ALDH1L1) , or both.
- GFAP glial fibrillary acidic protein
- ALDH1L1 Aldehyde Dehydrogenase 1 Family Member LI
- an astrocyte is a non-neuronal cell in a nervous system that expresses a glial fibrillary acidic protein (GFAP) promoter-driven transgene (e.g., red fluorescent protein (RFP) , Cre recombinase) .
- an astrocyte as described herein is not immunopositive for neuronal markers, e.g., Tuj1, NSE, NeuN, GAD67, VGluTl, or TH.
- an astrocyte as described herein is not immunopositive for oligodendrocyte markers, e.g., Oligodendrocyte Transcription Factor 2, OLIG2.
- an astrocyte as described herein is not immunopositive for microglia markers, e.g., transmembrane protein 119 (TMEM119) , CD45, ionized calcium binding adapter molecule 1 (Ibal) , CD68, CD40, F4/80, or CD11 Antigen-Like Family Member B (CDllb) .
- TMEM119 transmembrane protein 119
- Ibal ionized calcium binding adapter molecule 1
- CD68 CD68
- CD40 CD40
- F4/80 CD11 Antigen-Like Family Member B
- CDllb CD11 Antigen-Like Family Member B
- an astrocyte as described herein is not immunopositive for NG2 cell markers (e.g., Neural/glial antigen 2, NG2) .
- an astrocyte as described herein is not immunopositive for neural progenitor markers, e.g., Nestin, CXCR4, Musashi, Notch-1, SRY-Box 1 (SOX1) , SRY-Box 2 (SOX2) , stage-specific embryonic antigen 1 (SSEA-1, also called CD15) , or Vimentin.
- an astrocyte as described herein is not immunopositive for pluripotency markers, e.g., NANOG, octamer-binding transcription factor 4 (Oct-4) , SOX2, Kruppel Like Factor 4 (KLF4) , SSEA-1, or stage-specific embryonic antigen 4 (SSEA-4) .
- an astrocyte as described herein is not immunopositive for fibroblast markers (e.g. Fibronectin) .
- Astrocytes can include different types or classifications. The methods of the invention are applicable to different types of astrocytes.
- Non-limiting example of different types of astrocytes include type 1 astrocyte, which can be Ran2+, GFAP+, fibroblast growth factor receptor 3 positive (FGFR3+) , and A2B5.
- Type 1 astrocytes can arise from the tripotential glial restricted precursor cells (GRP) .
- Type 1 astrocytes may not arise from the bipotential 02A/0PC (oligodendrocyte, type 2 astrocyte precursor) cells.
- Another non limiting example includes type 2 astrocyte, which can be A2B5+, GFAP+, FGFR3-, and Ran2.
- Type 2 astrocytes can develop in vitro from either tripotential GRP or from bipotential 02A cells or in vivo when these progenitor cells are transplanted into lesion sites.
- Astrocytes that can be used in the method provided herein can be further classified based their anatomic phenotypes, for instance, protoplasmic astrocytes that can be found in grey matter and have many branching processes whose end-feet envelop synapses or fibrous astrocytes that can be found in white matter and can have long thin unbranched processes whose end-feet envelop nodes of Ranvier.
- Astrocytes that can be used in the methods provided herein can also include GluT type and GluR type.
- GluT type astrocytes can express glutamate transporters (EAAT1/SLC1A3 and EAAT2/SLC1A2) and respond to synaptic release of glutamate by transporter currents, while GluR type astrocytes can express glutamate receptors (mostly mGluR and AMPA type) and respond to synaptic release of glutamate by channel mediated currents and IP3-dependent Ca2+ transients.
- glutamate transporters EAAT1/SLC1A3 and EAAT2/SLC1A2
- glutamate receptors mostly mGluR and AMPA type
- the nucleic acid molecule targets a target sequence in a primate PTB gene sequence, such as a non-human primate PTB gene sequence or a human PTB gene sequence.
- a primate PTB gene sequence such as a non-human primate PTB gene sequence or a human PTB gene sequence.
- primate PTB gene sequences to be targeted include the human PTBP1 gene sequence (GenBank ID 5725) .
- RNA, siRNA, and guide RNA molecules can be designed and constructed to modulate (e.g., to decrease, to inhibit, or to overexpress) the expression of a primate PTB and/or nPTB.
- the nucleic acid molecule targets a target sequence in the primate PTB gene, transcript, or mRNA sequence that is conserved between humans and non-human primates.
- the nucleic acid molecule e.g. gRNA
- targets a target sequence in a primate PTB mRNA sequence such as a non-human primate PTB mRNA sequence or a human PTB mRNA sequence.
- the nucleic acid molecule e.g. gRNA
- the target sequence in the PTB gene, transcript, or mRNA is comprised in a PTB sequence that is at least 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%identical to SEQ ID NO: 87.
- a targeting the target sequence can cause an efficient suppression of the expression or activity of a primate PTB protein in a non-neuronal cell.
- the target sequence in the PTB gene, transcript, or mRNA is comprised in a PTB sequence that is at least 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%identical to SEQ ID NO: 87, wherein when the target sequence is targeted with a guide RNA (gRNA) , the guide sequence of which has at least 10 nucleotides overlap with at least one of SEQ ID NOs: 1-86, and which is co-expressed with CasRx in at least one of Cos7 and 293T cells, a relative expression of PTB mRNA of less than 0.55, 0.5, 0.45, 0.4, 0.35, 0.3, 0.25, 0.24, 0.23, 0.22, 0.21, 0.20, 0.19, 0.18, 0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02 or 0.015
- the target sequence in the PTB gene, transcript, or mRNA is comprised in a PTB sequence that is at least 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%identical to SEQ ID NO: 87, wherein when the target sequence is targeted with a guide RNA (gRNA) , the guide sequence of which comprises or consists of at least one of SEQ ID NOs: 1-86, and which is co-expressed with CasRx in at least one of Cos7 and 293T cells, a relative expression of PTB mRNA of less than 0.55, 0.5, 0.45, 0.4, 0.35, 0.3, 0.25, 0.24, 0.23, 0.22, 0.21, 0.20, 0.19, 0.18, 0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02 or 0.015 is observed, as compared to
- the target sequence in the PTB gene, transcript, or mRNA can comprise or consist of a contiguous sequence of at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides.
- the target sequence in the PTB gene, transcript, or mRNA can be less than 60, 55, 50, 45, 40, 35, 34, 33, 32, 31, 30, 29, 28, 27 or 26 nucleotides.
- the PTB sequence can be at least 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%identical to SEQ ID NO: 87.
- the target sequence in the PTB gene, transcript, or mRNA can comprise or consist of a contiguous sequence of at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides.
- the target sequence in the PTB gene, transcript, or mRNA can be less than 60, 55, 50, 45, 40, 35, 34, 33, 32, 31, 30, 29, 28, 27 or 26 nucleotides.
- the PTB sequence can be at least 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%identical to positions 951-1487 of SEQ ID NO: 87, wherein, when the target sequence is targeted with a guide RNA (gRNA) , the guide sequence of which comprises or consists of at least one of SEQ ID NOs: 38-68, and which is co-expressed with CasRx in at least one of Cos7 and 293T cells, a relative expression of PTB mRNA of less than 0.50, 0.45, 0.40, 0.35, 0.3, 0.25, 0.24, 0.23, 0.22, 0.21, 0.20, 0.19, 0.18, 0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02 or 0.015 is observed, as compared to a corresponding control cell expressing only Ca
- gRNA guide
- the target sequence in the PTB gene, transcript, or mRNA can comprise or consist of a contiguous sequence of at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides.
- the target sequence in the PTB gene, transcript, or mRNA can be less than 60, 55, 50, 45, 40, 35, 34, 33, 32, 31, 30, 29, 28, 27 or 26 nucleotides.
- the PTB sequence can be at least 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%identical to at least one of: positions 1000-1487 of SEQ ID NO: 87; positions 1050-1487 of SEQ ID NO: 87; positions 1100-1487 of SEQ ID NO: 87; positions 1150-1487 of SEQ ID NO: 87; positions 1200-1487 of SEQ ID NO: 87; positions 1250-1487 of SEQ ID NO: 87; positions 1300-1487 of SEQ ID NO: 87; positions 1350-1487 of SEQ ID NO: 87; positions 1400-1487 of SEQ ID NO: 87; positions 951-1400 of SEQ ID NO: 87; positions 951-1350 of SEQ ID NO: 87; positions 951-1300 of SEQ ID NO: 87; positions 951-1250 of SEQ ID NO: 87; positions 951-1200 of SEQ ID NO: 87; positions 9
- a nucleic acid molecule that targets a target sequence or its complement in a primate PTB gene, transcript or mRNA can be a guide RNA (gRNA) for a CRISPR/Cas family effector protein (Cas effector protein) .
- the composition can comprise a composition comprising: i) a Cas effector protein and the at least one gRNA that targets a target sequence or its complement in a primate PTB gene, transcript, or mRNA, or ii) at least one expression vector encoding a Cas effector protein and encoding the at least one gRNA.
- the invention provides for a composition comprising such compositions.
- the composition (e.g. comprising the Cas effector protein and the at least one gRNA, or the at least one expression vector) is comprised in a nanoparticle, such as a liposome.
- a nanoparticle such as a liposome.
- a liposome is a spherical vesicle having at least one lipid bilayer.
- a liposome can be used as a delivery vehicle for the administration of nanoparticles, nutrients, pharmaceuticals, and other cargo.
- a vector encoding a CRISPR/Cas family effector protein (Cas effector protein) and a guide RNA (gRNA) that targets a PTB gene sequence together encompass the composition.
- Cas effector protein CRISPR/Cas family effector protein
- gRNA guide RNA
- Non-viral transfection methods or viral transduction methods are utilized to introduce the composition.
- Non-viral transfection can refer to all cell transfection methods that are not mediated through a virus.
- Non-limiting examples of non-viral transfection include electroporation, microinjection, calcium phosphate precipitation, transfection with cationic polymers, such as DEAE-dextran followed by polyethylene glycol, transfection with dendrimers, liposome mediated transfection ( ′′lipofection′′ ) , microprojectile bombardment ( ′′gene gun′′ ) , fugene, direct sonic loading, cell squeezing, optical transfection, protoplast fusion, impalefection, magnetofection, nucleofection, and any combination thereof.
- the expression vector used to deliver the composition to the non-neuronal cell can be a gene therapy vector.
- the gene therapy vector can be a viral gene therapy vector, also known as a viral vector.
- viral vector methods can include the use of either DNA or RNA viral vectors. Examples of appropriate viral vectors can include adenovirus, lentivirus, adeno-associated virus (AAV) , poliovirus, poxvirus, herpes simplex virus (HSV) , an SV40, or murine Maloney-based virus vector.
- the vector is an AAV vector.
- a composition is administered in the form of AAV vector.
- a composition is administered in the form of lentiviral vector.
- a composition can be delivered to a non-neuronal cell using a lentivirus or AAV to express a Cas effector protein with gRNA against PTB/nPTB.
- methods provided herein comprise suppressing the expression or activity of PTB/nPTB in a non-neuronal cell (e.g., a glial cell or astrocyte) via a composition of a sufficient amount for reprogramming the non-neuronal cell to a mature neuron.
- the sufficient amount of composition can be determined empirically.
- the amount of composition can be determined by any type of assay that examines the activity of the composition in the non-neuronal cell.
- the sufficient amount of the composition can be determined by assessing the expression level of PTB/nPTB in an exemplary non-neuronal cell after administration of the agent, e.g., by Western blot.
- functional assays are utilized for assessing the activity of PTB/nPTB after delivery of the composition to an exemplary non-neuronal cell.
- other functional assays such as, immunostaining for neuronal markers or electrical recording for neuronal functional properties that examine downstream neuronal properties are used to determine a sufficient amount of composition.
- the composition is delivered in the form of a viral vector.
- a viral vector can comprise one or more copies of expression sequence coding for a composition; for example, a Cas effector protein can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more copies of a coding sequence for a gRNA against PTB/nPTB.
- a viral vector can be tittered to any appropriate amount for administration.
- the titer as determined by PCR, RT-PCR, or other methods can be at least about 105 viral particles/mL, 106 particles /mL, 107 particles /mL, 108 particles /mL, 109 particles /mL, 1010 particles/mL, 1011 particles /mL, 1012 particles/mL, 1013 particles /mL, 1014 particles/mL, or 1015 particles/mL.
- the titer of viral vector to be administered is at least about 1010 particles/mL, 1011 particles /mL, 1012 particles/mL, 1013 particles /mL, or 1014 particles/mL.
- the Cas effector protein in the composition is selected from the group consisting of: Cas13d, CasRx, Cas13X, CRISPR/Cas9, Cpf1, Cas9, Cas13a, Cas13b, Cas13c, Cas13Y and a functional domain thereof.
- the Cas effector protein is encoded by an ORF (from start codon to stop codon) of 4.5 kb or less, 4 kb or less, 3.5 kb or less, 3 kb or less, 2.5 kb or less, or 2.1 kb or less, or 1.5 kb or less.
- the Cas effector protein is modified to comprise a nuclear localization signal.
- the Cas effector protein is a DNA-targeting Cas effector protein.
- the Cas effector protein is a DNA-targeting Cas effector protein selected from the group consisting of: spCas9 or its variant, SaCas9 or its variant, Cpf1 or its variant, or a combination thereof.
- the Cas effector protein in the composition is an RNA-targeting Cas effector protein.
- An RNA-targeting Cas effector protein is herein understood as a Cas effector protein that uses its crRNA/gRNA to recognize and degrade target RNA sequences, rather than target DNA sequences.
- the Cas effector protein is the effector protein of a Type VI CRISPR-Cas system.
- the Cas effector protein contains 2 HEPN ribonuclease motifs, containing the RXXXXH-motif (see Anantharaman et al., 2013, Biol Direct. 2013; 8: 15) .
- the RNA-targeting Cas effector protein is selected from the group consisting of: Cas13a, Cas13b, Cas13c, Cas13d /CasRx, CRISPR/Cas9, Cpf1, Cas13X and Cas13Y and a functional domain thereof.
- the RNA-targeting Cas effector protein is encoded by an ORF (from start codon to stop codon) of 4.5 kb or less, 4 kb or less, 3.5 kb or less, 3 kb or less, 2.5 kb or less, or 2.1 kb or less, or 1.5 kb or less.
- the RNA-targeting Cas effector protein is modified to comprise a nuclear location signal.
- Cas13a previously known as C2c2; Cas13b; Cas13c; and Cas13d, also known as CasRx, are Class 2, Type VI effector proteins which introduce cleavage of single stranded and collateral RNA.
- Cas13X and Cas13Y are compact Cas enzymes which can tolerate single-nucleotide mismatches in RNA recognition.
- CasRx is an ortholog of Cas13d, which has the smallest size and exhibits high targeting specificity and efficiency, making it an option for in vivo therapeutic applications.
- a gRNA that targets a primate PTB mRNA sequence is used in a composition of the invention in combination with an RNA-targeting CRISPR/Cas family effector protein.
- the gRNA comprises a sequence that is complementary to a target sequence in a primate PTB mRNA, such as the human PTBP1 coding sequence (NM_002819; SEQ ID NO: 87) , preferably, the gRNA comprises a sequence that is complementary to a target sequence as herein defined above.
- the gRNA comprises a guide sequence that is complementary to a contiguous stretch of 14-60 nucleotides in a primate PTB mRNA sequence, such as SEQ ID NO: 87.
- the gRNA can comprise a guide sequence that is complementary to a contiguous stretch of at least 17 and no more than 60 nucleotides in a primate PTB mRNA sequence, such as SEQ ID NO: 87.
- the gRNA comprises a guide sequence that is complementary to a contiguous stretch of at least 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in a primate PTB mRNA sequence, such as SEQ ID NO: 87, and/or no more than 60, 55, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41 or 40 nucleotides in a primate PTB mRNA sequence, such as SEQ ID NO: 87.
- the gRNA comprises a guide sequence that is complementary to a contiguous stretch of 25-45 nucleotides in a primate PTB mRNA sequence, such as SEQ ID NO: 87.
- the guide sequence in the gRNA comprises or consists of at least 10, 11, 12, 13, 14, 15, 16, 17 contiguous nucleotides, or of all nucleotides of at least one of SEQ ID NOs: 1-86.
- the guide sequence in the gRNA comprises or consists of at least 10, 11, 12, 13, 14, 15, 16, 17 contiguous nucleotides, or of all nucleotides of at least one of SEQ ID NOs: 1-86, wherein when the gRNA is co-expressed with CasRx in at least one of Cos7 and 293T cells, a relative expression of PTB mRNA of less than 0.5, 0.45, 0.4, 0.35, 0.3, 0.25, 0.24, 0.23, 0.22, 0.21, 0.20, 0.19, 0.18, 0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02 or 0.015 is observed, as compared to a corresponding control cell expressing only CasRx.
- the guide sequence in the gRNA comprises or consists of at least one of SEQ ID NOs: 1-86. In one embodiment, the guide sequence in the gRNA comprises or consists of at least one of SEQ ID NOs: 1-86 and preferably produces a relative expression as determined in Table 1, that is no more than 0.5, 0.45, 0.4, 0.35, 0.3, 0.25, 0.24, 0.23, 0.22, 0.21, 0.20, 0.19, 0.18, 0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02 or 0.015.
- the guide sequence in the gRNA comprises or consists of at least one sequence selected from SEQ ID NOs: 56, 60, 47, 48, 43, 45, 40, 61, 50, 55, 41, 44, 42, 66, 49, 51, 46, 79, 07, 58, 53, 38, 64, 63, 59, 54, 62, 81, 83, 57, 05, 08, 70, 73, 76, 10, 04, 36, 52, 26, 67, 25, 39, 34, 80, 06, 23, 85, 24, 30, 02, 13, 03, 77, 31, 21, 69, 16, 75, 12, 78, 20, 74, 71, and 37.
- the guide sequence in the gRNA comprises or consists of at least one sequence selected from SEQ ID NOs: 56, 60, 47, 48, 43, 45, 40, 61, 50, 55, 41, 44, 42, 66, 49, 51, 46, 79, 07, 58, 53, 38, 64, 63, 59, 54, 62, 81, 83, 57, 05, 08, 70, 73, 76, 10, 04, 36, 52, 26, 67, 25, 39, 34, 80, 06, 23, 85, 24, 30, and 02.
- the guide sequence in the gRNA comprises or consists of at least one sequence selected from SEQ ID NOs: 56, 60, 47, 48, 43, 45, 40, 61, 50, 55, 41, 44, 42, 66, 49, 51, 46, 79, 07, 58, 53, 38, 64, 63, 59, 54, 62, 81, 83, 57, 05, 08, 70, 73, 76, 10, 04, 36, 52, 26, 67, and 25.
- the guide sequence in the gRNA comprises or consists of at least one sequence selected from SEQ ID NOs: 56, 60, 47, 48, 43, 45, 40, 61, 50, 55, 41, 44, 42, 66, 49, 51, 46, 79, 07, 58, 53, 38, 64, 63, 59, 54, 62, 81, 83, 57, 05, 08, 70, 73, and 76.
- the guide sequence in the gRNA comprises or consists of at least one sequence selected from SEQ ID NOs: 56, 60, 47, 48, 43, 45, 40, 61, 50, 55, 41, 44, 42, 66, 49, 51, 46, 79, 07, 58, 53, 38, 64, 63, and 59. In one embodiment, the guide sequence in the gRNA comprises or consists of at least one sequence selected from SEQ ID NOs: 56, 60, 47, 48, 43, 45, 40, 61, 50, 55, 41, 44, 42, 66, 49, 51, and 46.
- the guide sequence in the gRNA comprises or consists of at least one sequence selected from SEQ ID NOs: 56, 60, 47, 48, 43, 45, 40, 61, 50, 55, 41, 44, 42, 66, and 49. In one embodiment, the guide sequence in the gRNA comprises or consists of at least one sequence selected from SEQ ID NOs: 56, 60, 47, 48, 43, 45, 40, and 61. In one embodiment, the guide sequence in the gRNA comprises or consists of at least one sequence selected from SEQ ID NOs: 56, 60, 47, 48, 43, 45, and 40.
- the guide sequence in the gRNA comprises or consists of at least one sequence selected from SEQ ID NOs: 56, 60, 47, 48, 43, and 45. In one embodiment, the guide sequence in the gRNA comprises or consists of at least one sequence selected from SEQ ID NOs: 56, 60, 47, 48, and 43. In one embodiment, the guide sequence in the gRNA comprises or consists of at least one sequence selected from SEQ ID NOs: 56, 60, 47, and 48. In one embodiment, the guide sequence in the gRNA comprises or consists of at least one sequence selected from SEQ ID NOs: 56, 60, and 47. In one embodiment, the guide sequence in the gRNA comprises or consists of at least one sequence selected from SEQ ID NOs: 56 and 60.
- the guide sequence of the gRNA comprises or consists of at least one sequence selected from the SEQ ID NOs: 1-86. In another embodiment, the guide sequence of the gRNA comprises or consists of at least one sequence selected from the SEQ ID NO: 89-112.
- the gRNA targets a sequence in a primate PTB mRNA that corresponds to a sequence selected from the group consisting of: a) positions 59-91 of SEQ ID NO: 87; b) positions 303-349 of SEQ ID NO: 87; c) positions 422-451 of SEQ ID NO: 87; d) positions 460-489 of SEQ ID NO: 87; e) positions 542-576 of SEQ ID NO: 87; f) positions 646-681 of SEQ ID NO: 87; g) positions 706-769 of SEQ ID NO: 87; h) positions 773-806 of SEQ ID NO: 87; i) positions 1079-1139 of SEQ ID NO: 87; j) positions 1152-1184 of SEQ ID NO: 87; k) positions 1191-1254 of SEQ ID NO: 87; l) positions 1374-1434 of SEQ ID NO: 87; m) positions 951-1487 of SEQ ID NO: 87;
- a sequence in a primate PTB mRNA (other than SEQ ID NO: 87) that corresponds to a sequence in positions with respect to SEQ ID NO: 87, is a sequence that corresponds to those positions in SEQ ID NO: 87 in a nucleotide sequence alignment, preferably using a global Needleman Wunsch algorithm using the default settings (default Gap opening penalty is 10.0 and the default gap extension penalty is 0.5; default scoring matrix DNAFull) .
- the gRNA comprises or consists of a guide sequence that is complementary to a contiguous stretch of at least 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides, and/or no more than 60, 55, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, or 40 nucleotides in a primate PTB mRNA sequence that is at least 95, 96, 97, 98, 99, or 100%identical to positions 951-1487 of SEQ ID NO: 87, and wherein preferably, when the target sequence is targeted with a guide RNA (gRNA) , the guide sequence of which comprises or consists of at least one of SEQ ID NOs: 38-68, that is co-expressed with CasRx in at least one of Cos7 and 293T cells, a relative expression of PTB mRNA of less than 0.50, 0.45, 0.40, 0.35, 0.3, 0.25, 0.24, 0.23, 0.22, 0.21, 0.20
- the gRNA comprises or consists of a guide sequence that is complementary to a contiguous stretch of at least 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides, and/or no more than 60, 55, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, or 40 nucleotides in a primate PTB mRNA sequence that is at least 95, 96, 97, 98, 99, or 100%identical to at least one of: positions 1000-1487 of SEQ ID NO: 87; positions 1050-1487 of SEQ ID NO: 87; positions 1100-1487 of SEQ ID NO: 87; positions 1150-1487 of SEQ ID NO: 87; positions 1200-1487 of SEQ ID NO: 87; positions 1250-1487 of SEQ ID NO: 87; positions 1300-1487 of SEQ ID NO: 87; positions 1350-1487 of SEQ ID NO: 87; positions 1400-1487 of SEQ ID NO: 87; positions 951-1
- co-expression of a gRNA of the invention together with CasRx in a human or non-human primate cell causes a relative expression of PTB mRNA of no more than 0.9, 0.85, 0.8, 0.75, 0.7, 0.65, 0.6, 0.55, 0.5, 0.45, 0.4, 0.35, 0.3, 0.25, 0.24, 0.23, 0.22, 0.21, 0.20, 0.19, 0.18, 0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11, 0.10, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, or 0.01 as compared to a corresponding control cell expressing only CasRx.
- a gRNA of the invention can be assayed as described in the examples herein.
- transient co-transfection can be performed with 4 ⁇ g vectors expressing CAG-CasRx-P2A-GFP and 2 ⁇ g U6-gRNA-CMV-mCherry plasmid using Lipofectamine 3000 (or similar transfection reagent) .
- Cells transfected with only CAG-CasRx-P2A-GFP plasmid can be used as a control.
- GFP top 20% Two days after transient transfection, around 30K GFP and mCherry double-positive (GFP top 20%) cells are collected by fluorescence activated cell sorting (FACS) and lysed for qPCR analysis to determine the relative expression of PTB mRNA compared to a corresponding number of controls cells (sorted for GFP top 20%only) .
- FACS fluorescence activated cell sorting
- a composition e.g., Cas with PTB-targeting gRNA or polynucleotide encoding the same
- a composition e.g., Cas with nPTB-targeting gRNA or polynucleotide encoding the same
- a composition as provided herein directly suppress the expression level of PTB/nPTB, e.g., suppressing the transcription, translation, or protein stability of PTB and/or nPTB.
- composition as provided herein directly effects on the expression or activity of PTB/nPTB, without affecting other cellular signaling pathway.
- the composition comprising the composition comprises at least one gRNA that targets a primate PTB mRNA sequence or the at least one expression vector encodes at least one gRNA that targets a primate PTB mRNA sequence.
- the composition comprising the composition comprises no more than one gRNA that targets a primate PTB mRNA sequence or the at least one expression vector encodes no more than one gRNA that targets a primate PTB mRNA sequence.
- the composition comprising the composition comprises two, three, four five or six different gRNAs that target a primate PTB mRNA sequence or the at least one expression vector encodes two, three, four, five, or six different gRNAs that target a primate PTB mRNA sequence.
- contacting the non-neuronal cell with a composition comprising a composition as provided herein can be performed in any appropriate manner, depending on the type of non-neuronal cell to be reprogrammed, the environment in which the non-neuronal cell resides, and the desired cell reprogramming outcome.
- the non-neuronal cell is contacted with a composition comprising a composition as provided herein in the form of a polynucleotide encoding the encoding the Cas with PTB/nPTB-targeting gRNA.
- the non-neuronal cell is contacted with a composition comprising a composition as provided herein in the form of at least one expression vector encoding a Cas effector protein and encoding a gRNA that targets a primate PTB mRNA sequence.
- the at least one expression vector can comprise a nucleotide sequence encoding a Cas effector protein that is operably linked to a promoter that causes expression of the Cas effector protein in non-neuronal cell.
- the promoter that causes expression in non-neuronal cell can be a glial cell-specific promoter such as a GFAP promoter, an ALDH1L1 promoter, an EAAT1/GLAST promoter, a glutamine synthetase promoter, a S100 beta promoter, or an EAAT2/GLT1 promoter.
- the glial cell-specific promoter can be a Müller glia (MG) cell-specific promoter.
- the MG cell-specific promoter is selected from the group consisting of: the GFAP promoter, the ALDH1L1 promoter, GLAST (also known as Slc1a3) promoter and the Rlbp1 promoter.
- the glial cell-specific promoter or Müller glia (MG) cell-specific promoter can be a primate, human, or non-human primate promoter.
- a GFAP is a glial fibrillary acid protein, which is an intermediate filament protein found almost exclusively in astrocytes.
- ALDH1L1 or aldehyde dehydrogenase 1 family member 1
- EAAT1/GLAST/Slc1a3 also known as excitatory amino acid transporter 1
- Glutamine synthetase found in astrocytes, is the only enzyme capable of converting glutamate and ammonia to glutamine in the mammalian brain.
- S100 beta is a multifunctional protein found in large amounts in astrocytes.
- EAAT2/GLT1/Slc1a2 is a glutamate transporter.
- Rlbp1 also known as retinaldehyde-binding protein 1
- the at least one expression vector comprises at least one nucleotide sequence encoding a gRNA that targets a primate PTB mRNA sequence, which nucleotide sequence is operably linked to a promoter that causes expression of the gRNA in the non-neuronal cell.
- the promoter that is operably linked to a gRNA coding sequence (and that causes expression of the gRNA in the non-neuronal cell) can be a promoter from a U6 snRNA gene, such as a primate, human, or non-human primate U6 promoter.
- a nucleotide sequence encoding the Cas effector protein can be adapted to optimize its codon usage to that of the primate, human, or non-human primate non-neuronal host cell.
- the adaptiveness of a nucleotide sequence encoding a polypeptide to the codon usage of a host cell may be expressed as codon adaptation index (CAI) .
- CAI codon adaptation index
- the codon adaptation index is herein defined as a measurement of the relative adaptiveness of the codon usage of a gene towards the codon usage of highly expressed genes in a particular host cell or organism.
- the relative adaptiveness (w) of each codon is the ratio of the usage of each codon, to that of the most abundant codon for the same amino acid.
- CAI index is defined as the geometric mean of these relative adaptiveness values. Non-synonymous codons and termination codons (dependent on genetic code) are excluded. CAI values range from 0 to 1, with higher values indicating a higher proportion of the most abundant codons (see Sharp and Li, 1987, Nucleic Acids Research 15: 1281-1295; also see: Jansen et al., 2003, Nucleic Acids Res. 31 (8) : 2242-51) .
- An adapted nucleotide sequence encoding the Cas effector protein preferably has a CAI of at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9.
- Codon optimization methods for optimum gene expression in heterologous organisms are known in the art and have been previously described (see e.g., Welch et al., 2009, PLoS One 4: e7002; Gustafsson et al., 2004, Trends Biotechnol. 22: 346-353; Wu et al., 2007, Nucl. Acids Res. 35: D76-79; Villalobos et al., 2006, BMC Bioinformatics 7: 285; U.S. Patent Publication 2011/0111413; and U.S. Patent Publication 2008/0292918) .
- a composition comprising the PTB-targeting composition as defined above can comprise additional components, such as one or more non-PTB-targeting compositions, to further increase neuron conversion efficiency.
- additional components such as one or more non-PTB-targeting compositions
- the combination of the PTB-targeting compositions with one or more non-PTB-targeting compositions can act synergistically in increasing neuron conversion efficiency.
- a composition comprising a PTB-targeting composition as defined above can further comprise i) one or more dopamine neuron-associated factors, and/or ii) at least one expression vector for expression of one or more dopamine neuron-associated factors, preferably in a non-neuronal cell.
- the composition can be administered to a to a non-neuronal cell in the striatum for generating a functional dopaminergic neuron, whereby the composition can be administered to a glial cell in the striatum for generating a functional dopaminergic neuron.
- the one or more dopamine neuron-associated factors are selected from the group consisting of: Lmx1a, Lmx1b, FoxA2, Nurr1, Pitx3, Gata2, Gata3, FGF8, BMP, En1, En2, PET1, a Pax family protein, SHH, a Wnt family protein, and a TGF- ⁇ family protein. In one embodiment, the one or more dopamine neuron-associated factors are selected from the group consisting of: FoxA2, Lmx1a, and Nurr1.
- the one or more dopamine neuron-associated factors are FoxA2 alone, Lmx1a alone, Nurr1 alone, the combination of FoxA2 and Lmx1a, the combination of FoxA2 and Nurr1, the combination of Nurr1 and Lmx1a, or the combination of FoxA2, Lmx1a and Nurr1.
- Suitable primate and human amino acid and/or nucleotide sequences of these dopamine neuron-associated factors/genes can be accessed in publicly available databases. Construction and delivery of expression vectors for the expression of the one or more dopamine neuron-associated factors in the non-neuronal cell can be as described herein above for expression vectors of the PTB-targeting composition.
- a composition comprising a PTB-targeting composition as defined above further comprises i) one or more factors selected from ⁇ -catenin, OSK factors (also known as Oct4, Sox2, Klf4, and other factors involved in the cell re-cycling or epigenetic remodeling) , Crx, Brn3a, Brn3b, Math5, Nr2e3, and Nrl, and/or ii) at least one expression vector for expression of one or more factors selected from ⁇ -catenin, OSK factors (also known as Oct4, Sox2, Klf4 and other factors involved in the cell re-cycling or epigenetic remodeling) , Crx, Brn3a, Brn3b, Math5, Nr2e3, and Nrl, preferably in a non-neuronal cell.
- OSK factors also known as Oct4, Sox2, Klf4, and other factors involved in the cell re-cycling or epigenetic remodeling
- Crx also known as Oct4, Sox2, Klf4 and other factors involved in the cell re-cycling
- the one or more factors are selected from the group consisting of: ⁇ -catenin and the OSK factors, Oct4, Sox2, and Klf4.
- the one or more factors are a combination of ⁇ -catenin and Oct4, a combination of ⁇ -catenin and Sox2, a combination of ⁇ -catenin and Klf4, a combination of ⁇ -catenin and Oct4 with Sox2, a combination of ⁇ -catenin and Oct4 with Klf4, a combination of ⁇ -catenin and Sox2 with Klf4, a combination of ⁇ -catenin and all three OSK factors, Oct4, Sox2 and Klf4, or a combination of all three OSK factors, Oct4, Sox2 and Klf4 (without ⁇ -catenin) .
- such a composition comprising both the PTB-targeting composition and the factor is used for local administration to a non-neuronal cell in a mature retina. More preferably, the composition is administered to a to a non-neuronal cell in the mature retina for generating a functional retinal ganglion cell (RGC) neuron and/or a functional retinal photoreceptor, whereby it is further preferred that the composition is administered to a glial cell or Müller glia (MG) cell in the mature retina for generating a functional retinal ganglion cell (RGC) neuron and/or a functional retinal photoreceptor.
- the factor is ⁇ -catenin.
- Suitable primate and human amino acid and/or nucleotide sequences of ⁇ -catenin, Oct4, Sox2, Klf4, Crx, Brn3a, Brn3b, Math5, Nr2e3, and Nrl or their genes are known to the skilled person and can be accessed in publicly available databases. Construction and delivery of expression vectors for the expression of the one or more factors selected from ⁇ -catenin, Oct4, Sox2, Klf4, Crx, Brn3a, Brn3b, Math5, Nr2e3, and Nrl in the non-neuronal cell can be as described herein above for expression vectors of the PTB-targeting composition. Alternatively, a composition can have no co-factors expressed.
- the invention relates to a method of treating a neurological condition associated with degeneration of functional neurons in a region in the nervous system of a non-human primate or a human.
- the method can comprise the administration to a subject in need thereof an effective amount of a composition.
- one aspect of the invention provides methods of reprogramming a non-neuronal cell to a mature neuron.
- An exemplary method comprises providing a non-neuronal cell and contacting the non-neuronal cell with a composition comprising a composition (such as CRISPR/Cas effector with guide RNA (gRNA) or polynucleotide encoding the same) that suppresses expression and/or activity of PTB and/or nPTB in the non-neuronal cell, thereby reprogramming the non-neuronal cell to a mature neuron.
- gRNA CRISPR/Cas effector with guide RNA
- the methods and compositions not only convert cells in vitro but also directly in vivo in nervous system (such as in striatum, retina, inner ear, and spinal cord) .
- the invention pertains to a method of treating a neurological condition associated with degeneration of functional neurons in a region in the nervous system of a non-human primate or a human, the method comprising the administration to a non-neuronal cell in the region in the nervous system of a subject in need thereof, an effective amount of a composition comprising i) an RNA-targeting Cas effector protein and a guide RNA (gRNA) that targets a primate PTB mRNA sequence or ii) at least one expression vector encoding an RNA-targeting Cas effector protein and encoding a gRNA that targets a primate PTB mRNA sequence, to suppress the expression or activity of PTB in the non-neuronal cell, thereby allowing the non-neural cell to reprogram into a functional neuron.
- a composition comprising i) an RNA-targeting Cas effector protein and a guide RNA (gRNA) that targets a primate PTB mRNA sequence or ii) at least one expression
- the disclosure provides a method of reprogramming an astrocyte to a mature neuron.
- An exemplary method comprises providing the astrocyte to be reprogrammed and contacting the astrocyte with a composition comprising a composition (e.g., Cas with PTB/nPTB-targeting gRNA or polynucleotide encoding the same) that suppresses the expression or activity of PTB in the astrocyte for at least 1 day, thereby reprogramming the astrocyte to a mature neuron such as a dopamine neuron.
- a composition e.g., Cas with PTB/nPTB-targeting gRNA or polynucleotide encoding the same
- a single composition e.g., Cas with PTB-targeting gRNA
- a single composition that suppresses the expression or activity of PTB in an astrocyte can directly convert the astrocyte into a neuron such as a dopamine neuron.
- the astrocyte is in striatum.
- the invention provides a method of reprogramming an MG cell (e.g., one in mature retina) to an RGC neuron.
- An exemplary method comprises providing the MG cell to be reprogrammed and contacting the MG cell with a composition comprising a composition (e.g., Cas with PTB/nPTB-targeting gRNA or polynucleotide encoding the same) that suppresses the expression or activity of PTB and/or nPTB in the MG cell for at least 1 day, thereby reprogramming the MG cell to an RGC neuron.
- a composition e.g., Cas with PTB/nPTB-targeting gRNA or polynucleotide encoding the same
- a single composition e.g., Cas with PTB-targeting gRNA
- a single composition that suppresses the expression or activity of PTB in an MG cell can directly convert the MG cell into an RGC neuron.
- the MG cell is in mature retina.
- an exemplary method comprises providing a human non-neuronal cell to be reprogrammed; and contacting the human non-neuronal cell with a composition comprising a single composition (e.g., a Cas with PTB/nPTB-targeting gRNA) that yields a decrease in expression or activity of PTB in the human non-neuronal cell, and a decrease of expression or activity of nPTB after the expression or activity of PTB is decreased.
- the composition can lead to a sequential event as to the expression or activity levels of PTB and nPTB in a certain type of non-neuronal cell, e.g., human non-neuronal cell, e.g., human glial cell.
- the direct effect of contacting with the composition is a decrease of expression or activity of PTB in the non-neuronal cell.
- the decrease of expression or activity of PTB in the non-neuronal cell accompanies an initial increase of nPTB expression level in the non-neuronal cell.
- an initial nPTB expression level increases to a high nPTB expression level as expression or activity of PTB is suppressed.
- following the initial increase nPTB expression decreases from the high nPTB expression level to a low nPTB expression level.
- the low nPTB expression level is still higher than the initial nPTB expression level after expression or activity of PTB is suppressed.
- the nPTB expression level decreases after the initial increase spontaneously without external intervention other than the composition that suppresses the expression or activity of PTB.
- the subsequent decrease of nPTB expression level in the non-neuronal cell after PTB expression or activity can be decreased by the composition can be correlated with the direct conversion of the non-neuronal cell to a mature neuron by the composition.
- a single composition e.g., a Cas with PTB-targeting gRNA
- a single composition that suppresses the expression or activity of PTB does not induce the sequential event as described above in a human adult fibroblast cell, e.g., nPTB can experience the initial rise in expression level, but no subsequent decrease to a certain low level.
- the methods provided herein comprise reprogramming a plurality of non-neuronal cells into mature neurons at a high efficiency.
- At least 40%of the astrocytes are converted to mature neurons that are Map2 positive. In some embodiments, at least about 20%, 25%, 30%, 35%, 38%, 40%, 42%, 44%, 46%, 48%, 50%, 52%, 54%, 56%, 58%, 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, or 100%of the MG cells and/or astrocytes are converted to mature neurons that are positive for NeuN or Map2.
- At least 10 MG cells are converted to RGCs which express Brn3a or Rbpms in retinal ganglion cell layer (GCL) per 10 mm X 50 ⁇ m.
- at least about 1, 2, 4, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, or 60 MG cells are converted to RGCs which express Brn3a or Rbpms in retinal ganglion cell layer (GCL) per 10 mm X 50 ⁇ m.
- the methods comprise reprogramming astrocytes into mature neurons, and at least 40%, at least 60%, or at least 80%of the astrocytes are converted to mature neurons that are Map2 or NeuN positive. In some embodiments, at least 20%, at least 40%or at least 60%of the human astrocytes are converted to mature neurons that are Map2 or NeuN positive.
- the methods as provided herein comprise reprogramming a plurality of non-neuronal cells, e.g., human non-neuronal cells, e.g., human glial cells, or astrocytes, and at least about 20%, 25%, 30%, 35%, 38%, 40%, 42%, 44%, 46%, 48%, 50%, 52%, 54%, 56%, 58%, 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, or 99%of the non-neuronal cells, e.g., human non-neuronal cells, e.g., human glial cells, or astrocytes are reprogrammed to mature neurons.
- non-neuronal cells e.g., human non-neuronal cells, e.g., human glial cells, or astrocytes are reprogrammed
- the methods as provided herein reprogram about 20%, 25%, 30%, 35%, 38%, 40%, 42%, 44%, 46%, 48%, 50%, 52%, 54%, 56%, 58%, 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 92%94%, 96%98%99%, or 100%of the non-neuronal cells, e.g., human non-neuronal cells, e.g., human glial cells, MG cells, or astrocytes are reprogrammed to mature neurons.
- non-neuronal cells e.g., human non-neuronal cells, e.g., human glial cells, MG cells, or astrocytes are reprogrammed to mature neurons.
- a mature neuron is characterized by its expression of one or more neuronal markers selected from the group consisting of NeuN (neuronal nuclei antigen) , Map2 (microtubule-associated protein 2) , NSE (neuron specific enolase) , 160 kDa neurofilament medium, 200 kDa neurofilament heavy, PDS-95 (postsynaptic density protein 95) , Synapsin I, Synaptophysin, GAD67 (glutamate decarboxylase 67) , GAD65 (glutamate decarboxylase 67) , parvalbumin, DARPP32 (dopamine-and cAMP-regulated neuronal phosphoprotein 32) , vGLUT1 (vesicular glutamate transporter 1) , vGLUT2 (vesicular glutamate transporter 2) , acetylcholine, vesicular GABA transporter (VGAT) , and gamma-amin
- MG cells are converted into retinal photoreceptors characterized by the expression of one or more rod cell markers selected from rhodopsin and GNAT1, and/or characterized by expression of one or more cone cell markers selected from S-opsin, M-opsin, and mCAR.
- the expression of the markers mentioned above can be assessed. For example, immunostaining using antibodies against specific cell type markers as described herein can reveal whether or not the cell of interest expresses the corresponding cell type marker. Immunostaining under certain conditions can also uncover the subcellular distribution of the cell type marker, which can also be important for determining the developmental stage of the cell of interest. For instance, expression of Map2 can be found in various neurites (e.g., dendrites) in a post-mitotic mature neuron, but which absent in axon of the neuron. Expression of voltage-gated sodium channels (e.g., a subunits Navi.
- voltage-gated sodium channels e.g., a subunits Navi.
- b subunits can be another example because they can be clustered in a mature neuron at axon initial segment, where an action potential can be initiated, and/or in the Node of Ranvier.
- other techniques such as, but not limited to, flow cytometry, mass spectrometry, in situ hybridization, RT-PCR, and microarrays, can also be used for assessing expression of specific cell type markers.
- Certain aspects of the present disclosure provide methods that comprise reprogramming a plurality of non-neuronal cells, and at least about 20%, 25%, 30%, 35%, 38%, 40%, 42%, 44%, 46%, 48%, 50%, 52%, 54%, 56%, 58%, 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, or 99%of the non-neuronal cells, e.g., glial cells, MG cells, or astrocytes are reprogrammed to functional neurons.
- the non-neuronal cells e.g., glial cells, MG cells, or astrocytes are reprogrammed to functional neurons.
- the methods provided herein reprogram at least 20%of the non-neuronal cells, e.g., glial cells, MG cells, or astrocytes, are reprogrammed to functional neurons. In some embodiments, the methods provided herein reprogram about 20%, 25%, 30%, 35%, 38%, 40%, 42%, 44%, 46%, 48%, 50%, 52%, 54%, 56%, 58%, 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%92%94%, 96%, 98%, 99%, or 100%of the non-neuronal cells, e.g., glial cells, MG cells, or astrocytes, are reprogrammed to functional neurons.
- the non-neuronal cells e.g., glial cells, MG cells, or astrocytes
- the methods provided herein comprise the use of a combination of i) a PTB-targeting composition as defined herein, and ii) one or more non-PTB-targeting compositions as defined herein, the combination increases the neuron conversion efficiency by at least a factor 1.1, 1.2, 1.5, 2.0, 3.0, 4.0, 5.0, 8.0, 10, 15, or 20.
- a functional neuron is characterized by their ability to form a neuronal network, to send and receive neuronal signals, or both.
- functional neurons fire action potentials.
- functional neurons establish synaptic connections with other neurons.
- a functional neuron can be a postsynaptic neuron in a synapse, e.g., having its dendritic termini (dendritic spines) forming postsynaptic compartments in synapses with another neuron.
- a functional neuron can be a presynaptic neuron in a synapse, e.g., having axonal terminal forming presynaptic terminal in synapses with another neuron.
- a functional neuron can form synapses with another neuron which can include, but are not limited to, axoaxonic, axodendritic, and axosomatic synapses.
- a functional neuron can form synapses with another neuron which can be excitatory (e.g., glutamatergic) , inhibitory (e.g., GABAergic) , modulatory, or any combination thereof.
- a functional neuron forms synapses with another neuron which are glutamatergic, GABAergic, cholinergic, adrenergic, dopaminergic, or any other appropriate type.
- a functional neuron can release neurotransmitters such as, but not limited to, glutamate, GABA, acetylcholine, aspartate, D-serine, glycine, nitric oxide (NO) , carbon monoxide (CO) , hydrogen sulfide (H2S) , dopamine, norepinephrine (also known as noradrenaline) , epinephrine (adrenaline) , histamine, serotonin, phenethylamine, N-methylphenethylamine, tyramine, 3-iodothyronamine, octopamine, tryptamine, somatostatin, substance P, opioid peptides, adenosine triphosphate (ATP) , adenosine, and anandamide.
- neurotransmitters such as, but not limited to, glutamate, GABA, acetylcholine, aspartate, D-serine
- a functional neuron can elicit a postsynaptic response to a neurotransmitter released by a presynaptic neuron into the synaptic cleft.
- the postsynaptic response of a functional neuron as generated in the method provided herein can be either excitatory, inhibitory, or any combination thereof, depending on the type of neurotransmitter receptor the functional neuron expresses.
- the functional neuron expresses ionic neurotransmitter receptors, e.g., ionic glutamate receptors and ionic GABA receptors.
- Ionic glutamate receptors can include, but not limited to, a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) -type glutamate receptors (e.g., GluAl/GRIAl; GluA2/GRIA2; GluA3/GRIA3; GluA4 /GRIA4 ) , delta receptors (e.g., GluDl/GRIDl; GluD2/GRID2) , kainate receptors (e.g., GluKl/GRIKl; GluK2/GRIK2; GluK3/GRIK3 ; GluK4/GRIK4; GluK5/GRIK5) and iV-methyl-D-aspartate (NMDA) receptors (e.g., GluNl/GRINl; GluN2A/GRIN2A; GluN2B/GRIN2B; GluN2C/GRIN2C; GluN2D/GR
- Ionic GABA receptors can include, but are not limited to, GABAA receptor.
- the functional neuron expresses metabolic neurotransmitter receptors, e.g., metabolic glutamate receptors (e.g., mGluRi, mGluRs, mGluR , mGluR , mGluRi, mGluRe, mGluR , mGluRs) , and metabolic GABA receptors (e.g., GABAB receptor) .
- metabolic neurotransmitter receptors e.g., metabolic glutamate receptors (e.g., mGluRi, mGluRs, mGluR , mGluR , mGluRi, mGluRe, mGluR , mGluRs)
- metabolic GABA receptors e.g., GABAB receptor
- the functional neuron expresses a type of dopamine receptor, either Dl-like family dopamine receptor, e.g., D1 and D5 receptor (DIR and D5R) , or D2-like family dopamine receptor, e.g., D2, D3, and D4 receptors (D2R, D3R, and D4R) .
- a functional neuron as provided herein forms electrical synapse with another neuron (e.g., a gap junction) .
- a function neuron as provided herein forms either chemical or electrical synapse (s) with itself, as known as an autapse.
- the characteristics of a functional neuron can be assessed.
- the electrical properties of a functional neuron such as firing of action potential and postsynaptic response to neurotransmitter release can be examined by techniques such as patch clamp recording (e.g., current clamp and voltage clamp recordings) , intracellular recording, and extracellular recording (e.g., tetrode recording, single-wire recording, and filed potential recording) .
- patch clamp recording e.g., current clamp and voltage clamp recordings
- intracellular recording e.g., intracellular recording, and extracellular recording (e.g., tetrode recording, single-wire recording, and filed potential recording)
- Specific properties of a functional neuron e.g., expression of ion channels and resting membrane potential
- patch clamp recording where different variants of patch clamp recording can be applied for different purposes, such as cell-attached patch, inside-out patch, outside-out patch, whole-cell recording, perforated patch, or loose patch.
- AMPA-type glutamate receptor-mediated postsynaptic current can be assessed by AMPA receptor agonists, e.g., AMPA, or antagonists, e.g., 2, 3-dihydroxy-6-nitro-7-sulfamoyl-benzoquinoxaline (NBQX) or 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX) .
- AMPA receptor agonists e.g., AMPA
- antagonists e.g., 2, 3-dihydroxy-6-nitro-7-sulfamoyl-benzoquinoxaline (NBQX) or 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX) .
- NMDA-type glutamate receptor-mediated postsynaptic current can be assessed by NMDA receptor agonists, e.g., NMDA and glycine, or antagonists, e.g., AP5 and ketamine.
- NMDA receptor agonists e.g., NMDA and glycine
- antagonists e.g., AP5 and ketamine.
- functional neurons are examined by techniques other than electrical approaches.
- various fluorescent dyes or genetically encoded fluorescent proteins and imaging techniques can be utilized for monitoring electrical signals conveyed or transmitted by a functional neuron.
- calcium-dependent fluorescent dyes e.g., calcium indicators
- calcium-dependent fluorescent proteins such as, but not limited to, Cameleons, FIP-CBSM, Pericams, GCaMP, TN-L15, TN-humTnC, TN-XL, TNXXL, and Twitchs
- voltage-sensitive dyes that can change their spectral properties in response to voltage changes can also be used for monitoring neuronal activities.
- Neurotransmitter release can be an important aspect of a functional neuron.
- the methods provided herein can comprise reprograming of a non-neuronal cell to a functional neuron that releases a certain type of neurotransmitter.
- the functional neuron releases neurotransmitters such as, but not limited to, glutamate, GABA, acetylcholine, aspartate, D-serine, glycine, nitric oxide (NO) , carbon monoxide (CO) , hydrogen sulfide (H2S) , dopamine, norepinephrine (also known as noradrenaline) , epinephrine (adrenaline) , histamine, serotonin, phenethylamine, N-methylphenethylamine, tyramine, 3-iodothyronamine, octopamine, tryptamine, somatostatin, substance P, opioid peptides, adenosine triphosphat
- the functional neuron releases dopamine as a major neurotransmitter. In some embodiments, the functional neuron releases more than one type of neurotransmitter. In some embodiments, the functional neuron releases neurotransmitters in response to an action potential. In some embodiments, the functional neuron releases neurotransmitters in response to graded electrical potential (e.g., membrane potential changes that do not exceed a threshold for eliciting an action potential) . In some embodiments, the functional neuron exhibits neurotransmitter release at a basal level (e.g., spontaneous neurotransmitter release) .
- a basal level e.g., spontaneous neurotransmitter release
- Neurotransmitter release as described herein from a functional neuron can be assessed by various techniques.
- imaging approaches can be used for characterizing a functional neuron′s neurotransmitter release, for instance, by imaging a genetically encoded fluorescent fusion molecule comprising a vesicular protein, one can monitor the process of synaptic vesicles being fused to presynaptic membrane.
- a high-performance liquid chromatography (HPLC) probe can be used to measure the amount of dopamine in a culture dish or in a brain region where a functional neuron projects its axon.
- the level of dopamine as detected by HPLC can indicate the presynaptic activity of a functional neuron.
- such an assessment can be coupled with stimulation of the functional neuron in order to change its membrane potential, e.g., to make it elicit action potential.
- the present disclosure provides a method of generating a functional neuron in vivo.
- An exemplary method comprises administering to a region in the nervous system, e.g., mature retina, inner ear, or a region in the brain or spinal cord (e.g., striatum) , of a subject a composition comprising a composition (e.g., a Cas effector protein and a gRNA targeting or complementing to PTB and/or nPTB, or a polynucleotide encoding the same) in a non-neuronal cell (e.g., a glial cell or astrocyte) in the region in the nervous system, and allowing the non-neuronal cell to reprogram into the functional neuron.
- the composition suppresses the expression or activity of PTB and/or nPTB.
- the methods provided herein comprise direct administration of a composition (e.g., a Cas effector protein and a gRNA targeting /complementing to PTB and/or nPTB or polynucleotide encoding the same) into a region in the nervous system (e.g., mature retina, inner ear, or a region in the brain or spinal cord (e.g., striatum) ) of a subject.
- a composition e.g., a Cas effector protein and a gRNA targeting /complementing to PTB and/or nPTB or polynucleotide encoding the same
- a region in the nervous system e.g., mature retina, inner ear, or a region in the brain or spinal cord (e.g., striatum)
- a subject e.g., a striatum
- the composition (e.g., a Cas effector protein and a gRNA targeting or complementing PTB and/or nPTB or a polynucleotide encoding the same) is delivered locally to a region in the nervous system (e.g., mature retina or a region in the brain or spinal cord (e.g., striatum) ) .
- a composition comprising a composition, such as a viral vector (e.g. AAV vector) , is administered to the subject or organism by stereotaxic or convection enhanced delivery to a brain region (e.g., striatum) .
- a specific brain region e.g., striatum
- a composition as provided herein is delivered systemically to a subject or to a region in nervous system, e.g., brain (e.g., striatum) or spinal cord, of a subject, e.g., delivered to cerebrospinal fluid or cerebral ventricles, and the composition comprises one or more agents that are configured to relocate the composition to a particular region in the nervous system (e.g., striatum) or a particular type of cells in the nervous system of the subject.
- the composition used in the methods provided herein comprises a virus that expresses a Cas effector and an anti-PTB or anti-nPTB gRNA, and the methods comprise injection of the virus in a desired brain region stereotaxically.
- the virus comprises adenovirus, lentivirus, adeno-associated virus (AAV) , poliovirus, herpes simplex virus (HSV) , or murine Maloney-based virus.
- AAV that can be used in the methods provided herein can be any appropriate serotype of AAV, such as, but not limited to, AAV2, AAV5, AAV6, AAV7, AAV8, and AAV9.
- the methods comprise delivering an AAV2-or AAV9-based viral vector that expresses an agent that suppresses expression or activity of PTB and/or nPTB in a non-neuronal cell in a region in nervous system, e.g., brain (e.g., striatum) or spinal cord.
- an AAV2-or AAV9-based viral vector that expresses an agent that suppresses expression or activity of PTB and/or nPTB in a non-neuronal cell in a region in nervous system, e.g., brain (e.g., striatum) or spinal cord.
- the methods provided herein comprise reprogramming a variety of non-neuronal cells to mature neurons.
- the methods provided herein comprise administering to a region in the nervous system, e.g., the brain (e.g., striatum) or spinal cord, of a subject a composition comprising a composition that suppresses the expression or activity of PTB and/or nPTB in a variety of non-neuronal cells (e.g., glial cells) , allowing the non-neuronal cells to reprogram into functional neurons.
- Types of glial cells can include, but are not limited to, astrocytes, oligodendrocytes, NG2 cells, satellite cells, microglial cells, radial glial cells, Schwann cells, precentral gyrus cells, or ependymal cells.
- the methods provided herein comprise reprogramming astrocyte in a region in the nervous system, e.g., brain (e.g., striatum) or spinal cord, of a subject into a functional neuron.
- Methods provided herein can comprise reprogramming a non-neuronal cell in a specific brain region (e.g., striatum) into a functional neuron.
- exemplary brain regions include the hindbrain, midbrain, and forebrain.
- the methods provided herein comprise administering to a midbrain, striatum, or cortex of a subject a composition comprising a composition that suppresses the expression or activity of PTB in a non-neuronal cell in mature retina or in the striatum and allowing the non-neuronal cell to reprogram into the functional neuron.
- the methods provided herein comprise administering to mature retina or in the striatum of a subject a composition comprising a composition that suppresses the expression or activity of PTB/nPTB in a non-neuronal cell in the mature retina or in the striatum and allowing the non-neuronal cell to reprogram into the functional neuron.
- the methods provided herein comprise reprogramming a non-neuronal cell into a functional neuron, wherein the non-neuronal cell is located in a brain region, such as, but not limited to, medulla oblongata, medullary pyramids, olivary body, inferior olivary nucleus, rostral ventrolateral medulla, caudal ventrolateral medulla, solitary nucleus, respiratory center-respiratory groups, dorsal respiratory group, ventral respiratory group or apneustic centre, pre-botzinger complex, botzinger complex, retrotrapezoid nucleus, nucleus retrofacialis, nucleus retroambiguus, nucleus para-ambiguus, paramedian reticular nucleus, gigantocellular reticular nucleus, parafacial zone, cuneate nucleus, gracile nucleus, perihypoglossal nuclei, intercalated nucle
- ventral anterior nucleus anterodorsal nucleus, anteromedial nucleus, medial nuclear group, medial dorsal nucleus, midline nuclear group, paratenial nucleus, reuniens nucleus, rhomboidal nucleus, intralaminar nuclear group, centromedian nucleus, parafascicular nucleus, paracentral nucleus, central lateral nucleus, lateral nuclear group, lateral dorsal nucleus, lateral posterior nucleus, pulvinar, ventral nuclear group, ventral anterior nucleus, ventral lateral nucleus, ventral posterior nucleus, ventral posterior lateral nucleus, ventral posterior medial nucleus, metathalamus, medial geniculate body, lateral geniculate body, thalamic reticular nucleus, hypothalamus (limbic system) (hpa axis) , anterior, medial area, parts of preoptic area, medial preoptic nucleus, suprachiasmatic
- the invention provides a method of generating a dopaminergic neuron in vivo.
- the method can comprise administering to the striatum in the brain of a subject a composition comprising a composition (e.g., a Cas effector protein and a coding sequence for gRNA against PTB and/or nPTB) that suppresses expression or activity of PTB and/or nPTB in a non-neuronal cell in the brain (e.g., a glial cell or astrocyte) , and allowing the non-neuronal cell to reprogram into the dopaminergic neuron.
- a composition e.g., a Cas effector protein and a coding sequence for gRNA against PTB and/or nPTB
- a non-neuronal cell in the brain e.g., a glial cell or astrocyte
- the method comprises administering to the putamen of the subject a composition comprising a composition (e.g., a Cas effector protein and a coding sequence for gRNA against PTB and/or nPTB) that suppresses expression or activity of PTB and/or nPTB in a non-neuronal cell in the brain (e.g., a glial cell or astrocyte) , and allowing the non-neuronal cell to reprogram into the dopaminergic neuron.
- the method comprises administering exclusively to the putamen of the subject (e.g. avoiding administration to the caudate nucleus and/or other parts of the striatum) and allowing the non-neuronal cell to reprogram into the dopaminergic neuron.
- the invention provides a method of generating a retinal ganglion cell (RGC) neuron or a functional retinal photoreceptor in vivo.
- An exemplary method comprises administering to the mature retina of a subject a composition comprising a composition (e.g., a Cas effector protein and a coding sequence for gRNA against PTB and/or nPTB) that suppresses expression or activity of PTB and/or nPTB in a non-neuronal cell in the mature retina (e.g., a glial cell or MG cell) , and allowing the non-neuronal cell to reprogram into the RGC neuron or the functional retinal photoreceptor.
- a composition e.g., a Cas effector protein and a coding sequence for gRNA against PTB and/or nPTB
- a non-neuronal cell in the mature retina e.g., a glial cell or MG cell
- the methods provided herein comprise administering to a region in the nervous system, e.g., brain or spinal cord, of a subject a composition comprising a cell programming agent (e.g., a Cas effector protein and a coding sequence for gRNA against PTB and/or nPTB) that suppresses the expression or activity of PTB and/or nPTB in a non-neuronal cell in the region, and allowing the non-neuronal cell to reprogram into a functional neuron of a subtype that is predominant in the region.
- a cell programming agent e.g., a Cas effector protein and a coding sequence for gRNA against PTB and/or nPTB
- the methods provided herein can take advantage of local induction signals in a region, e.g., a specific brain region, when reprogramming a non-neuronal cell into a functional neuron in vivo.
- local signals in the striatum may induce the conversion of non-neuronal cells with PTB/nPTB suppressed into dopamine neurons.
- Local neurons, non-neuronal cells, e.g., astrocytes, microglia, and/or other local constituents of the striatum can contribute to the subtype specification of the neuron that is generated from the non-neuronal cell under the induction of the composition.
- the methods provided herein comprise administering to a brain region (e.g., striatum) of a subject a composition comprising a composition (e.g., a Cas effector protein and a coding sequence for gRNA against PTB and/or nPTB) that suppresses the expression or activity of PTB/nPTB in a plurality of non-neuronal cell in the brain region, and the methods further comprise reprogramming at least about 5%, 10%, 20%, 25%, 30%, 35%, 38%, 40%, 42%, 44%, 46%, 48%, 50%, 52%, 54%, 56%, 58%, 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, or 99%of the non-neuronal cells to dopaminergic neurons.
- a composition e.g., a Cas effector protein and
- Expression of PTB/nPTB can be suppressed by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800-fold, at least about 900-fold, at least about 1000-fold, at least about 5000-fold, at least about 10,000-fold, at least about 100,000-fold, or at least about 1,000,000-fold.
- Expression of PTB/nPTB can be suppressed by at least about 5%, 10%, 20%, 25%, 30%, 35%, 38%, 40%, 42%, 44%, 46%, 48%, 50%, 52%, 54%, 56%, 58%, 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, or 99%. Expression can be suppressed by 100%.
- the methods provided herein comprise administering to the mature retina or a brain region (e.g., striatum) of a subject a composition comprising a composition (e.g., a Cas effector protein and a coding sequence for gRNA against PTB and/or nPTB) that suppresses the expression or activity of PTB/nPTB in a plurality of non-neuronal cell in the brain region, so at least about 5%, 10%, 20%, 25%, 30%, 35%, 38%, 40%, 42%, 44%, 46%, 48%, 50%, 52%, 54%, 56%, 58%, 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, or 99%of the functional neurons generated by the methods are RGC or dopaminergic, respectively.
- a composition e.g., a Cas effector protein and
- the dopaminergic neuron generated in the methods provided herein expresses one or more markers of dopaminergic neurons, including, but not limited to, dopamine, tyrosine hydroxylase (TH) , dopamine transporter (DAT) , vesicular monoamine transporter 2 (VMAT2) , engrailed homeobox 1 (Enl) , Nuclear receptor related-1 (Nurrl) , G protein-regulated inward-rectifier potassium channel 2 (Girk2) , forkhead box A2 (FoxA2) , orthodenticle homeobox 2 (OTX2) , and/or LIM homeobox transcription factor 1 alpha (Lmx1a) .
- dopamine tyrosine hydroxylase
- DAT dopamine transporter
- VMAT2 vesicular monoamine transporter 2
- Enl engrailed homeobox 1
- Enl Nuclear receptor related-1
- Girk2 G protein-regulated inward-rectifier potassium channel 2
- OX2 orthoden
- the dopamine neuron generated in the methods provided herein exhibit Ih current, which can be mediated by Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels.
- Ih current can be characterized as a slowly activating, inward current, which can be activated by hyperpolarizing steps. For instance, under voltage clamp and the holding potential Vh is -40 mV, an inward slowly activating current can be triggered in a dopamine neuron, with a reversal potential close to -30 mV.
- the activation curve of Ih current characteristic of a dopamine neuron generated in the methods provided herein can range from -50 to -120 mV with a mid-activation point of -84-1 mV.
- the dopaminergic neurons generated in the methods provided herein have gene expression profile similar to a native dopaminergic neuron. In some embodiments, the dopaminergic neurons generated in the methods provided herein release dopamine as a neurotransmitter.
- a dopaminergic neuron generated in the methods provided herein can be of any subtype of dopaminergic neuron, including, but not limited to, A9 (e.g., immunopositive for Girk2) , A10 (e.g., immunopositive for calbindin-D28 k) , A11, A12, A13, A16, Aaq, and telencephalic dopamine neurons.
- A9 e.g., immunopositive for Girk2
- A10 e.g., immunopositive for calbindin-D28 k
- A11, A12, A13, A16, Aaq telencephalic dopamine neurons.
- the methods provided herein comprise reprogramming a non-neuronal cell in a region in the nervous system, e.g., mature retina or a region of the brain or spinal cord (e.g., striatum) , of a subject to a functional neuron.
- the functional neuron as discussed here is integrated into the neural network in the nervous system.
- the reprogrammed functional neuron can form synaptic connections with local neurons, e.g., neurons that are adjacent to the reprogrammed functional neurons.
- synaptic connections between the reprogrammed neuron and neighboring primary neuron e.g., glutamatergic neurons
- GABAergic interneurons e.g., GABAergic interneurons
- other neighboring neurons e.g., dopaminergic neuron, adrenergic neurons, or cholinergic neurons
- the reprogrammed functional neuron can be a presynaptic neuron, a postsynaptic neuron, or both.
- the reprogrammed functional neuron sends axonal projections to remote brain regions.
- a reprogrammed functional neuron can integrate itself into one or more existing neural pathways in the brain or spinal cord, for instance, but not limited to, superior longitudinal fasciculus, arcuate fasciculus, uncinate fasciculus, perforant pathway, thalamocortical radiations, corpus callosum, anterior commissure, amygdalofugal pathway, interthalamic adhesion, posterior commissure, habenular commissure, fornix, mammillotegmental fasciculus, incertohypothalamic pathway, cerebral peduncle, medial forebrain bundle, medial longitudinal fasciculus, myoclonic triangle, mesocortical pathway, mesolimbic pathway, nigrostriatal pathway, tuberoinfundibular pathway, extrapyramidal system, pyramidal tract, corticospinal tract or cerebrospinal fibers, lateral corticospinal tract, anterior corticospinal tract, corticopontine fibers, frontopontine
- the local cellular environment can be correlated with the projections of a functional neuron generated according to some embodiments of the present disclosure.
- a functional neuron generated in striatum according to some embodiments of the methods provided herein can be affected by other cells in the local environment of striatum.
- the present disclosure provides a method of treating a neurological condition or disease.
- neurological conditions and diseases include: Alzheimer’s disease, amyotrophic lateral sclerosis, ataxia, Bell’s palsy, brain tumors, spinal cord injury, epilepsy, seizures, cerebral aneurysms, Guillain-Barre Syndrome, multiple sclerosis, Parkinson’s disease, stroke, Friedreich ataxia, Huntington’s disease, schizophrenia, depression, drug addition, blindness, deafness, Lewy body disease, motor neuron disease, movement disorder, choreoathetosis, dyskinesias, bipolar disorder, Autism spectrum disorder, spinal muscular atrophy, or head injury.
- the neurological condition or disease can be associated with degeneration of functional neurons in a region in the nervous system.
- the methods provided herein can also find use in treating or ameliorating one or more symptoms of neurodegenerative diseases including, but not limited to, autosomal dominant cerebellar ataxia, autosomal recessive spastic ataxia of Charlevoix-Saguenay, Corticobasal degeneration, Corticobasal syndrome, Creutzfeldt-Jakob disease, fragile X-associated tremor/ataxia syndrome, frontotemporal dementia and parkinsonism linked to chromosome 17, Kufor-Rakeb syndrome, Lyme disease, Machado-Joseph disease, Niemann-Pick disease, pontocerebellar hypoplasia, Refsum disease, pyruvate dehydrogenase complex deficiency, Sandhoff disease, Shy-Drager syndrome, Tay-Sachs disease, and Wobbly hedgehog syndrome.
- neurodegenerative diseases including, but not limited to, autosomal dominant cerebellar ataxia, autosomal recessive spastic ataxi
- the neurological condition is associated with degeneration of functional neurons in the mature retina of a subject.
- neurological conditions associated with the retina include, but are not limited to, glaucoma, age-related RGC loss, optic nerve injury, retinal ischemia, Leber’s congenital amaurosis (LCA) , retinitis pigmentosa (RP) , and Leber’s hereditary optic neuropathy.
- ′′neurodegeneration′′ or its grammatical equivalents can refer to the progressive loss of structure, function, or both of neurons, including death of neuron.
- Neurodegeneration can be due to any type of mechanisms.
- a neurological condition of the methods provided herein are applicable to can be of any etiology.
- a neurological condition can be inherited or sporadic, can be due to genetic mutations, protein misfolding, oxidative stress, or environment exposures (e.g., toxins or drugs of abuse) .
- the methods provided herein treat a neurological condition associated with degeneration of dopaminergic neurons in a brain region. In some embodiments, the methods provided herein treat a neurological condition associated with degeneration of RGC neurons in the mature retina. In other embodiments, the methods provided herein treat a neurological condition associated with degeneration of any type of neurons, such as, but not limited to, glutamatergic neurons, GABAergic neurons, cholinergic neurons, adrenergic neurons, dopaminergic neurons, or any other appropriate type neurons that release neurotransmitter aspartate, D-serine, glycine, nitric oxide (NO) , carbon monoxide (CO) , hydrogen sulfide (H2S) , norepinephrine (also known as noradrenaline) , histamine, serotonin, phenethylamine, N-methylphenethylamine, tyramine, 3-iodothyronamine, octopamine, trypt
- the methods provided herein can find use in treating a neurological condition associated with neuronal degeneration in any region, such as, but limited to, midbrain regions (e.g., substantial nigra or ventral tegmental area) , forebrain regions, hindbrain regions, or spinal cord.
- the methods provided herein can comprise reprogramming non-neuronal cells to functional neurons in any appropriate region (s) in the nervous system in order to treat a neurological condition associated with neuronal degeneration.
- Parkinson′s disease is a neuro-degenerative disease with early prominent functional impairment or death of dopaminergic neurons in the substantia nigra pars compacta (SNpc) .
- the resultant dopamine deficiency within the basal ganglia can lead to a movement disorder characterized by classical parkinsonian motor symptoms.
- Parkinson′s disease can also be associated with numerous non-motor symptoms.
- One standard for diagnosis of Parkinson′s disease can be the presence of SNpc degeneration and Lewy pathology at post-mortem pathological examination.
- Lewy pathology can include abnormal aggregates of asynuclein protein, called Lewy bodies and Lewy neurites.
- Parkinson′s disease can exhibit a number of symptoms, including motor symptoms and non-motor symptoms. Methods provided herein can treat or ameliorate one or more of these motor or non-motor symptoms associated with Parkinson′s disease.
- Motor symptoms of Parkinson′s disease can include bradykinesia (slowness) , stiffness, impaired balance, shuffling gait, and postural instability.
- Parkinson′s disease Motor features in patients with Parkinson′s disease can be heterogeneous, which has prompted attempts to classify subtypes of the disease, for instance, tremor-dominant Parkinson′s disease (with a relative absence of other motor symptoms) , non-tremor-dominant Parkinson′s disease (which can include phenotypes described as akinetic-rigid syndrome and postural instability gait disorder) , and an additional subgroup with a mixed or indeterminate phenotype with several motor symptoms of comparable severity.
- Non-motor symptoms of Parkinson′s disease can include olfactory dysfunction, cognitive impairment, psychiatric symptoms (e.g., depression) , sleep disorders, autonomic dysfunction, pain, and fatigue. These symptoms can be common in early Parkinson′s disease.
- Non-motor features can also be frequently present in Parkinson′s disease before the onset of the classical motor symptoms.
- This premotor or prodromal phase of the disease can be characterized by impaired olfaction, constipation, depression, excessive daytime sleepiness, or rapid eye movement sleep behavior disorder.
- methods provided herein mitigate or slow the progression of Parkinson′s disease.
- Progression of Parkinson′s disease can be characterized by worsening of motor features.
- complications related to long-term symptomatic treatment including motor and non-motor fluctuations, dyskinesia, and psychosis.
- One pathological feature of Parkinson′s disease can be loss of dopaminergic neurons within the substantial nigra, e.g., substantial nigra pars compacta (SNpc) .
- methods provided herein replenish dopamine (secreted from converted dopamine neuron in the striatum) diminished due to loss of dopamine neuron in substantial nigra (e.g., SNpc) of a patient.
- Neuronal loss in Parkinson′s disease can also occur in many other brain regions, including the locus ceruleus, nucleus basalis of Meynert, pedunculopontine nucleus, raphe nucleus, dorsal motor nucleus of the vagus, amygdala, and hypothalamus.
- methods of treating or ameliorating one or more symptoms of Parkinson′s disease in a subject as provided herein include reprogramming non-neuronal cells to functional neurons in brain regions experiencing neuronal loss in a patient with Parkinson′s disease.
- Parkinson′s disease can find use in treating Parkinson′s disease of different etiology.
- Parkinson′s disease as a result of one or more genetic mutations, such as, but not limited to, mutations in genes SNCA, LRRK2 , VPS35 , EIF4G1, DNAJC13, CHCHD2 , Parkin, PINK1, DJ-1, ATP13A2, C9ORF72, FBX07, PLA2G6, POLG1, SCA2, SCA3, SYNJ1, RAB39B, and possibly one or more genes affected in 22qll. 2 microdeletion syndrome.
- Parkinson′s disease with no known genetic traits.
- the one or more symptoms of Parkinson′s disease the methods provided herein can ameliorate can include not only the motor symptoms and non-symptoms as described above, but also pathological features at other levels. For example, reduction in dopamine signaling in the brain of a patient with Parkinson′s disease can be reversed or mitigated by methods provided herein by replenishing functional dopamine neurons, which can be integrated into the neural circuitry and reconstruct the dopamine neuron projections to appropriate brain regions.
- the present disclosure also provides methods of restoring dopamine release in subject with a decreased amount of dopamine biogenesis compared to a normal level.
- An exemplary method comprises reprogramming a non-neuronal cell in a brain region of the subject (e.g., striatum) , and allowing the non-neuronal cell to reprogram into a dopaminergic neuron, thereby restoring at least 50%of the decreased amount of dopamine.
- the reprogramming is performed by administering to the brain region of the subject (e.g., striatum) a composition comprising a composition that suppresses the expression or activity of PTB/nPTB in a non-neuronal cell (e.g., an astrocyte) in the brain region.
- the methods provided herein restore at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%of the decreased amount of dopamine.
- the methods provided herein restore about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100%of the decreased amount of dopamine. In some embodiments, the methods provided herein restore at least about 50%of the decreased amount of dopamine.
- Methods provided herein can comprise suppressing the expression or activity of PTB and/or nPTB in a non-neuronal cell for a certain period of time sufficient for reprogramming the non-neuronal cell to a mature neuron.
- exemplary methods comprise contacting the non-neuronal cell with a composition that suppresses the expression or activity of PTB and/or nPTB in the non-neuronal cell for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 2 months, at least 3 months, at least 4 months, at least 5 months, or at least 1 year, thereby reprogramming the non-neuronal cell to a mature neuron.
- suppression of PTB or nPTB expression or activity is sequential. For example, the expression or activity of PTB is first suppressed for any one of the above-mentioned time periods before the expression or activity of nPTB is suppressed. In certain embodiments, suppression of PTB and nPTB expression or activity is concurrent.
- exemplary methods comprise contacting the non-neuronal cell with a composition that suppresses the expression or activity of PTB in the non-neuronal cell for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 3 weeks, 4 weeks, 5 weeks, 2 months, 3 months, 4 months, or 5 months, before contacting the non-neuronal cell with a composition that suppresses the expression or activity of nPTB in the non-neuronal cell, thereby reprogramming the non-neuronal cell to a mature neuron.
- the methods provided herein comprise administering the composition only once, e.g., adding the composition to a cell culture comprising the non-neuronal cell, or delivering the composition to a brain region in a subject comprising the non-neuronal cell (e.g., the striatum) , once and the composition can remain active, suppressing expression or activity of PTB and/or nPTB in the non-neuronal cell for a desirable amount of time.
- a desirable amount of time can be for at least 1 day, at least 2 days, at least 4 days, or at least 10 days.
- the composition comprises an AAV vector expressing a Cas effector and a coding sequence for an anti-PTB gRNA
- the design of the AAV vector can enable it to remain transcriptionally active for an extended period of time.
- the methods provided herein comprise administering the composition more than once, e.g., at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 12 times, at least 15 times, at least 20 times or more.
- the methods provided herein further comprise administering at least one immunosuppressant.
- An immunosuppressant is a drug that inhibits or prevents activity of the immune system.
- the immunosuppressant can be administered prior to, simultaneously with, or after the administration of the composition, for example, when the composition comprises an AAV vector expressing a Cas effector and a coding sequence for an anti-PTB gRNA.
- Suitable immunosuppressants include, but are not limited to, corticosteroids (e.g. prednisone, prednisolone, dexamethasone, etc. ) , calcineurin inhibitors (e.g. cyclosporine, tacrolimus, etc.
- mTOR inhibitors sirolimus, everolimus, etc.
- IMDH inhibitors azathioprine, mycophenolate, etc.
- antibodies e.g. basiliximab, rituximab, alemtuzumab, etc.
- interferons e.g. IFN- ⁇ , IFN- ⁇ , etc.
- Janus kinase inhibitors e.g. tofacitinib, etc.
- biologics anakinra, etc.
- the immunosuppressant is administered about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 3 weeks, 4 weeks, 5 weeks, 2 months, 3 months, 4 months, or 5 months, before and/or after administration of the composition.
- the immunosuppressant is administered about simultaneously with the administration of the composition.
- compositions comprising a composition in an amount effective to reprogram a mammalian non-neuronal cell to a mature neuron by suppressing the expression or activity of PTB/nPTB in the non-neuronal cell.
- An exemplary pharmaceutical composition can further comprise another component, such as a pharmaceutically acceptable carrier, stabilizer, dilutent, dispersing agent, suspending agent, thickening agent or excipient.
- a composition as provided herein can be a Cas effector protein and a coding sequence for a gRNA against PTB/nPTB.
- a pharmaceutical composition provided herein can comprise one or more carriers or excipients, including but not limited to: buffers, carbohydrates, mannitol, proteins, peptides or amino acids such as glycine, antioxidants, bacteriostats, chelating agents, suspending agents, thickening agents and/or preservatives.
- carriers or excipients including but not limited to: buffers, carbohydrates, mannitol, proteins, peptides or amino acids such as glycine, antioxidants, bacteriostats, chelating agents, suspending agents, thickening agents and/or preservatives.
- a pharmaceutical composition can comprise water, oils (e.g., petroleum oil, animal oil, vegetable oil, synthetic oil , peanut oil, soybean oil, mineral oil, or sesame oil) , saline solutions, aqueous dextrose and glycerol solutions, flavoring agents, coloring agents, detackifiers and other acceptable additives, binders, or other pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH buffering agents, tonicity adjusting agents, emulsifying agents, or wetting agents, etc.
- oils e.g., petroleum oil, animal oil, vegetable oil, synthetic oil , peanut oil, soybean oil, mineral oil, or sesame oil
- saline solutions e.g., aqueous dextrose and glycerol solutions
- flavoring agents e.g., coloring agents, detackifiers and other acceptable additives, binders, or other pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH buffering agents, tonicity adjusting agents,
- excipients can include, but are not limited to, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, and ethanol.
- the composition is substantially free of preservatives.
- the composition contains at least one preservative.
- a pharmaceutically-acceptable carrier can be present at about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 5
- a pharmaceutically-acceptable excipient can be present at about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%,
- Dosage forms can include, but are not limited to, feed, food, pellets, lozenges, liquids, elixirs, aerosols, inhalants, sprays, powders, tablets, pills, capsules, gels, nanoparticles, microgels, suppositories, aqueous suspensions, oily suspensions, ointments, patches, lotions, emulsions, creams, drops, dispersible powders or granules, emulsions in hard or soft capsules, syrups, phytoceuticals, nutraceuticals, or any combination thereof.
- a pharmaceutical composition can be formulated for injection, inhalation, parenteral administration, intravenous administration, subcutaneous administration, intramuscular administration, intradermal administration, topical administration, opthalamic administration, nasal administration, or oral administration.
- a pharmaceutical composition can be administered in a local or systemic manner (e.g., via injection of the compound directly into an organ or in the form of an intermediate release formulation) .
- a pharmaceutical composition can be administered to the nervous system.
- a pharmaceutical composition can be administered to a region of the nervous system (e.g., the mature retina, the inner ear, or the stratium of the brain) .
- the pharmaceutical composition comprising an AAV vector encoding a Cas effector and a coding sequence for a gRNA against PTB/nPTB can be injected into the mature retina, or the striatum of a subject′s brain.
- the composition is administered and directly affects functional neurons.
- the composition is administered to non-functional neurons and reprograms them into functional neurons, thereby replenishing the degenerated neurons in the region.
- a subject can be an animal.
- An animal can be a mammal.
- An animal can be a human.
- An animal can be a non-human primate, such as, but not limited to, rhesus macaques, crab-eating macaques, stump-tailed macaques, pig-tailed macaques, squirrel monkeys, owl monkeys, baboons, chimpanzees, marmosets and spider monkeys.
- An animal can be a bovine, canine, caprine, cervine, cricetine, feline, equine, lapine, murine, musteline, or ovine.
- An animal can be a research animal, a genetically modified animal, or any other appropriate type of animal.
- compositions can comprise any appropriate carrier or excipient, depending on the type of composition and the administration route the composition is designed for.
- a composition comprising a composition as provided herein can be formulated for parenteral administration and can be presented in unit dose form in ampoules, pre-filled syringes, small volume infusion or in multi dose containers with an added preservative.
- the composition can take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, for example solutions in aqueous polyethylene glycol.
- a vehicle for injectable formulations, can be chosen from those known in the art to be suitable, including aqueous solutions or oil suspensions; emulsions with sesame oil, corn oil, cottonseed oil, or peanut oil; elixirs; mannitol; dextrose; a sterile aqueous solution; or similar pharmaceutical vehicles.
- the formulation can also comprise polymer compositions which are biocompatible, biodegradable, such as poly (lactic-co-glycolic) acid. These materials can be made into micro or nanospheres, loaded with a drug and further coated or derivatized to provide superior sustained release performance.
- Vehicles suitable for periocular or intraocular injection include, for example, suspensions of active agent in injection grade water, liposomes, and vehicles suitable for lipophilic substances.
- a composition as provided herein can further comprise additional agents besides a composition and a pharmaceutically acceptable carrier or excipient.
- additional agents can be provided for promoting neuronal survival.
- additional agents can be provided for monitoring pharmacodynamics purpose.
- a composition comprises additional agents as a penetration enhancer or for sustained or controlled release of the active ingredient, e.g., a composition.
- a composition provided herein can be administered in a therapeutically effective amount.
- a ′′therapeutically effective amount′′ of a composition of the disclosure will vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the composition to elicit a desired response in the individual.
- a therapeutically effective amount can also be one in which any toxic or detrimental effects of the composition are outweighed by the therapeutically beneficial effects.
- a therapeutically effective amount of composition as provided herein can be an amount of composition that converts a certain proportion of astrocytes in a brain region that experiences neuronal loss, conversion of such proportion of astrocytes to functional neurons in the brain region is sufficient to ameliorate or treating the disease or condition associated with the neuronal loss in the brain region, and meanwhile, such proportion of astrocytes does not exceed a threshold level that can lead to aversive effects that can overweigh the beneficial effects brought by the neuronal conversion, for instance, due to excessive reduction in the number of astrocytes in the brain region as a direct consequence of the neuronal conversion.
- a composition provided herein can be administered to a subject in a dosage volume of about 0.0005, 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.7, 0.8, 0.9, 1.0 mL, or more.
- the composition can be administered as a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more dose course regimen. Sometimes, the composition can be administered as a 2, 3, or 4 dose course regimen. Sometimes the composition can be administered as a 1 dose course regimen.
- the administration of the first dose (e.g., an AAV vector encoding a Cas effector and a gRNA against PTB) and second dose (e.g., an AAV vector encoding a Cas effector and a gRNA against nPTB) of a multi dose course regimen can be separated by about 0 days, 1 day, 2 days, 5 days, 7 days, 14 days, 21 days, 30 days, 2 months, 4 months, 6 months, 9 months, 1 year, 1.5 years, 2 years, 3 years, 4 years, 5 years, 10 years, 20 years, or more.
- a composition described herein can be administered to a subject once a day, once a week, once two weeks, once a month, a year, twice a year, three times a year, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more years. Sometimes, the composition can be administered to a subject every 2, 3, 4, 5, 6, 7, or more years. Sometimes, the composition can be administered to a subject once.
- An exemplary method can comprise reprogramming a non-neuronal cell to a neuron in vitro and transplanting the reprogrammed neuron into the brain region in a subject.
- in vitro reprogramming can be performed according to the methods provided herein.
- An exemplary composition can comprise a neuron reprogrammed according to any embodiment of the methods provided herein.
- a method provided herein comprises reprogramming a non-neuronal cell to a neuron in vivo and explanting the reprogrammed neuron.
- the explant comprises brain tissue comprising the reprogramed neuron.
- the explant is transplanted into the brain region of a subject.
- the transplantation of neurons reprogrammed according to the methods provided herein can be used to replenish degenerated neurons in a subject suffering a condition associated with neuronal loss.
- brain tissue e.g., explant
- Such brain tissue can be alive.
- brain tissue can be fixed by any appropriate fixative.
- Brain tissue can be used for transplantation, medical research, basic research, or any type of purpose.
- the disclosure demonstrates that the method is applicable to disease models of neurodegeneration.
- the disclosure shows that astrocyte-to-neuron conversion strategy can work in a chemical-induced Parkinson′s disease model.
- the methods and compositions can convert astrocytes to neurons including dopaminergic, glutamatergic, and GABAergic neurons, these neurons are able to form synapses in the brain, and remarkably, the converted neurons can efficiently reconstruct the lesioned nigrostriatal pathway to correct measurable Parkinson′s phenotypes.
- the effectiveness of this method was demonstrated both in astrocytes in culture (human and mouse) as well as in vivo in a mouse Parkinson′s disease model. Therefore, this strategy has the potential to cure Parkinson′s disease, which can also be applied to a wide range of neurodegenerative diseases (e.g., other neurological diseases associated with neuronal dysfunction) .
- neurodegenerative diseases e.g., other neurological diseases associated with neuronal dysfunction
- the approach of the disclosure exploits the genetic foundation of a neuronal maturation program already present, but latent, in both mammalian astrocytes that progressively produce mature neurons once they are reprogrammed by PTB suppression.
- These findings provide a clinically feasible approach to generate neurons from local astrocytes in mammalian brain using a single dose of a vector comprising coding sequence for a Cas effector and a gRNA against PTB/nPTB.
- the phenotypes of PTB/nPTB knockdown-induced neurons can be a function of the context in which they are produced and/or the astrocytes from which they are derived.
- the disclosure demonstrates the potent conversion of astrocytes to neurons (e.g., dopamine neurons in the striatum) . More particularly, the disclosure shows that in a primate model, the strategy efficiently can convert astrocytes to neurons, thus satisfying all five factors for in vivo reprogramming.
- the data provided herein show that PTB reduction in the primate brain can convert astrocytes to dopamine neurons (e.g., dopaminergic neurons) .
- C57BL/6 mice were purchased from Shanghai SLAC Laboratory. The mice were housed in a light/dark cycle room with water and food. All animal experiments were performed and approved by the Animal Care and Use Committee of the CEBSIT, Chinese Academy of Sciences, Shanghai, China.
- the Cos7, 293T and N2a cell lines were obtained from Cell bank of Shanghai Institute of Biochemistry and Cell Biology (SIBCB) , Chinese Academy of Sciences (CAS) , and cultured in DMEM with 10%FBS and 1%penicillin/streptomycin in a 37°C incubator under 5%CO 2 .
- Transient transfection was conducted with 4 ⁇ g vectors expressing CAG-CasRx-P2A-GFP/CAG-Cas13X-P2A-GFP + 2 ⁇ g U6-gRNA-CMV-mCherry plasmid using Lipofectamine 3000.
- CAG-CasRx-P2A-GFP/CAG-Cas13X-P2A-GFP plasmid was used as a control.
- Two days after transient transfection around 30K GFP and mCherry double-positive (GFP top 20%) cells were collected by fluorescence activated cell sorting (FACS) and lysed for qPCR analysis.
- FACS fluorescence activated cell sorting
- RNA-seq Cos7 cells were cultured in 10-cm dishes. Around 100k positive cells (GFP top 20%) were isolated by FACS, RNA was extracted and reverse transcribed to cDNA, which was then used for RNA-seq. Analysis of the RNA-seq data was performed and presented as the mean of all repeats.
- the DNA sequence of CasRx is:
- the guide sequences (i.e. the sequence that is complementary to the PTB mRNA sequence to be targeted) in the guide RNAs as used are shown in Table 1.
- Guide sequences of guide RNAs (Cas13d) and the relative expression level (vs control without gRNA) achieved by the respective guide RNAs.
- High efficiency gRNAs (bold, ⁇ 0.12) , almost all located in the key region (positions 951-1487 of SEQ ID NO: 87, which is SEQ ID NO: 88; see Table 2 and Figure 1) , are indicated in bold print.
- High efficiency gRNAs (bold) all located in the key region (positions 951-1487 of SEQ ID NO: 87, which is SEQ ID NO: 88; see also Figure 2) , are indicated in bold print.
- the sequence of Cas13X is:
- AAV8 was used in this study. Stereotactic injections in the mice were performed as previously described (Zhou et al., 2014, Elife 3, e02536, doi: 10.7554/eLife. 02536) . The mice were placed in a stereotactic frame. Next, the skin over the skull was shaven and opened using a razor. A craniotomy with coordinates (AP +0.8 mm, ML ⁇ 1.6 mm) was made over the boundary of frontal and parietal bones, allowing the placement of an injection micropipette ( ⁇ 20 ⁇ m outside diameter at the tip) .
- the viral solution containing either AAV-GFAP-CasRx-Ptbp1+AAV-GFAP-mCherry or AAV-GFAP-CasRx+AAV-GFAP-mCherry was injected slowly ( ⁇ 0.3 ⁇ l/min) .
- Mice were injected in the striatum (AP +0.8 mm, ML ⁇ 1.6 mm and DV 2.6 mm) with AAVs (> 1 X 1012 vg/ml, 1 ⁇ L, mice aged 8-10 weeks) .
- Macaca fascicularis the enrolled animals were rated into three stages (severe, moderate, and mild) based on their motor symptoms. In each stage, the assignment into treatment and control groups was blindly.
- the immunosuppressant dexamethasone or Tacrolimus was administrated via intramuscular injection to reduce immunological reaction.
- both sides of the putamen were injected with 60-80 ⁇ L (around 1 X 1013 vg/ml) AAV-GFAP-CasRx-Ptbp1+AAV-GFAP-mCherry and AAV-GFAP-CasRx+AAV-GFAP-mCherry, respectively.
- the volume ratio between GFAP-mCherry and GFAP-CasRx or GFAP-CasRx-Ptbp1 were 1: 20.
- the viral solution was injected into putamen of both hemispheres using a 100ul syringe and a syringe pump. After the surgery, animals were treated with Ceftriaxone sodium and Meloxicam to avoid infection and painfulness. Daily administration of Dexamethasone or Tacrolimus was performed for about 1 week after surgery.
- the brains were perfused and fixed with 4%paraformaldehyde (PFA) overnight, and kept in 30%sucrose for at least 12 hours for mice and 2 weeks for Macaca fascicularis. Brains were sectioned after embedding and freezing, and slices with the thickness of 30 ⁇ m for mice and 30 ⁇ m for Macaca fascicularis were used for immunofluorescence staining. Brain sections were rinsed thoroughly with 0.1 M phosphate buffer (PB) .
- PB phosphate buffer
- Rabbit-anti-NeuN (1: 500, 24307S, Cell Signaling Technology)
- Guinea Pig anti-NeuN antibody (1: 500, ABN90, Millipore)
- Mouse anti-Flag (1: 2000, F3165, Sigma)
- Rabbit anti-TH anti-body (1: 500, AB152, Millipore)
- Rat anti-DAT (1: 100, MAB369, Millipore)
- Rabbit anti-RBPMS (Proteintech, Cat# 15187-1-AP) .
- AAV-GFAP-GFP-Cre 0.2 ⁇ l
- AAV-GFAP-CasRx-Ptbp1 0.4 ⁇ l
- AAV-GFAP-GFP-Cre 0.2 ⁇ l
- AAV-GFAP-CasRx 0.4 ml
- PBS 0.4 ⁇ l
- AAV-GFAP-GFP-Cre 0.2 ⁇ l
- AAV-GFAP-CasRx-Ptbp1 0.4 ⁇ l
- AAV-GFAP- ⁇ -catenin 0.4 ⁇ l
- AAV-GFAP-tdTomato + AAV-GFAP-CasRx-Ptbp1 or AAV-GFAP-tdTomato + AAV-GFAP-CasRx were delivered.
- the eyes, optic nerves, and brain tissue were collected.
- Tissue samples were then fixed with 4%paraformaldehyde (PFA) , and then maintained in the solution containing 30%sucrose. After embedding, the eyes were sectioned, and slices were washed and covered with mountant (Life Technology) . The images were collected using Olympus FV3000 microscope.
- PFA paraffin wax
- each animal was moved to a transparent cage (1.2m X 1.2m X 1.2m) with foods (apple and peanuts to induce animal movement) in each corner and recorded by 5 camaras (4 on the floor and 1 on the top) for 1.5h without human interference.
- the animal keeper walked around the cage for 3 times and then 3 times of pole provoking (sticked a pole into the cage and swung it several times) toward to animal.
- PET-CT was typically performed before and after virus injection.
- PET-CT scanning before MPTP administration. Monkeys were fasted for 12 hours and then habituated for more than 1 hour before imaging. Intramuscular injection of propofol or Zoletil50 were used to induce stable anesthesia before and during scanning. After anesthesia, each animal was intravenously injected with a PET tracer, [18F] DOPA, 30 minutes before emission scanning. The animals were placed on the PET scanner (Discovery IQ Gen 2, GE healthcare) and CT scan was performed for attenuation correction, followed by a 10-minute emission scan. The emission data were reconstructed using Ultra-TOF algorithm with an attenuation correction and no scatter correction.
- the regions of interest (ROIs) were manually drawn on the bilateral putamenand cerebellum.
- the analysis of PET data was performed using RadiAnt DICOM Viewer (Medixant, Poland) .
- T1-and T2-weighted MR images were obtained using a 3-tesla MRI scanner (Discovery TM MR750w GEM 3.0T, GE Healthcare) .
- the analysis of MRI data was performed using RadiAnt DICOM Viewer (Medixant, Poland) .
- CSF samples cerebrospinal fluid (CSF) samples from monkeys in three time points (before MPTP administration, after MPTP but before AAV injection and after AAV injection) and analyzed them using high precision liquid chromatography (HPLC) .
- CSF samples were obtained via lumbar puncture during monkey anesthesia in the same time period (9 to 12 am) and then frozen in -80°C refrigerator. All three samples of each monkey were examined at the same batch to avoid reading variability.
- HPLC UPLC (Shimadzu) and MS (Triple Quad6500+) (Model No: LCMS021)
- the targeting site of gRNA 60 is conserved in the Macaca Fascicularis, human Ptbp1 and mouse gene and enabled the potent downregulation of Ptbp1 gene in human 293T, Monkey Cos7 cells and mouse N2a cells, thus it was used in the following experiments ( Figures 3) .
- Figures 3 To determine the targeting specificity of this strategy, we performed RNA-seq and found that Ptbp1 was specifically downregulated in Cos7 cells ( Figure 4) .
- a recent study showed that Ptbp1 knockdown could convert striatal astrocytes into dopamine neurons in mice, we next examined whether Ptbp1 knockdown in the nonhuman primate striatal astrocytes could locally convert astrocytes into dopamine neurons in vivo.
- mCherry+ cells expressed mature neuron markers NeuN were observed after injected with AAV-GFAP-mCherry and AAV-GFAP-CasRx-Ptbp1 but not in the control striatum injected with AAV-GFAP-CasRx ( Figure 5C) .
- a large fraction of mCherry+ cells expressed dopamine neuron marker DAT at one month after AAV injection ( Figure 5D) .
- astrocyte-to-dopamine neuron conversion could be achieved in the putamen of PD monkey, we performed immunofluorescence staining to observe a large fraction mCherry+ cells expressed the dopamine neuron marker tyrosine hydroxylase (TH) in the putamen injected with AAV-GFAP-mCherry and AAV-GFAP-CasRx-Ptbp1, but not in the putamen injected with control AAVs (AAV-GFAP-mCherry and AAV-GFAP-CasRx) (Figure 8A) .
- TH dopamine neuron marker tyrosine hydroxylase
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Abstract
The invention described herein provides methods and compositions for treating certain diseases, using a composition for regulating a primate PTBP gene expression comprising a Cas effector protein and a guide RNA (gRNA) to target a target sequence or its complement of the primate PTBP gene or a transcript thereof, which suppresses the expression of the primate PTBP gene.
Description
BRIEF SUMMARY
Provided herein is a composition for regulating a primate PTBP gene expression comprising: (1) a Cas effector protein, or a nucleic acid encoding the same, (2) a guide RNA (gRNA) that targets a target sequence, or its complement, of the primate PTBP gene or a transcript thereof, or a nucleic acid encoding the same, wherein the composition is capable of suppressing the expression of the primate PTBP gene. In some embodiments, the target sequence is located within a key region of the primate PTBP gene. In some embodiments, wherein the key region is SEQ ID NO: 88. In some embodiments, the Cas effector protein is an RNA-targeting Cas effector protein.
In some embodiments, the RNA-targeting Cas effector protein is selected from the group consisting of Cas13d, CasRx, Cas13X, Cas13a, Cas13b, Cas13c, Cas13Y, and a functional domain thereof. In some embodiments, the Cas effector protein is CasRx. In some embodiments, the Cas effector protein is Cas13X.
In some embodiments, the gRNA sequence has at least 90%sequence identity to SEQ ID NOs: 1-86. In some embodiments, the gRNA sequence has at least 90%sequence identity to SEQ ID NOs: 89-112. In some embodiments, the nucleic acid encoding the Cas effector protein is located on an expression vector. In some embodiments, the expression vector is a gene therapy vector. In some embodiments, the gene therapy vector is a viral gene therapy vector. In some embodiments, the viral gene therapy vector is selected from the group consisting of an adeno-associated virus (AAV) vector, an adenovirus vector, a lentivirus vector, a retrovirus vector, a herpes virus, a SV40 vector, a poxvirus vector, and a combination thereof. In some embodiments, the expression vector is comprised in a nanoparticle.
In some embodiments, the expression vector further comprises a glial cell-specific promoter that causes expression of the Cas effector protein in a non-neuronal cell. In some embodiments, the glial cell-specific promoter is selected from the group consisting of a GFAP promoter, a ALDH1L1 promoter, a EAAT1/GLAST promoter, a glutamine synthetase promoter, a S100 beta promoter and a EAAT2/GLT-1 promoter. In some embodiments, the glial cell-specific promoter is a Müller glia (MG) cell-specific promoter. In some embodiments, the MG cell-specific promoter is selected from the group consisting of a GFAP promoter, a ALDH1L1 promoter, a Glast (also known as Slc1a3) promoter and a Rlbp1 promoter. In some embodiments, the nucleic acid encoding the gRNA is located on an expression vector. In some embodiments, expression vector further comprises a promoter that causes expression of the gRNA in a non-neuronal cell. In some embodiments, the promoter that causes expression of the gRNA in the non-neuronal cell is a U6 promoter.
In some embodiments, the primate PTBP gene is located in a non-neuronal cell. In some embodiments, the non-neuronal cell is located in mature retina, striatum, substantia nigra, inner ear, spinal cord, prefrontal cortex, motor cortex, or ventral tegmental area (VTA) . In some embodiments, the non-neuronal cell located in the striatum is in putamen. In some embodiments, the non-neuronal cell is a glial cell. In some embodiments, the glial cell is an astrocyte.
The composition disclosed hereof further comprising one or more dopamine neuron-associated factors, or an expression vector for expression of the one or more dopamine neuron-associated factors thereof. In some embodiments, the one or more dopamine neuron-associated factors are selected from the group consisting of: Lmx1a, Lmx1b, FoxA2, Nurr1, Pitx3, Gata2, Gata3, FGF8, BMP, En1, En2, PET1, a Pax family protein, SHH, a Wnt family protein, and a TGF-β family protein. The composition disclosed hereof further comprising one or more factors selected from β-catenin, Oct4, Sox2, Klf4, Crx, Brn3a, Brn3b, Math5, Nr2e3 and Nrl, or an expression vector for expression of the one or more factors thereof.
The composition disclosed hereof further comprising an immunosuppressant. In some embodiments, the immunosuppressant is selected from the group consisting of a corticosteroid, a calcineurin inhibitor, an mTOR inhibitor, an IMDH inhibitor, an immune-suppressive antibody, an interferon, a Janus kinase inhibitor and an anakinra.
Described herein is a method of treating a disease comprising administering an effective amount of the composition disclosed thereof to a subject in need thereof. In some embodiments, the composition suppresses the expression or activity of the primate PTBP protein in a non-neuronal cell, thereby allowing the non-neuronal cell to reprogram into a functional neuron. In some embodiments, the disease is a neurological condition associated with degeneration of functional neurons. In some embodiments, the neurological condition is a neurological condition associated with degeneration of functional neurons in the mature retina which is selected from the group consisting of: glaucoma, age-related RGC loss, optic nerve injury, retinal ischemia, LCA (Leber′s congenital amaurosis) , RP (Retinitis pigmentosa) disease and Leber′s hereditary optic neuropathy. In some embodiments, the condition associated with degeneration of functional neurons is selected from the group consisting of Parkinson′s disease, Alzheimer′s disease, Huntington′s disease, Schizophrenia, depression, drug addiction, stroke, movement disorder, spinal cord injury, choreoathetosis, dyskinesias, bipolar disorder, and Autism spectrum disorder (ASD) . In some embodiments, the composition is administered to a glial cell or a MG cell in a mature retina to generate a functional retinal ganglion cell (RGC) neuron or a functional retinal photoreceptor. In some embodiments, an expression level of PTBP mRNA is less than 40%compared to a corresponding control. In some embodiments, an expression level of PTBP mRNA is less than 50%compared to a corresponding control. In some embodiments, there are no co-factors expressed.
INCORPORATION BY REFERENCE
All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.
Figures 1A-B depict a screen of efficient gRNAs. Figure 1A depicts downregulation of Ptbp1 mRNA with 86 gRNAs in human 293T cells, two days after transient transfection of plasmids encoding CasRx and gRNAs. Note that the gRNA 60 was used in the following experiments. Figure 1B depicts targeting sites of 86 gRNAs in human Ptbp1 gene, as well as the information of knockdown efficiency (red, < 0.12; green, > 0.1) . Key region indicates the targeting region with high knockdown efficiency. The key region corresponds to positions 951-1487 of SEQ ID NO: 87, which are position 1-536 of SEQ ID NO: 88.
Figures 2A-B show confirmation of the key region using another RNA-targeting Cas13 protein. Figure 2A shows downregulation of Ptbp1 mRNA with 24 gRNAs in human 293T cells, two days after transient transfection of plasmids encoding CasRx and gRNAs. Note that gRNAs targeting the key region were highlighted as red dots. Figure 2B shows targeting sites of 24 gRNAs in the human Ptbp1 gene, as well as the information of knockdown efficiency (red, < 0.75; green, > 0.75) . Key region indicates the targeting region with high knockdown efficiency. The key region corresponds to positions 951-1487 of SEQ ID NO: 87, which are positions 1-536 of SEQ ID NO: 88.
Figure 3A-C shows that gRNA 60 showed high knockdown efficiency in human 293T cells, monkey Cos7 cells, and mouse N2a cells. All values are presented as mean ± SEM.; unpaired t test; *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 4 shows expression levels in log2 (fragments per kilobase per million mapped reads [FPKM] + 1) values of all detected genes in RNA sequencing (RNA-seq) libraries of CasRx-Ptbp1 (y axis) compared to CasRx control (x axis) , showing that Ptbp1 was specifically downregulated. Cos7 cells, n = 3 independent replicates for both groups. The gRNA 60 was used.
Figures 5A-D shows a conversion of astrocytes into dopamine neurons in the striatum of mice. Figure 5A shows a schematic illustration of the AAV vectors and injection strategy. Vector 1 (AAV-GFAP-mCherry) encodes mCherry driven by the astrocyte-specific promoter GFAP; Vector 2 (AAV-GFAP-CasRx) encodes CasRx; and Vector 3 (AAV-GFAP-CasRx-Ptbp1) encodes CasRx and gRNAs. The striatum was either injected with AAV-GFAP-CasRx-Ptbp1 or control vector AAV-GFAP-CasRx together with AAV-GFAP-mCherry. Astrocyte-to-dopamine neuron conversion is evaluated around 2-3 weeks post-injection. ST, striatum. Figure 5B shows representative images showing that mCherry (Vector 1) and CasRx (Fused with Flag, Vector 3) were specifically expressed in the mouse astrocytes. Note that GFAP is the astrocyte-specific marker. Figure 5C shows confocal images showing that converted mCherry+NeuN+ cells (white arrowheads) were observed in mice 2 weeks after Vector 1 + 3 injection, but not in the striatum injected with control AAVs. Note that NeuN is neuron-specific marker. Figure 5D shows confocal images showing that converted mCherry+DAT+ cells (white arrowheads) were observed in mice one month after Vector 1 + 3 injection, but not in the control striatum. Note that DAT is the mature dopamine neuron-specific marker.
Figures 6A-E depict an increase of MG-to-RGC conversion efficiency by co-injecting β-catenin in middle-aged mice. Figure 6A shows a schematic showing MG-to-RGC conversion. Vector I (AAV-GFAP-GFP-Cre) expresses Cre recombinase and GFP, which were driven by the MG-specific promoter GFAP. Vector II (AAV-GFAP-CasRx) expresses CasRx, Vector III (AAV-GFAP-beta-Catenin) expresses beta-Catenin, Vector IV (AAV-GFAP-CasRx-Ptbp1) expresses CasRx and gRNAs. To induce MG-to-RGC conversion, retinas (Ai9 mice, 4-5 months old) were injected with AAV-GFAP-CasRx-Ptbp1 or control AAV-GFAP-CasRx together with AAV-GFAP-GFP-Cre. Induction of RGC conversion was examined 2-3 weeks post-injection. ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Figure 6B-E shows representative images indicating colocalization of RBPMS+tdTomato+ cells in the GCL, and tdTomato+ optic nerve for AAV-GFAP-GFP-Cre + AAV-GFAP-CasRx, AAV-GFAP-GFP-Cre + AAV-GFAP-β-catenin, AAV-GFAP-GFP-Cre + AAV-GFAP-CasRx-Ptbp1, and AAV-GFAP-GFP-Cre + AAV-GFAP-CasRx-Ptbp1 + AAV-GFAP-β-catenin groups. Note that AAV-GFAP-GFP-Cre + AAV-GFAP-CasRx and AAV-GFAP-GFP-Cre + AAV-GFAP-β-catenin are control groups. Yellow arrowhead showing the colocalization of tdTomato and RBPMS in the retinas injected with AAV-GFAP-GFP-Cre, AAV-GFAP-CasRx-Ptbp1 and AAV-GFAP-β-catenin. RBPMS is a specific marker for RGCs. The gRNA 60 was used.
Figures 7A-B depicts the conversion of astrocytes into dopamine neurons in the putamen of non-PD monkeys. Figure 7A shows representative images showing that the expression of mCherry+ colocalized with GFAP in the putamen injected with control AAVs. Figure 7B shows representative images showing a mCherry+TH+ cell in the Macaca fascicularis putamen injected with Vector 1 and 3 (yellow arrowhead) , but not in the putamen injected with control AAVs. The gRNA 60 was used.
Figures 8A-C show the induction of dopaminergic neurons in a PD model monkey. Figure 8A shows confocal images of mCherry+ cells with (AAV-GFAP-CasRx-Ptbp1) or without (AAV-GFAP-CasRx) DAT and TH signal (white arrowheads) . Note that TH is the most classical dopaminergic neuron marker, and DAT is the marker of mature dopaminergic neurons. Figure 8B shows confocal images of mCherry+ cells with (AAV-GFAP-CasRx-Ptbp1) or without (AAV-GFAP- CasRx) GIRK2 and TH signal (white arrowheads) . Note that GIRK2 is the specific marker of substantia nigra (A9 region) dopaminergic neurons. Figure 8C shows confocal images of mCherry+ cells with (AAV-GFAP-CasRx-Ptbp1) or without (AAV-GFAP-CasRx) VMAT2 and TH signal (white arrowheads) . Note that the expression of VMAT2 is essential for dopamine packaging, storage, and release. For all images, the scale bars were 50 μm.
Figure 9 shows how induced dopamine neurons alleviated the symptoms of a PD model monkey. The open field test was used to evaluate the behavioral recover ratio of the PD model monkeys, at 6 months and 9 months after injection of AAV-GFAP-CasRx-Ptbp1 +_AAV-GFAP-mCherry (red line) or control AAVs (AAV-GFAP-CasRx + AAV-GFAP-mCherry, blue line) .
Figure 10 shows how induced dopaminergic neurons improved the density of PET-CT signal in putamen of a PD model monkey. The tracer, [18F] DOPA, was used to detect the intensity of dopaminergic neurons’ signal of the PD model monkeys, at before or around one year after injection of AAV-GFAP-CasRx +AAV-GFAP-mCherry (Figures 10A) or AAV-GFAP-CasRx-Ptbp1 + AAV-GFAP-mCherry (Figures 10B) . Figures 10C shows the quantification result of PET-CT. Figures 10D shows the MRI images of the same monkey with the same planes shown in Figures 10B for anatomy structure validation. All three views of sagittal (left panel) , horizontal (middle panel) and coronal (right panel) planes were presented. Both the red arrows and the intersections indicated the putamen, the brain region for virus injection. Data was quantified as the change ratio of SUVR (n= SUVR of after injection/SUVR of before injection) , and the calculation formular of SUVR was provided in method part.
Figure 11 shows how induced dopamine neurons increased the concentration of dopamine in cerebrospinal fluid (CSF) of a PD monkey. The HPLC was used to evaluate the dopamine level of the PD monkeys, at before or after virus injection of AAV-GFAP-CasRx-Ptbp1 + AAV-GFAP-mCherry (red line) or control AAVs (AAV-GFAP-CasRx + AAV-GFAP-mCherry, blue line) . Data was quantified as the percentage of dopamine concentration (n= concentration of different time points/concentration of healthy) .
Neurodegenerative diseases are devastating diseases associated with the progressive loss of neurons in various parts of the nervous system. On the other hand, regenerative medicine has great promise for treating neurodegenerative diseases that lead to cell (e.g., neuron) loss. One approach employs cell replacement, while another utilizes cellular trans-differentiation.
Trans-differentiation takes advantage of the existing cellular plasticity of endogenous cells to generate new cell types. One challenge for this approach, however, is to identify efficient strategies to convert certain target cells to a desired cell type (e.g., neurons) , not only in culture but more importantly in their in vivo native contexts, particularly at a desired location (e.g., a tissue or organ type) .
Down regulation of a single gene, polypyrimidine tract-binding protein 1 (also known as Ptbp1, PTBP1, PTBP, or PTB) , in the striatum can directly convert astrocytes into dopaminergic neurons in mice and alleviate symptoms in a mouse model of Parkinson′s disease. However, it is unknown whether this strategy can be applied in nonhuman primates or in humans. Many experimental therapies showing the effectiveness in rodent models may be inefficient or impractical in nonhuman primates or humans; these strategies may fail for many reasons, including intrinsic biological differences or lack of scalability. Research in nonhuman primates is a key step and can be of great value in addressing these issues before clinical trials.
Definitions
The terminology used herein is for the purpose of describing particular cases only and is not intended to be limiting. As used herein, the singular forms “a” , “an, ” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms “including” , “includes” , “having” , “has” , “with” , or variants thereof are used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising. ”
The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the given value. Where particular values are described in the application and claims, unless otherwise stated the term “about” should be assumed to mean an acceptable error range for the particular value.
As used herein, the term ″and/or″ indicates that one or more of the stated cases may occur, alone or in combination with at least one of the stated cases, up to with all of the stated cases.
As used herein, with ″at least″ a particular value means that particular value or more. For example, ″at least 2″ is understood to be the same as ″2 or more″ i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, ..., etc.
″Astrocyte″ generally refers to characteristic star-shaped glial cells in the brain and spinal cord that are characterized by one or more of: star shape; expression of markers like glial fibrillary acidic protein (GFAP) , aldehyde dehydrogenase 1 family member LI (ALDH1L1) , excitatory amino acid transporter 1 /glutamate aspartate transporter (EAAT1/GLAST) , glutamine synthetase, S100 beta, or excitatory amino acid transporter 1 /glutamate transporter 1 (EAAT2/GLT-1) ; participation of the blood-brain barrier together with endothelial cells; transmitter uptake and release; regulation of ionic concentration in extracellular space; reaction to neuronal injury and participation in nervous system repair; and metabolic support of surrounding neurons. In certain embodiments, an astrocyte refers to a non-neuronal cell in a nervous system that expresses glial fibrillary acidic protein (GFAP) , Aldehyde Dehydrogenase 1 Family Member L1 (ALDH1L1) , or both. In certain embodiments, an astrocyte refers to a non-neuronal cell in a nervous system that expresses a glial fibrillary acidic protein (GFAP) promoter-driven transgene (e.g., red fluorescent protein (RFP) , Cre recombinase) . In certain embodiments, a Müller glia (MG) refers to a non-neuronal glial cell found in the retina and that expresses a MG-specific promoter-driven transgene (e.g., red fluorescent protein (RFP) ) . MG-specific promoters include promoters from GFAP, Glast (also known as Slc1a3) , and Rlbp1.
A ″BRN2 transcription factor″ or ″Brain-2 transcription factor, ″ also called ″POU domain, class 3, transcription factor 2″ ( ″POU3F2″ ) or ″Oct-7, ″ can refer to a class III POU domain transcription factor, having a DNA-binding POU domain that consists of an N-terminal POU-specific domain of about 75 amino acids and a C-terminal POU-homeodomain of about 60 amino acids, which are linked via a linker comprising a short α-helical fold, and which can be predominantly expressed in the central nervous system.
The term ″composition″ generally refers to an agent that reprograms a differentiated non-neuronal cell to a neuronal cell through inhibiting the expression and/or function of PTB and/or nPTB. In a specific embodiment, the composition refers to a CRISPR/Cas effector protein (which may or may not include any variants, derivatives, functional equivalents or fragments thereof) with a guide RNA (gRNA) complementary to a PTB mRNA or to a nPTB mRNA, and can knock down the expression and/or activity of PTB or nPTB, to an extent sufficient to convert a non-neuronal cell to a neuronal cell, preferably in vivo at a local microenvironment where the converted neuron is expected to be functional. The composition may also refer to a polynucleotide encoding such CRISPR/Cas effector protein as defined above and/or the guide RNA (gRNA) . The polynucleotide may include a mRNA for the Cas effector, as defined above. The polynucleotide may also include a DNA encoding the Cas effector as defined above and/or the gRNA complementary to PTB/nPTB mRNA. The DNA encoding the Cas effector as defined above and/or the gRNA may be part of a vector, including a viral vector (e.g., an adeno-associated viral (AAV) vector or a lentiviral vector, or any of the other viral vectors described herein) . In the case of AAV, any AAV with tropism for glial cell or non-neuronal cell in the CNS and/or PNS can be used, including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, etc. Also in the case of AAV, any Cas effector as defined above can be used, so long as the coding sequence for the Cas effector is smaller than the packaging capacity of AAV, such as 4.7 kb, 4.5 kb, 4.0 kb, 3.5 kb, 3.0 kb, 2.5 kb, 2.0 kb, 1.5 kb or less. Exemplary Cas effectors that may be used include Cas13a, Cas13b, Cas13c, Cas13d, CasRx, Cas13X, Cas13Y, Cpf1, Cas9, and functional equivalents or fragments thereof. The term ″composition″ may also be used interchangeably with the Cas effector with the gRNA, or polynucleotide (e.g., DNA or vector) encoding the same.
The Cas effector protein that can be used with the invention described herein includes CRISPR-Cas Class 2 systems utilizing a single large Cas protein to degrade target nucleic acids (e.g., mRNA) . Suitable Class 2 Cas effectors may include Type II Cas effectors such as Cas9 (e.g., Streptococcus pyogenes SpCas9 and S. thermophilus Cas9) . The suitable Cas effector may also be Class 2, Type V Cas proteins, including Cas12a (formerly known as Cpf1, such as Francisella novicida Cpf1 and Prevotella Cpf1) , C2c1 and C2c3, which lack an HNH nuclease but have RuvC nuclease activity. Particularly suitable Cas effector proteins may include Class 2, type VI Cas proteins, including Cas13 (also known as C2c2) , Cas13a, Cas13b, Cas13c, Cas13d /CasRx, Cas13X, and Cas13Y, each of which is an RNA-guided RNase (i.e., these Cas proteins use their crRNA to recognize target RNA sequences, rather than target DNA sequences as in Cas9 and Cas12a) . Overall, the CRISPR/Cas13 systems can achieve higher RNA digestion efficiency compared to the traditional RNAi and CRISPRi technologies, while simultaneously exhibiting much less off-target cleavage compared to RNAi. In one embodiment, the composition of the invention is or encodes a Cas effector protein that, together with its canonical gRNA, targets PTB or nPTB mRNA. In another embodiment, the Cas effector targets PTB or nPTB DNA.
The term ″contacting″ cells with a composition of the disclosure refers to placing the composition (e.g., a compound, a nucleic acid, a viral vector, etc. ) in a location that will allow it to touch the cell in order to produce ″contacted″ cells. The contacting may be accomplished using any suitable method. For example, in one embodiment, contacting is by adding the compound to a culture of cells. Contacting may also be accomplished by injection or delivering the composition to a location within a body such that the composition ″contacts″ the targeted cell type.
The term ″differentiation, ″ ″differentiate, ″ ″converting, ″ or ″inducing differentiation″ are used interchangeably to refer to changing the default cell type (genotype and/or phenotype) to a non-default cell type (genotype and/or phenotype) . Thus ″inducing differentiation in an astrocyte cell″ can refer to inducing the cell to change its morphology from that of an astrocyte to that of a neuronal cell type (e.g., a change in gene expression as determined by genetic analysis such as a microarray) and/or phenotype (e.g., a change in protein expression) .
As used herein, ″an effective amount″ can refer to the amount of an agent required to ameliorate the symptoms of a disease relative to an untreated patient. The effective amount (s) of active agent (s) used to practice the present invention for therapeutic treatment of a disease (e.g., a cancer) varies depending upon the manner of administration, age, body weight, and/or general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an ″effective″ amount, which may be determined as genome copies per kilogram (GC/kg) . Thus, in connection with the administration of a drug which, in the context of the current disclosure, is ″effective against″ a disease or condition indicates that administration in a clinically appropriate manner results in a beneficial effect for at least a statistically significant fraction of patients, such as an improvement of symptoms, a cure, a reduction in at least one disease sign or symptom, extension of life, improvement in quality of life, or other effect generally recognized as positive by medical doctors familiar with treating the particular type of disease or condition.
The term ″expression control sequence″ is intended to include, at a minimum, a sequence whose presence is designed to influence expression, and can also include additional advantageous components. For example, leader sequences and fusion partner sequences can be expression control sequences. The term can also include the design of a nucleic acid sequence such that undesirable potential initiation codons in and out of frame are removed from the sequence. It can also include the design of the nucleic acid sequence such that undesirable potential splice sites are removed. It can include sequences or polyadenylation sequences (pA) which direct the addition of a polyA tail (i.e., a string of adenine residues at the 3′-end of an mRNA, commonly referred to as polyA sequences) . It also can be designed to enhance mRNA stability. Expression control sequences which affect the transcription and translation stability, e.g., promoters, as well as sequences which affect the translation, e.g., Kozak sequences, are known in the art per se. Expression control sequences can be of such nature as to modulate the nucleotide sequence to which they are operably linked such that lower expression levels or higher expression levels are achieved.
The term ″gene″ means a DNA fragment comprising a region (transcribed region) , which is transcribed into an RNA molecule (e.g., an mRNA) in a cell, operably linked to suitable regulatory regions (e.g., a promoter) . A gene can comprise several operably linked fragments, such as a promoter, a 5′ leader sequence, a coding region, and/or a 3′-nontranslated sequence (3′-end) comprising a polyadenylation site. ″Expression of a gene″ refers to the process wherein a DNA region, which is operably linked to appropriate regulatory regions, such as a promoter, is transcribed into a RNA, which is biologically active, i.e., which is capable of being translated into a biologically active protein or peptide.
The term ″glial cell″ can generally refer to a type of supportive cell in the central nervous system (e.g., the brain and spinal cord) or the peripheral nervous system. In some embodiments, glial cells do not conduct electrical impulses or exhibit action potential. In some embodiments, glial cells do not transmit information with each other, or with neurons via synaptic connection or electrical signals. In a nervous system or in an in vitro culture system, glial cells can surround neurons and provide support for and insulation between neurons. Non-limiting examples of glial cells include oligodendrocytes, astrocytes, Müller Glia, ependymal cells, Schwann cells, microglia, spiral ganglion glial cells, and satellite cells.
A ″guide sequence″ is to be understood herein as a sequence that directs an RNA or DNA guided endonuclease to a specific site in an RNA or DNA molecule. In the context of a guide RNA (gRNA) -Cas complex, a ″guide sequence″ is further understood herein as the section of gRNA (or crRNA) , which is required for targeting a gRNA-Cas complex to a specific site in the target RNA or DNA molecule. The ″guide sequence″ in a gRNA is complementary to a specific site in the target RNA or DNA molecule; said site is the ″target sequence″ .
The term ″homologous, ″ when used to indicate the relationship between a given (recombinant) nucleic acid or polypeptide molecule and a given host organism or host cell, is understood to mean that, in nature, the nucleic acid or polypeptide molecule is produced by a host cell or organisms of the same species, preferably of the same variety or strain. If homologous to a host cell, a nucleic acid sequence encoding a polypeptide will typically (but not necessarily) be operably linked to another (heterologous) promoter sequence and, if applicable, an additional (heterologous) secretory signal sequence and/or terminator sequence than in its natural environment. It is understood that the regulatory sequences, signal sequences, terminator sequences, etc. may also be homologous to the host cell. In this context, the use of only ″homologous″ sequence elements allows the construction of ″self-cloned″ genetically modified organisms (GMOs) (self-cloning is defined herein as in European Directive 98/81/EC Annex II) . When used to indicate the relatedness of two nucleic acid sequences, the term ″homologous″ means that one single-stranded nucleic acid sequence may hybridize to a complementary single-stranded nucleic acid sequence. The degree of hybridization may depend on a number of factors including the amount of identity between the sequences and the hybridization conditions such as temperature and salt concentration.
The terms ″heterologous″ and ″exogenous, ″ when used with respect to a nucleic acid (DNA or RNA) or protein, refer to a nucleic acid or protein that does not occur naturally as part of the organism, cell, genome or DNA or RNA sequence in which it is present, or that is found in a cell or location or locations in the genome or DNA or RNA sequence that differ from that in which it is found in nature. Heterologous and exogenous nucleic acids or proteins are not endogenous to the cell into which they are introduced but have been obtained from another cell or are synthetically or recombinantly produced. Generally, though not necessarily, such nucleic acids encode proteins, i.e., exogenous proteins, that are not normally produced by the cell in which the DNA is transcribed or expressed. Similarly, exogenous RNA encodes for proteins not normally expressed in the cell in which the exogenous RNA is present. Heterologous/exogenous nucleic acids and proteins may also be referred to as foreign nucleic acids or proteins. Any nucleic acid or protein that one of skill in the art would recognize as foreign to the cell in which it is expressed is herein encompassed by the terms heterologous or exogenous nucleic acid or protein. The terms heterologous and exogenous also apply to non-natural combinations of nucleic acid or amino acid sequences, e.g., combinations where at least two of the combined sequences are foreign with respect to each other.
A ″microRNA″ or ″miRNA″ refers to a non-coding nucleic acid (RNA) sequence that binds to at least partially complementary nucleic acid sequence (mRNAs) and negatively regulates the expression of the target mRNA at the post-transcriptional level. A microRNA is typically processed from a ″precursor″ miRNA having a double-stranded, hairpin loop structure to a ″mature″ form. Typically, a mature microRNA sequence is about 19-25 nucleotides in length.
″miR-9″ is a short non-coding RNA gene involved in gene regulation and highly conserved from Drosophila and mice to humans. The mature ~21nt miRNAs are processed from hairpin precursor sequences by the Dicer enzyme. miR-9 can be one of the most highly expressed microRNAs in the developing and adult vertebrate brain. Key transcriptional regulators such as FoxGl, Hesl or Tlx, can be direct targets of miR-9, placing it at the core of the gene network controlling the neuronal progenitor state.
The term ″neuron″ or ″neuronal cell″ as used herein can have the ordinary meaning one skilled in the art would appreciate. In some embodiments, neuron can refer to an electrically excitable cell that can receive, process, and transmit information through electrical signals (e.g., membrane potential discharges) and chemical signals (e.g., synaptic transmission of neurotransmitters) . As one skilled in the art would appreciate, the chemical signals (e.g., based on release and recognition of neurotransmitters) transduced between neurons can occur via specialized connections called synapses.
The term ″mature neuron″ can refer to a differentiated neuron. In some embodiments, a neuron is said to be a mature neuron if it expresses one or more markers of mature neurons, e.g., microtubule-associated protein 2 (MAP2) and neuronal nuclei (NeuN) , neuron specific enolase (NSE) , 160 kDa neurofilament medium, 200 kDa neurofilament heavy, postsynaptic density protein 95 (PDS-95) , synapsin I, synaptophysin, glutamate decarboxylase 67 (GAD67) , glutamate decarboxylase 67 (GAD65) , parvalbumin, dopamine-and cAMP-regulated neuronal phosphoprotein 32 (DARPP32) , vesicular glutamate transporter 1 (vGLUT1) , vesicular glutamate transporter 2 (vGLUT2) , acetylcholine, tyrosine hydroxylase (TH) , etc.
The term ″functional neuron″ can refer to a neuron that is able to send or receive information through chemical or electrical signals. In some embodiments, a functional neuron exhibits one or more functional properties of a mature neuron that exists in a normal nervous system, including, but not limited to: excitability (e.g., ability to exhibit action potential, or a rapid rise and subsequent fall in voltage or membrane potential across a cellular membrane) , forming synaptic connections with other neurons, pre-synaptic neurotransmitter release, or post-synaptic response (e.g., excitatory postsynaptic current or inhibitory postsynaptic current) .
In some embodiments, a functional neuron is characterized in its expression of one or more markers of functional neurons, including, but not limited to, synapsin, synaptophysin, glutamate decarboxylase 67 (GAD67) , glutamate decarboxylase 67 (GAD65) , parvalbumin, dopamine-and cAMP-regulated neuronal phosphoprotein 32 (DARPP32) , vesicular glutamate transporter 1 (vGLUT1) , vesicular glutamate transporter 2 (vGLUT2) , acetylcholine, tyrosine hydroxylase (TH) , dopamine, vesicular GABA transporter (VGAT) , and gamma-aminobutyric acid (GABA) .
The term ″non-neuronal cell″ can refer to any type of cell that is not a neuron. An exemplary non-neuronal cell is a cell that is of a cellular lineage other than a neuronal lineage (e.g., a hematopoietic lineage) . In some embodiments, a non-neuronal cell is a cell of neuronal lineage but not a neuron, for example, a glial cell. In some embodiments, a non-neuronal cell is somatic cell that is not neuron, such as, but not limited to, a glial cell, an adult primary fibroblast, an embryonic fibroblast, an epithelial cell, a melanocyte, a keratinocyte, an adipocyte, a blood cell, a bone marrow stromal cell, a Langerhans cell, a muscle cell, a rectal cell, or a chondrocyte. In some embodiments, a non-neuronal cell is from a non-neuronal cell line, such as, but not limited to, a glioblastoma cell line, a Hela cell line, a NT2 cell line, an ARPE19 cell line, or a N2A cell line.
″Cell lineage″ or ″lineage″ can denote the developmental history of a tissue or organ from the fertilized embryo. ″Neuronal lineage″ can refer to the developmental history from a neural stem cell to a mature neuron, including the various stages along this process (also known as neurogenesis) , such as, but not limited to, neural stem cells (neuroepithelial cells, radial glial cells) , neural progenitors (e.g., intermediate neuronal precursors) , neurons, astrocytes, oligodendrocytes, and microglia.
As used herein, the term ″non-naturally occurring″ when used in reference to an organism means that the organism has at least one genetic alternation that is not normally found in a naturally occurring strain of the referenced species, including wild-type strains of the referenced species. Genetic alterations include, for example, modifications introducing expressible nucleic acids encoding proteins or enzymes, other nucleic acid additions, nucleic acid deletions, nucleic acid substitutions, or other functional disruption of the organism′s genetic material. Such modifications include, for example, coding regions and functional fragments thereof for heterologous or homologous polypeptides for the referenced species. Additional modifications include, for example, non-coding regulatory regions in which the modifications alter expression of a gene or operon. Genetic modifications to nucleic acid molecules encoding enzymes, or functional fragments thereof, can confer a biochemical reaction capability or a metabolic pathway capability to the non-naturally occurring organism that is altered from its naturally occurring state.
The terms ″nucleic acid″ and ″polynucleotide, ″ as used interchangeably herein, can refer to deoxyribonucleotides or ribonucleotides and polymers thereof in either single-or double-stranded form. The term can encompass nucleic acids containing known nucleotide analogs or modified backbone residues or linkages which are synthetic, naturally occurring, or non-naturally occurring; which have similar binding properties as the reference nucleic acid; and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, locked nucleic acids (LNAs) , and peptide-nucleic acids (PNAs) .
A ″nucleic acid construct″ or ″nucleic acid vector″ is herein understood to mean a man-made nucleic acid molecule resulting from the use of recombinant DNA technology. The term ″nucleic acid construct″ therefore does not include naturally-occurring nucleic acid molecules, although a nucleic acid construct may comprise naturally occurring nucleic acid molecules or fragments thereof. A ″vector″ is a nucleic acid construct (typically DNA or RNA) that serves to transfer an exogenous nucleic acid sequence (i.e., DNA or RNA) into a host cell. A vector is preferably maintained in the host by at least one of: autonomous replication or integration into the host cell′s genome. The terms ″expression vector″ or ″expression construct″ refer to nucleotide sequences that are capable of affecting expression of a gene in host cells or host organisms compatible with such sequences. These expression vectors typically include at least one ″expression cassette″ that is the functional unit capable of affecting expression of a sequence encoding a product to be expressed, and wherein the coding sequence is operably linked to the appropriate expression control sequences which at least comprise a suitable transcription regulatory sequence and/or 3′ transcription termination signals. Additional factors necessary for or helpful in affecting expression, such as expression enhancer elements, may also be present. The expression vector can be introduced into a suitable host cell and can be able to affect expression of the coding sequence in an in vitro cell culture of the host cell. An expression vector can be suitable for expression of viral proteins and/or nucleic acids, particularly recombinant AAV proteins and/or nucleic acids.
″Oligodendrocyte″ can refer to a type of glial cell that can create the myelin sheath that surrounds a neuronal axon to provide support and insulation to axons in the central nervous system. An oligodendrocyte can also be characterized in its expression of PDGF receptor alpha (PDGFR-α) , SOXIO, neural/glial antigen 2 (NG2) , Olig1, Olig2, Olig3, oligodendrocyte specific protein (OSP) , Myelin basic protein (MBP) , or myelin oligodendrocyte glycoprotein (MOG) .
″Polypyrimidine tract binding protein″ or ″PTB″ and its homolog, neural PTB (nPTB) , are both ubiquitous RNA-binding proteins. PTB can also be called polypyrimidine tract-binding protein 1, and in humans is encoded by the PTBP1 gene. PTBP1 belongs to the subfamily of ubiquitously expressed heterogeneous nuclear ribonucleoproteins (hnRNPs) .
The hnRNPs are RNA-binding proteins that complex with heterogeneous nuclear RNA (hnRNA) . These proteins are associated with pre-mRNAs in the nucleus and appear to influence pre-mRNA processing and other aspects of mRNA metabolism and transport. PTB can have four repeats of quasi-RNA recognition motif (RRM) domains that bind RNAs. Consistent with its widespread expression, PTB can contribute to the repression of a large number of alternative splicing events. PTB can recognize short RNA motifs, such as UCUU and UCUCU, located within a pyrimidine-rich context and is often associated with the polypyrimidine tract upstream of the 3′ splice site of both constitutive and alternative exons. In some cases, binding sites for PTB can also include exonic sequences and sequences in introns downstream of regulated exons.
In most alternative splicing systems regulated by PTB, repression can be achieved through the interaction of PTB with multiple PTB binding sites surrounding the alternative exon. In some cases, repression can involve a single PTB binding site. Splicing repression by PTB can occur by a direct competition between PTB and U2AF65, which, in turn, can preclude the assembly of the U2 snRNP on the branch point. In some cases, splicing repression by PTB can involve PTB binding sites located on both sides of alternative exons and can result from cooperative interactions between PTB molecules that would loop out the RNA, thereby making the splice sites inaccessible to the splicing machinery. Splicing repression by PTB can also involve multimerization of PTB from a high-affinity binding site which can create a repressive wave that covers the alternative exon and prevents its recognition.
PTB can be widely expressed in non-neuronal cells, while nPTB can be restricted to neurons. PTB and nPTB can undergo a programmed switch during neuronal differentiation. For example, during neuronal differentiation, PTB is gradually downregulated at the neuronal induction stage; coincidentally or consequentially, nPTB levels are gradually up-regulated to a peak level. Later, when the neuronal differentiation enters into neuronal maturation stage, nPTB level experiences reduction after its initial rise and then returns to a relatively low level as compared to its peak level during neuronal differentiation, when the cell develops into a mature neuron.
The terms ″protein, ″ ″peptide, ″ and ″polypeptide″ are used interchangeably, and can refer to an amino acid polymer or a set of two or more interacting or bound amino acid polymers, without reference to a specific mode of action, size, 3-dimensional structure, or origin.
As used herein, the term ″promoter″ or ″transcription regulatory sequence″ refers to a nucleic acid fragment that functions to control the transcription of one or more coding sequences and which is located upstream with respect to the direction of transcription of the transcription initiation site of the coding sequence. A promoter can be structurally identified by the presence of a binding site for a DNA-dependent RNA polymerase, transcription initiation sites, and/or any other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, enhancers, and any other sequence or sequences of nucleotides known to one of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter. A ″constitutive″ promoter is a promoter that is active in most tissues under most environmental and developmental conditions. An ″inducible″ promoter is a promoter that is environmentally or developmentally regulated, e.g. by the application of a chemical inducer or biological entity.
As used herein, the term ″operably linked″ refers to a linkage of polynucleotide (or polypeptide) elements in a functional relationship. A nucleic acid can be ″operably linked″ when it is placed into a functional relationship with another nucleic acid sequence. For instance, a transcription regulatory sequence can be operably linked to a coding sequence if it affects the transcription of the coding sequence. Operably linked can mean that the DNA sequences being linked are contiguous and, where necessary to join two protein encoding regions, contiguous and in reading frame. An expression control sequence is ″operably linked″ to a nucleotide sequence when the expression control sequence controls and regulates the transcription and/or the translation of the nucleotide sequence. Thus, an expression control sequence can include promoters, enhancers, internal ribosome entry sites (IRES) , transcription terminators, a start codon in front of a protein-encoding gene, splicing signals for introns, and stop codons.
The term ″reprogramming″ or ″trans-differentiation″ can refer to the generation of a cell of a certain lineage (e.g., a neuronal cell) from a different type of cell (e.g., a fibroblast cell) without an intermediate process of de-differentiating the cell into a cell exhibiting pluripotent stem cell characteristics.
″Pluripotent″ can refer to the ability of a cell to form all lineages of the body or soma (i.e., the embryo proper) . Exemplary ″pluripotent stem cells″ can include embryonic stem cells and induced pluripotent stem cells.
The terms ″sequence identity, ″ ″homology, ″ and the like are used interchangeably herein. Sequence identity is herein defined as a relationship between two or more amino acid (polypeptide or protein) sequences and/or two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences. In the art, ″identity″ also means the degree of sequence relatedness between amino acid or nucleic acid sequences, as the case may be, as determined by the match between strings of such sequences. ″Similarity″ between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide. ″Identity″ and ″similarity″ can be readily calculated by known methods.
″Sequence identity″ and ″sequence similarity″ can be determined by alignment of two peptide or two nucleotide sequences using global or local alignment algorithms, depending on the length of the two sequences. Sequences of similar lengths are preferably aligned using global alignment algorithms (e.g., Needleman Wunsch) which align the sequences optimally over the entire length, while sequences of substantially different lengths are preferably aligned using local alignment algorithms (e.g., Smith Waterman) . Sequences may then be referred to as ″substantially identical″ or ″essentially similar″ when they (when optimally aligned by, for example, the programs GAP or BESTFIT using default parameters) share at least a certain minimal percentage of sequence identity. GAP uses the Needleman and Wunsch global alignment algorithm to align two sequences over their entire length (full length) , maximizing the number of matches and minimizing the number of gaps. A global alignment is suitably used to determine sequence identity when the two sequences have similar lengths. The GAP default parameters can be used, with a gap creation penalty = 50 (nucleotides) /8 (proteins) and gap extension penalty = 3 (nucleotides) /2 (proteins) . For nucleotides the default scoring matrix used is nwsgapdna and for proteins the default scoring matrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919) . Sequence alignments and scores for percentage sequence identity may be determined using computer programs, such as the GCG Wisconsin Package, Version 10.3, available from Accelrys Inc., 9685 Scranton Road, San Diego, CA 92121-3752 USA, or using open source software, such as the program ″needle″ (using the global Needleman Wunsch algorithm) or ″water″ (using the local Smith Waterman algorithm) in EmbossWIN version 2.10.0, using the same parameters as for GAP above, or using the default settings (both for ′needle′ and for ′water′ and both for protein and for DNA alignments, the default Gap opening penalty is 10.0 and the default gap extension penalty is 0.5; default scoring matrices are Blossum62 for proteins and DNAFull for DNA) . When sequences have a substantially different overall length, local alignments, such as those using the Smith Waterman algorithm, are preferred. Alternatively, percentage similarity or identity may be determined by searching against public databases, using algorithms such as FASTA or BLAST, etc. Thus, the nucleic acid and protein sequences of the present invention can be further used as a ″query sequence″ to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the BLASTn and BLASTx programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215: 403-10. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to nucleic acid molecules of the invention. BLAST protein searches can be performed with the BLASTx program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25 (17) : 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., BLASTx and BLASTn) can be used. See the homepage of the National Center for Biotechnology Information at www. ncbi. nlm. nih. gov/.
The terms ″subject″ and ″patient″ as used interchangeably can refer to, except where indicated, mammals such as humans and non-human primates, as well as rabbits, rats, mice, goats, pigs, and other mammalian species. The term does not necessarily indicate that the subject has been diagnosed with a particular disease, but instead can refer to an individual under medical supervision.
A ″target sequence″ denotes an order of nucleotides within a nucleic acid that is to be targeted (e.g., wherein an alteration is to be introduced or to be detected) . In the context of a guide RNA (gRNA) -CAS complex, a ″target sequence″ is further to be understood herein as the section within the RNA or DNA molecule that is to be targeted by the gRNA-CAS complex by its complementarity to the ″guide sequence″ in the gRNA. Likewise, an antisense oligonucleotide or miRNA is targeted by its complementarity to the ″target sequence″ within the RNA or DNA molecule that is to be targeted. For example, the target sequence can be an order of nucleotides comprised by a first strand of a DNA duplex. mammalian species that can benefit from the disclosed methods and composition can include, but are not limited to, primates, such as apes, chimpanzees, orangutans, and humans.
A ″vector″ is a nucleic acid that can be capable of transporting another nucleic acid into a cell. A vector can be capable of directing expression of a protein or proteins encoded by one or more genes, or a microRNA encoded by a polynucleotide, carried by the vector when it is present in the appropriate environment.
The use of a substance as a medicament as described in this document can also be interpreted as the use of said substance in the manufacture of a medicament. Similarly, whenever a substance is used for treatment or as a medicament, it can also be used for the manufacture of a medicament for treatment. Products for use as a medicament described herein can be used in methods of treatments, wherein such methods of treatment comprise the administration of the product for use.
A ″viral vector″ is a viral-derived nucleic acid that can be capable of transporting another nucleic acid into a cell. A viral vector can be capable of directing expression of a protein or proteins encoded by one or more genes, or a microRNA encoded by a polynucleotide, carried by the vector when it is present in the appropriate environment. Examples of viral vectors include, but are not limited to, retroviral, adenoviral, lentiviral and adeno-associated viral vectors.
General compositions
Described herein is a composition for regulating a primate PTBP gene expression comprising: a Cas effector protein, or a nucleic acid encoding the same, and a guide RNA (gRNA) that targets a target sequence, or its complement, of the primate PTBP gene or a transcript thereof, or a nucleic acid encoding the same. In some embodiments, the composition described herein is capable of suppressing the expression of the primate PTBP gene. A composition described herein can comprise a nucleic acid molecule that targets a target sequence or its complement in a primate PTB gene, transcript, or mRNA in order to suppress the expression or activity of PTB in the non-neuronal cell. This suppression allows the non-neural cell to reprogram into a functional neuron.
In one embodiment, the nucleic acid molecule in the composition comprising the composition can be a nucleic acid molecule that targets a target sequence in a primate PTB gene, transcript, or mRNA; or in case of a double-stranded PT, DNA, or RNA sequence, the nucleic acid molecule can target either one of the two complementary strands. Targeting of the target sequence (or its complement) in a primate PTB gene, transcript, or mRNA by the nucleic acid molecule is understood to mean that at least a part of the nucleic acid molecule is substantially complementary to the target sequence in the primate PTB nucleic acid, such that it can base pair with the primate PTB nucleic acid, under, for example, physiological conditions, so as to exerts its biological effect, i.e. to suppress the expression or activity of PTB in the non-neuronal cell.
Target sequences can be identified within a primate or human PTB gene, transcript, or mRNA efficiency target sequence, the targeting of which by the nucleic acid molecules of the invention ensures high efficiency suppression of the expression or activity of PTB in the non-neuronal cell.
In one embodiment, the composition comprises: i) a CRISPR/Cas effector protein and a gRNA complementary to a Polypyrimidine Tract Binding protein (PTB) mRNA or ii) at least one expression vector encoding a CRISPR/Cas effector protein and a guide RNA (gRNA) complementary to a PTB mRNA.
According to some embodiments of the disclosure, a single composition (e.g., Cas/gRNA) that suppresses the expression and/or activity of PTB/nPTB in a non-human primate or human non-neuronal cell (e.g., MG cell in mature retina or astrocyte in striatum) can directly convert the non-neuronal cell into a mature neuron (e.g., a retinal ganglion cell (RGC) neuron, a retinal photoreceptor, or dopamine neuron, respectively) . In some embodiments, the direct conversion of a non-neuronal cell into a neuron by a single composition (e.g., Cas/gRNA) can mean that the conversion of the non-neuronal cell into the neuron requires no other intervention than contacting with the single composition.
According to the disclosure, in some cases, PTB reduction can induce a number of key neuronal differentiation factors. For example, without wishing to be bound to a certain theory, PTB and nPTB can be involved in two separate but intertwined loops, that can be important in neuronal differentiation. PTB can suppress a neuronal induction loop in which the microRNA miR-124 can inhibit the transcriptional repressor RE1-Silencing Transcription factor (REST) , which in turn can block the induction of miR-124 and many neuronal-specific genes (loop I) . During a normal neuronal differentiation process, PTB can be gradually downregulated, and the PTB down-regulation can thus induce the expression of nPTB, which is part of a second loop for neuronal maturation that includes the transcription activator Brn2 and miR-9 (loop II) . In loop II, nPTB can inhibit Brn2 and consequentially can inhibit miR-9, and miR-9 in turn can inhibit nPTB.
According to some embodiments, the expression level of miR-9 or Brn2 in a non-neuronal cell can affect the conversion of the non-neuronal cell into a mature neuron by a composition that suppresses the expression or activity of PTB in the non-neuronal cell. For example, a human adult fibroblast cell can have a low expression level of miR-9 and Brn2. In some embodiments, a single agent that suppresses the expression or activity of PTB in a human adult fibroblast cell can induce the human adult fibroblast cell to differentiate into a neuron-like cell, e.g., expression of Tuj1 protein, but not into a mature neuron, e.g., expression of NeuN protein or other markers of a mature neuron.
Without wishing to be bound by a particular theory, the subject method and composition in some embodiments are particularly effective in creating a reinforcing feedback loop in molecular changes that direct the conversion of a non-neuronal cell into a neuron. Without wishing to be bound by a particular theory, when PTB expression or activity is initially downregulated by an exogenous anti-PTB agent, which can in turn lead to upregulation of miR-124 level.
Without wishing to be bound by a particular theory, in some cases, because miR-124 can target and inhibit the expression of PTB, the upregulated miR-124 can thus reinforce the inhibition of PTB in the cell; such a positive reinforcing effect can be long-lasting, even though in some cases, the anti-PTB agent, e.g., an antisense oligonucleotide against PTB, may be present and active merely temporarily in the cell.
According to some embodiments of the disclosure, a single composition (e.g., a Cas with PTB/nPTB-targeting gRNA or a polynucleotide encoding the same) that suppresses the expression or activity of PTB/nPTB in a human non-neuronal cell can directly convert the non-neuronal cell into a mature neuron, optionally when the human non-neuronal cell expresses miR-9 or Brn2 at a level that is higher than that expressed in a human adult fibroblast.
An exemplary human non-neuronal cell that can be used in the method provided herein expresses miR-9 or Brn2 at a level that is at least two times higher than that expressed in a human adult fibroblast. In some embodiments, the human non-neuronal cell expresses miR-9 or Brn2 at a level that is at least about 1.2, 1.5, 2, 5, 10, 15, 20 or 50 times higher than that expressed in a human adult fibroblast.
In some embodiments, a single composition that suppresses the expression or activity of PTB/nPTB (e.g., Cas with PTB/nPTB-targeting gRNA) in a human non-neuronal cell can directly convert the non-neuronal cell into a mature neuron, when the human non-neuronal cell expresses both miR-9 and Brn2 at a level that is higher than that expressed in a human adult fibroblast.
An exemplary human non-neuronal cell that can be used in the method as provided herein express both miR-9 and Brn2 at a level that is at least two times higher than that expressed in a human adult fibroblast. In some embodiments, the human non-neuronal cell expresses both miR-9 and Brn2 at a level that is at least about 1.2, 1.5, 2, 5, 10, 15, 20 or 50 times higher than that expressed in a human adult fibroblast.
In some embodiments, a single composition (e.g., Cas with PTB/nPTB-targeting gRNA) that suppresses the expression or activity of PTB/nPTB in a human non-neuronal cell can directly convert the non-neuronal cell into a mature neuron, when the human non-neuronal cell expresses endogenous miR-9 or endogenous Brn2 at a level that is higher than that expressed in a human adult fibroblast. In some embodiments, no exogenous miR-9 is introduced into the human non-neuronal cell. In some embodiments, no exogenous Brn2 is introduced into the human non-neuronal cell.
In some embodiments, the expression level of miR-9 or Brn 2 in a non-neuronal cell can be assessed by any technique one skilled in the art would appreciate. For example, the expression level of miR-9 in a cell can be measured by reverse transcription (RT) -polymerase chain reaction (PCR) , miRNA array, RNA sequencing (RNA-seq) , and multiplex miRNA assays. Expression levels of miR-9 can also be assayed by in situ methods like in situ hybridization. Expression levels of Brn2 as a protein can be assayed by conventional techniques, like Western blot, enzyme-linked immunosorbent assay (ELISA) , and immunostaining, or by other techniques, such as, but not limited to, protein microarray and spectrometry methods (e.g., high performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC/MS) ) . In some embodiments, information on the expression level of miR-9 in a cell or a certain type of tissue/cells can be obtained by referring to publicly available databases for microRNAs, such as, but not limited to, Human MiRNA Expression Database (HMED) , miRGator 3.0, miRmine, and PhenomiR. In some embodiments, information on the expression level of miR-9 in a cell or a certain type of tissue/cells can be obtained by referring to publicly available databases for protein expression, including, but not limited to, The Human Protein Atlas, GeMDBJ Proteomics, Human Proteinpedia, and Kahn Dynamic Proteomics Database.
In some embodiments, in a human astrocyte, a single composition (e.g., Cas with PTB-targeting gRNA) that suppresses the expression or activity of PTB in the human astrocyte leads to immediate decrease in expression or activity of PTB, an initial increase in expression level of nPTB, and a subsequent decrease in expression level of nPTB. In some embodiments, a single composition (e.g., Cas with PTB-targeting gRNA) that suppresses the expression or activity of PTB directly converts a human astrocyte to a mature neuron. In some embodiments, the expression level of miR-9 or Brn2 in the non-neuronal cell can be correlated with whether or not nPTB expression level in the non-neuronal cell decreases after the initial increase following PTB expression or activity is suppressed by a composition. For instance, in human astrocytes, where miR-9 or Brn2 is expressed at a higher level than a human adult fibroblast, nPTB expression level in the non-neuronal cells decreases after the initial increase following PTB expression or activity is suppressed by a composition, while in human adult fibroblast, as described above, in some cases, the subsequent decrease in nPTB expression level may not happen.
Non-neuronal cells
According to some embodiments, an exemplary non-neuronal cell that can be reprogrammed into a mature neuron herein can include a glial cell, such as, but not limited, an astrocyte, an oligodendrocyte, an ependymal cell, a microglia, a Muller glia, a spiral ganglion glial cell, a Schwan cell, a NG2 cell, and a satellite cell. In some embodiments, a glial cell can be a primate glial cell, for instance, a human glial cell or a non-human primate glial cell. Preferably the glial cell is a primate astrocyte, for instance, a human astrocyte or a non-human primate astrocyte.
In some embodiments, a glial cell that can be used herein is a glial cell isolated from a brain. In some embodiments, a glial cell is a glial cell in a cell culture, for instance, divided from a parental glial cell. In some embodiments, a glial cell as provided herein is a glial cell differentiated from a different type of cell under external induction, for instance, differentiated in vitro from a neuronal stem cell in a culture medium containing differentiation factors, or differentiated from an induced pluripotent stem cell. In some other embodiments, a glial cell is a glial cell in a nervous system, for example, a MG cell in the mature retina, or an astrocyte residing in a brain region, such as in the striatum.
In some embodiments, an astrocyte that can be herein is a glial cell that is of a star-shape in brain or spinal cord. In some embodiments, an astrocyte expresses one or more of well-recognized astrocyte markers, including, but not limited to, glial fibrillary acidic protein (GFAP) and aldehyde dehydrogenase 1 family member LI (ALDH1L1) , excitatory amino acid transporter 1 /glutamate aspartate transporter (EAAT1/GLAST) , glutamine synthetase, S100 beta, or excitatory amino acid transporter 1 /glutamate transporter 1 (EAAT2/GLT-1) . In some embodiments, an astrocyte expresses glial fibrillary acidic protein (GFAP) , Aldehyde Dehydrogenase 1 Family Member LI (ALDH1L1) , or both. In certain embodiments, an astrocyte is a non-neuronal cell in a nervous system that expresses a glial fibrillary acidic protein (GFAP) promoter-driven transgene (e.g., red fluorescent protein (RFP) , Cre recombinase) . In some embodiments, an astrocyte as described herein is not immunopositive for neuronal markers, e.g., Tuj1, NSE, NeuN, GAD67, VGluTl, or TH. In some embodiments, an astrocyte as described herein is not immunopositive for oligodendrocyte markers, e.g., Oligodendrocyte Transcription Factor 2, OLIG2. In some embodiments, an astrocyte as described herein is not immunopositive for microglia markers, e.g., transmembrane protein 119 (TMEM119) , CD45, ionized calcium binding adapter molecule 1 (Ibal) , CD68, CD40, F4/80, or CD11 Antigen-Like Family Member B (CDllb) . In some embodiments, an astrocyte as described herein is not immunopositive for NG2 cell markers (e.g., Neural/glial antigen 2, NG2) . In some embodiments, an astrocyte as described herein is not immunopositive for neural progenitor markers, e.g., Nestin, CXCR4, Musashi, Notch-1, SRY-Box 1 (SOX1) , SRY-Box 2 (SOX2) , stage-specific embryonic antigen 1 (SSEA-1, also called CD15) , or Vimentin. In some embodiments, an astrocyte as described herein is not immunopositive for pluripotency markers, e.g., NANOG, octamer-binding transcription factor 4 (Oct-4) , SOX2, Kruppel Like Factor 4 (KLF4) , SSEA-1, or stage-specific embryonic antigen 4 (SSEA-4) . In some embodiments, an astrocyte as described herein is not immunopositive for fibroblast markers (e.g. Fibronectin) .
Astrocytes can include different types or classifications. The methods of the invention are applicable to different types of astrocytes. Non-limiting example of different types of astrocytes include type 1 astrocyte, which can be Ran2+, GFAP+, fibroblast growth factor receptor 3 positive (FGFR3+) , and A2B5. Type 1 astrocytes can arise from the tripotential glial restricted precursor cells (GRP) . Type 1 astrocytes may not arise from the bipotential 02A/0PC (oligodendrocyte, type 2 astrocyte precursor) cells. Another non limiting example includes type 2 astrocyte, which can be A2B5+, GFAP+, FGFR3-, and Ran2. Type 2 astrocytes can develop in vitro from either tripotential GRP or from bipotential 02A cells or in vivo when these progenitor cells are transplanted into lesion sites. Astrocytes that can be used in the method provided herein can be further classified based their anatomic phenotypes, for instance, protoplasmic astrocytes that can be found in grey matter and have many branching processes whose end-feet envelop synapses or fibrous astrocytes that can be found in white matter and can have long thin unbranched processes whose end-feet envelop nodes of Ranvier. Astrocytes that can be used in the methods provided herein can also include GluT type and GluR type. GluT type astrocytes can express glutamate transporters (EAAT1/SLC1A3 and EAAT2/SLC1A2) and respond to synaptic release of glutamate by transporter currents, while GluR type astrocytes can express glutamate receptors (mostly mGluR and AMPA type) and respond to synaptic release of glutamate by channel mediated currents and IP3-dependent Ca2+ transients.
Target sequences
In certain embodiments, the nucleic acid molecule (e.g. a gRNA) targets a target sequence in a primate PTB gene sequence, such as a non-human primate PTB gene sequence or a human PTB gene sequence. Examples of primate PTB gene sequences to be targeted include the human PTBP1 gene sequence (GenBank ID 5725) .
The sequences of primate and human PTB and nPTB are known (see e.g., Romanelli et al. (2005) Gene, August 15: 356: 11-8; Robinson et al., PLoS One. 2008 Mar. 12; 3 (3) : e1801. doi: 10.1371/journal. pone. 0001801; Makeyev et al., Mol. Cell (2007) August 3; 27 (3) : 435-48) . miRNA, siRNA, and guide RNA molecules can be designed and constructed to modulate (e.g., to decrease, to inhibit, or to overexpress) the expression of a primate PTB and/or nPTB.
In some embodiments, the nucleic acid molecule (e.g. gRNA) targets a target sequence in the primate PTB gene, transcript, or mRNA sequence that is conserved between humans and non-human primates. In certain embodiments, the nucleic acid molecule (e.g. gRNA) targets a target sequence in a primate PTB mRNA sequence, such as a non-human primate PTB mRNA sequence or a human PTB mRNA sequence. In some embodiment, the nucleic acid molecule (e.g. gRNA) targets a target sequence in the protein coding sequence in the primate mRNA sequence.
In certain embodiments, the target sequence in the PTB gene, transcript, or mRNA is comprised in a PTB sequence that is at least 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%identical to SEQ ID NO: 87. Alternatively, or in addition to, a targeting the target sequence can cause an efficient suppression of the expression or activity of a primate PTB protein in a non-neuronal cell. In one embodiment, the target sequence in the PTB gene, transcript, or mRNA is comprised in a PTB sequence that is at least 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%identical to SEQ ID NO: 87, wherein when the target sequence is targeted with a guide RNA (gRNA) , the guide sequence of which has at least 10 nucleotides overlap with at least one of SEQ ID NOs: 1-86, and which is co-expressed with CasRx in at least one of Cos7 and 293T cells, a relative expression of PTB mRNA of less than 0.55, 0.5, 0.45, 0.4, 0.35, 0.3, 0.25, 0.24, 0.23, 0.22, 0.21, 0.20, 0.19, 0.18, 0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02 or 0.015 is observed, as compared to a corresponding control cell expressing only CasRx. Alternatively, the target sequence in the PTB gene, transcript, or mRNA is comprised in a PTB sequence that is at least 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%identical to SEQ ID NO: 87, wherein when the target sequence is targeted with a guide RNA (gRNA) , the guide sequence of which comprises or consists of at least one of SEQ ID NOs: 1-86, and which is co-expressed with CasRx in at least one of Cos7 and 293T cells, a relative expression of PTB mRNA of less than 0.55, 0.5, 0.45, 0.4, 0.35, 0.3, 0.25, 0.24, 0.23, 0.22, 0.21, 0.20, 0.19, 0.18, 0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02 or 0.015 is observed, as compared to a corresponding control cell expressing only CasRx.
In one embodiment, the target sequence in the PTB gene, transcript, or mRNA can comprise or consist of a contiguous sequence of at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides. Alternatively, or in addition to, the target sequence in the PTB gene, transcript, or mRNA can be less than 60, 55, 50, 45, 40, 35, 34, 33, 32, 31, 30, 29, 28, 27 or 26 nucleotides. Alternatively, or in addition to, the PTB sequence can be at least 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%identical to SEQ ID NO: 87.
In one embodiment, the target sequence in the PTB gene, transcript, or mRNA can comprise or consist of a contiguous sequence of at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides. Alternatively or in addition to, the target sequence in the PTB gene, transcript, or mRNA can be less than 60, 55, 50, 45, 40, 35, 34, 33, 32, 31, 30, 29, 28, 27 or 26 nucleotides. Alternatively, or in addition to, the PTB sequence can be at least 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%identical to positions 951-1487 of SEQ ID NO: 87, wherein, when the target sequence is targeted with a guide RNA (gRNA) , the guide sequence of which comprises or consists of at least one of SEQ ID NOs: 38-68, and which is co-expressed with CasRx in at least one of Cos7 and 293T cells, a relative expression of PTB mRNA of less than 0.50, 0.45, 0.40, 0.35, 0.3, 0.25, 0.24, 0.23, 0.22, 0.21, 0.20, 0.19, 0.18, 0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02 or 0.015 is observed, as compared to a corresponding control cell expressing only CasRx.
In one embodiment, the target sequence in the PTB gene, transcript, or mRNA can comprise or consist of a contiguous sequence of at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides. Alternatively, or in addition to, the target sequence in the PTB gene, transcript, or mRNA can be less than 60, 55, 50, 45, 40, 35, 34, 33, 32, 31, 30, 29, 28, 27 or 26 nucleotides. Alternatively, or in addition to, the PTB sequence can be at least 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%identical to at least one of: positions 1000-1487 of SEQ ID NO: 87; positions 1050-1487 of SEQ ID NO: 87; positions 1100-1487 of SEQ ID NO: 87; positions 1150-1487 of SEQ ID NO: 87; positions 1200-1487 of SEQ ID NO: 87; positions 1250-1487 of SEQ ID NO: 87; positions 1300-1487 of SEQ ID NO: 87; positions 1350-1487 of SEQ ID NO: 87; positions 1400-1487 of SEQ ID NO: 87; positions 951-1400 of SEQ ID NO: 87; positions 951-1350 of SEQ ID NO: 87; positions 951-1300 of SEQ ID NO: 87; positions 951-1250 of SEQ ID NO: 87; positions 951-1200 of SEQ ID NO: 87; positions 951-1150 of SEQ ID NO: 87; positions 951-1100 of SEQ ID NO: 87; positions 951-1050 of SEQ ID NO: 87; and positions 951-1000 of SEQ ID NO: 87; and wherein when the target sequence is targeted with a guide RNA (gRNA) , the guide sequence of which comprises or consists of at least one of SEQ ID NOs: 38-68, and which is co-expressed with CasRx in at least one of Cos7 and 293T cells, a relative expression of PTB mRNA of less than 0.50, 0.45, 0.40, 0.35, 0.3, 0.25, 0.24, 0.23, 0.22, 0.21, 0.20, 0.19, 0.18, 0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02 or 0.015 is observed, as compared to a corresponding control cell expressing only CasRx.
In an embodiment, a nucleic acid molecule that targets a target sequence or its complement in a primate PTB gene, transcript or mRNA can be a guide RNA (gRNA) for a CRISPR/Cas family effector protein (Cas effector protein) . In one embodiment, the composition can comprise a composition comprising: i) a Cas effector protein and the at least one gRNA that targets a target sequence or its complement in a primate PTB gene, transcript, or mRNA, or ii) at least one expression vector encoding a Cas effector protein and encoding the at least one gRNA. Likewise, the invention provides for a composition comprising such compositions.
Vectors
In some embodiments, the composition (e.g. comprising the Cas effector protein and the at least one gRNA, or the at least one expression vector) is comprised in a nanoparticle, such as a liposome. A liposome is a spherical vesicle having at least one lipid bilayer. A liposome can be used as a delivery vehicle for the administration of nanoparticles, nutrients, pharmaceuticals, and other cargo.
In one embodiment, a vector encoding a CRISPR/Cas family effector protein (Cas effector protein) and a guide RNA (gRNA) that targets a PTB gene sequence together encompass the composition.
In these configurations, non-viral transfection methods or viral transduction methods are utilized to introduce the composition. Non-viral transfection can refer to all cell transfection methods that are not mediated through a virus. Non-limiting examples of non-viral transfection include electroporation, microinjection, calcium phosphate precipitation, transfection with cationic polymers, such as DEAE-dextran followed by polyethylene glycol, transfection with dendrimers, liposome mediated transfection ( ″lipofection″ ) , microprojectile bombardment ( ″gene gun″ ) , fugene, direct sonic loading, cell squeezing, optical transfection, protoplast fusion, impalefection, magnetofection, nucleofection, and any combination thereof.
In some embodiments, the expression vector used to deliver the composition to the non-neuronal cell can be a gene therapy vector. In some embodiments, the gene therapy vector can be a viral gene therapy vector, also known as a viral vector. As provided herein, viral vector methods can include the use of either DNA or RNA viral vectors. Examples of appropriate viral vectors can include adenovirus, lentivirus, adeno-associated virus (AAV) , poliovirus, poxvirus, herpes simplex virus (HSV) , an SV40, or murine Maloney-based virus vector.
In some embodiments, the vector is an AAV vector. In some embodiments, a composition is administered in the form of AAV vector. In some embodiments, a composition is administered in the form of lentiviral vector. For example, a composition can be delivered to a non-neuronal cell using a lentivirus or AAV to express a Cas effector protein with gRNA against PTB/nPTB.
According to some embodiments of the disclosure, methods provided herein comprise suppressing the expression or activity of PTB/nPTB in a non-neuronal cell (e.g., a glial cell or astrocyte) via a composition of a sufficient amount for reprogramming the non-neuronal cell to a mature neuron. The sufficient amount of composition can be determined empirically. In some embodiments, the amount of composition can be determined by any type of assay that examines the activity of the composition in the non-neuronal cell.
For example, when the composition is configured to suppress the expression of PTB/nPTB in the non-neuronal cell, the sufficient amount of the composition can be determined by assessing the expression level of PTB/nPTB in an exemplary non-neuronal cell after administration of the agent, e.g., by Western blot. In some embodiments, functional assays are utilized for assessing the activity of PTB/nPTB after delivery of the composition to an exemplary non-neuronal cell. In some embodiments, other functional assays, such as, immunostaining for neuronal markers or electrical recording for neuronal functional properties that examine downstream neuronal properties are used to determine a sufficient amount of composition.
In some embodiments, the composition is delivered in the form of a viral vector. A viral vector can comprise one or more copies of expression sequence coding for a composition; for example, a Cas effector protein can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more copies of a coding sequence for a gRNA against PTB/nPTB.
A viral vector can be tittered to any appropriate amount for administration. For example, the titer as determined by PCR, RT-PCR, or other methods can be at least about 105 viral particles/mL, 106 particles /mL, 107 particles /mL, 108 particles /mL, 109 particles /mL, 1010 particles/mL, 1011 particles /mL, 1012 particles/mL, 1013 particles /mL, 1014 particles/mL, or 1015 particles/mL. In some embodiments, the titer of viral vector to be administered is at least about 1010 particles/mL, 1011 particles /mL, 1012 particles/mL, 1013 particles /mL, or 1014 particles/mL.
Cas effector protein
In some embodiments, the Cas effector protein in the composition is selected from the group consisting of: Cas13d, CasRx, Cas13X, CRISPR/Cas9, Cpf1, Cas9, Cas13a, Cas13b, Cas13c, Cas13Y and a functional domain thereof. In certain embodiments, the Cas effector protein is encoded by an ORF (from start codon to stop codon) of 4.5 kb or less, 4 kb or less, 3.5 kb or less, 3 kb or less, 2.5 kb or less, or 2.1 kb or less, or 1.5 kb or less. In some embodiments, the Cas effector protein is modified to comprise a nuclear localization signal.
In some embodiments, the Cas effector protein is a DNA-targeting Cas effector protein. In some embodiment, the Cas effector protein is a DNA-targeting Cas effector protein selected from the group consisting of: spCas9 or its variant, SaCas9 or its variant, Cpf1 or its variant, or a combination thereof. In certain embodiments, the Cas effector protein in the composition is an RNA-targeting Cas effector protein. An RNA-targeting Cas effector protein is herein understood as a Cas effector protein that uses its crRNA/gRNA to recognize and degrade target RNA sequences, rather than target DNA sequences. In some embodiments, the Cas effector protein is the effector protein of a Type VI CRISPR-Cas system. In some embodiments, the Cas effector protein contains 2 HEPN ribonuclease motifs, containing the RXXXXH-motif (see Anantharaman et al., 2013, Biol Direct. 2013; 8: 15) . In some embodiments, the RNA-targeting Cas effector protein is selected from the group consisting of: Cas13a, Cas13b, Cas13c, Cas13d /CasRx, CRISPR/Cas9, Cpf1, Cas13X and Cas13Y and a functional domain thereof. In certain embodiments, the RNA-targeting Cas effector protein is encoded by an ORF (from start codon to stop codon) of 4.5 kb or less, 4 kb or less, 3.5 kb or less, 3 kb or less, 2.5 kb or less, or 2.1 kb or less, or 1.5 kb or less. In some embodiments, the RNA-targeting Cas effector protein is modified to comprise a nuclear location signal.
Cas13a, previously known as C2c2; Cas13b; Cas13c; and Cas13d, also known as CasRx, are Class 2, Type VI effector proteins which introduce cleavage of single stranded and collateral RNA. Cas13X and Cas13Y are compact Cas enzymes which can tolerate single-nucleotide mismatches in RNA recognition. CasRx is an ortholog of Cas13d, which has the smallest size and exhibits high targeting specificity and efficiency, making it an option for in vivo therapeutic applications.
Guide RNA
A gRNA that targets a primate PTB mRNA sequence is used in a composition of the invention in combination with an RNA-targeting CRISPR/Cas family effector protein. Thus, in some embodiments, the gRNA comprises a sequence that is complementary to a target sequence in a primate PTB mRNA, such as the human PTBP1 coding sequence (NM_002819; SEQ ID NO: 87) , preferably, the gRNA comprises a sequence that is complementary to a target sequence as herein defined above.
In one embodiment, the gRNA comprises a guide sequence that is complementary to a contiguous stretch of 14-60 nucleotides in a primate PTB mRNA sequence, such as SEQ ID NO: 87. The gRNA can comprise a guide sequence that is complementary to a contiguous stretch of at least 17 and no more than 60 nucleotides in a primate PTB mRNA sequence, such as SEQ ID NO: 87. In some certain embodiments, the gRNA comprises a guide sequence that is complementary to a contiguous stretch of at least 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in a primate PTB mRNA sequence, such as SEQ ID NO: 87, and/or no more than 60, 55, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41 or 40 nucleotides in a primate PTB mRNA sequence, such as SEQ ID NO: 87. In some embodiments, the gRNA comprises a guide sequence that is complementary to a contiguous stretch of 25-45 nucleotides in a primate PTB mRNA sequence, such as SEQ ID NO: 87.
In one embodiment, the guide sequence in the gRNA comprises or consists of at least 10, 11, 12, 13, 14, 15, 16, 17 contiguous nucleotides, or of all nucleotides of at least one of SEQ ID NOs: 1-86.
In one embodiment, the guide sequence in the gRNA comprises or consists of at least 10, 11, 12, 13, 14, 15, 16, 17 contiguous nucleotides, or of all nucleotides of at least one of SEQ ID NOs: 1-86, wherein when the gRNA is co-expressed with CasRx in at least one of Cos7 and 293T cells, a relative expression of PTB mRNA of less than 0.5, 0.45, 0.4, 0.35, 0.3, 0.25, 0.24, 0.23, 0.22, 0.21, 0.20, 0.19, 0.18, 0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02 or 0.015 is observed, as compared to a corresponding control cell expressing only CasRx.
In one embodiment, the guide sequence in the gRNA comprises or consists of at least one of SEQ ID NOs: 1-86. In one embodiment, the guide sequence in the gRNA comprises or consists of at least one of SEQ ID NOs: 1-86 and preferably produces a relative expression as determined in Table 1, that is no more than 0.5, 0.45, 0.4, 0.35, 0.3, 0.25, 0.24, 0.23, 0.22, 0.21, 0.20, 0.19, 0.18, 0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02 or 0.015.
In one embodiment, the guide sequence in the gRNA comprises or consists of at least one sequence selected from SEQ ID NOs: 56, 60, 47, 48, 43, 45, 40, 61, 50, 55, 41, 44, 42, 66, 49, 51, 46, 79, 07, 58, 53, 38, 64, 63, 59, 54, 62, 81, 83, 57, 05, 08, 70, 73, 76, 10, 04, 36, 52, 26, 67, 25, 39, 34, 80, 06, 23, 85, 24, 30, 02, 13, 03, 77, 31, 21, 69, 16, 75, 12, 78, 20, 74, 71, and 37. In one embodiment, the guide sequence in the gRNA comprises or consists of at least one sequence selected from SEQ ID NOs: 56, 60, 47, 48, 43, 45, 40, 61, 50, 55, 41, 44, 42, 66, 49, 51, 46, 79, 07, 58, 53, 38, 64, 63, 59, 54, 62, 81, 83, 57, 05, 08, 70, 73, 76, 10, 04, 36, 52, 26, 67, 25, 39, 34, 80, 06, 23, 85, 24, 30, and 02. In one embodiment, the guide sequence in the gRNA comprises or consists of at least one sequence selected from SEQ ID NOs: 56, 60, 47, 48, 43, 45, 40, 61, 50, 55, 41, 44, 42, 66, 49, 51, 46, 79, 07, 58, 53, 38, 64, 63, 59, 54, 62, 81, 83, 57, 05, 08, 70, 73, 76, 10, 04, 36, 52, 26, 67, and 25. In one embodiment, the guide sequence in the gRNA comprises or consists of at least one sequence selected from SEQ ID NOs: 56, 60, 47, 48, 43, 45, 40, 61, 50, 55, 41, 44, 42, 66, 49, 51, 46, 79, 07, 58, 53, 38, 64, 63, 59, 54, 62, 81, 83, 57, 05, 08, 70, 73, and 76. In one embodiment, the guide sequence in the gRNA comprises or consists of at least one sequence selected from SEQ ID NOs: 56, 60, 47, 48, 43, 45, 40, 61, 50, 55, 41, 44, 42, 66, 49, 51, 46, 79, 07, 58, 53, 38, 64, 63, and 59. In one embodiment, the guide sequence in the gRNA comprises or consists of at least one sequence selected from SEQ ID NOs: 56, 60, 47, 48, 43, 45, 40, 61, 50, 55, 41, 44, 42, 66, 49, 51, and 46. In one embodiment, the guide sequence in the gRNA comprises or consists of at least one sequence selected from SEQ ID NOs: 56, 60, 47, 48, 43, 45, 40, 61, 50, 55, 41, 44, 42, 66, and 49. In one embodiment, the guide sequence in the gRNA comprises or consists of at least one sequence selected from SEQ ID NOs: 56, 60, 47, 48, 43, 45, 40, and 61. In one embodiment, the guide sequence in the gRNA comprises or consists of at least one sequence selected from SEQ ID NOs: 56, 60, 47, 48, 43, 45, and 40. In one embodiment, the guide sequence in the gRNA comprises or consists of at least one sequence selected from SEQ ID NOs: 56, 60, 47, 48, 43, and 45. In one embodiment, the guide sequence in the gRNA comprises or consists of at least one sequence selected from SEQ ID NOs: 56, 60, 47, 48, and 43. In one embodiment, the guide sequence in the gRNA comprises or consists of at least one sequence selected from SEQ ID NOs: 56, 60, 47, and 48. In one embodiment, the guide sequence in the gRNA comprises or consists of at least one sequence selected from SEQ ID NOs: 56, 60, and 47. In one embodiment, the guide sequence in the gRNA comprises or consists of at least one sequence selected from SEQ ID NOs: 56 and 60. In another embodiment, the guide sequence of the gRNA comprises or consists of at least one sequence selected from the SEQ ID NOs: 1-86. In another embodiment, the guide sequence of the gRNA comprises or consists of at least one sequence selected from the SEQ ID NO: 89-112.
In certain embodiments, the gRNA targets a sequence in a primate PTB mRNA that corresponds to a sequence selected from the group consisting of: a) positions 59-91 of SEQ ID NO: 87; b) positions 303-349 of SEQ ID NO: 87; c) positions 422-451 of SEQ ID NO: 87; d) positions 460-489 of SEQ ID NO: 87; e) positions 542-576 of SEQ ID NO: 87; f) positions 646-681 of SEQ ID NO: 87; g) positions 706-769 of SEQ ID NO: 87; h) positions 773-806 of SEQ ID NO: 87; i) positions 1079-1139 of SEQ ID NO: 87; j) positions 1152-1184 of SEQ ID NO: 87; k) positions 1191-1254 of SEQ ID NO: 87; l) positions 1374-1434 of SEQ ID NO: 87; m) positions 951-1487 of SEQ ID NO: 87; n) positions 1502-1575 of SEQ ID NO: 87; and, o) positions 1626-1669 of SEQ ID NO: 87. It is herein understood that a sequence in a primate PTB mRNA (other than SEQ ID NO: 87) that corresponds to a sequence in positions with respect to SEQ ID NO: 87, is a sequence that corresponds to those positions in SEQ ID NO: 87 in a nucleotide sequence alignment, preferably using a global Needleman Wunsch algorithm using the default settings (default Gap opening penalty is 10.0 and the default gap extension penalty is 0.5; default scoring matrix DNAFull) .
In some certain embodiments, the gRNA comprises or consists of a guide sequence that is complementary to a contiguous stretch of at least 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides, and/or no more than 60, 55, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, or 40 nucleotides in a primate PTB mRNA sequence that is at least 95, 96, 97, 98, 99, or 100%identical to positions 951-1487 of SEQ ID NO: 87, and wherein preferably, when the target sequence is targeted with a guide RNA (gRNA) , the guide sequence of which comprises or consists of at least one of SEQ ID NOs: 38-68, that is co-expressed with CasRx in at least one of Cos7 and 293T cells, a relative expression of PTB mRNA of less than 0.50, 0.45, 0.40, 0.35, 0.3, 0.25, 0.24, 0.23, 0.22, 0.21, 0.20, 0.19, 0.18, 0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, or 0.015 is observed, as compared to a corresponding control cell expressing only CasRx.
In one embodiment, the gRNA comprises or consists of a guide sequence that is complementary to a contiguous stretch of at least 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides, and/or no more than 60, 55, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, or 40 nucleotides in a primate PTB mRNA sequence that is at least 95, 96, 97, 98, 99, or 100%identical to at least one of: positions 1000-1487 of SEQ ID NO: 87; positions 1050-1487 of SEQ ID NO: 87; positions 1100-1487 of SEQ ID NO: 87; positions 1150-1487 of SEQ ID NO: 87; positions 1200-1487 of SEQ ID NO: 87; positions 1250-1487 of SEQ ID NO: 87; positions 1300-1487 of SEQ ID NO: 87; positions 1350-1487 of SEQ ID NO: 87; positions 1400-1487 of SEQ ID NO: 87; positions 951-1400 of SEQ ID NO: 87; positions 951-1350 of SEQ ID NO: 87; positions 951-1300 of SEQ ID NO: 87; positions 951-1250 of SEQ ID NO: 87; positions 951-1200 of SEQ ID NO: 87; positions 951-1150 of SEQ ID NO: 87; positions 951-1100 of SEQ ID NO: 87; positions 951-1050 of SEQ ID NO: 87; and positions 951-1000 of SEQ ID NO: 87; and wherein preferably, when the target sequence is targeted with a guide RNA (gRNA) , the guide sequence of which comprises or consists of at least one of SEQ ID NOs: 38-68, that is co-expressed with CasRx in at least one of Cos7 and 293T cells, a relative expression of PTB mRNA of less than 0.50, 0.45, 0.40, 0.35, 0.3, 0.25, 0.24, 0.23, 0.22, 0.21, 0.20, 0.19, 0.18, 0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, or 0.015 is observed, as compared to a corresponding control cell expressing only CasRx.
In some embodiments, co-expression of a gRNA of the invention together with CasRx in a human or non-human primate cell (e.g. in at least one of Cos7 and 293T cells) causes a relative expression of PTB mRNA of no more than 0.9, 0.85, 0.8, 0.75, 0.7, 0.65, 0.6, 0.55, 0.5, 0.45, 0.4, 0.35, 0.3, 0.25, 0.24, 0.23, 0.22, 0.21, 0.20, 0.19, 0.18, 0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11, 0.10, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, or 0.01 as compared to a corresponding control cell expressing only CasRx. The relative expression of a gRNA of the invention can be assayed as described in the examples herein. For example, transient co-transfection can be performed with 4 μg vectors expressing CAG-CasRx-P2A-GFP and 2 μg U6-gRNA-CMV-mCherry plasmid using Lipofectamine 3000 (or similar transfection reagent) . Cells transfected with only CAG-CasRx-P2A-GFP plasmid can be used as a control. Two days after transient transfection, around 30K GFP and mCherry double-positive (GFP top 20%) cells are collected by fluorescence activated cell sorting (FACS) and lysed for qPCR analysis to determine the relative expression of PTB mRNA compared to a corresponding number of controls cells (sorted for GFP top 20%only) .
As provided herein, a composition (e.g., Cas with PTB-targeting gRNA or polynucleotide encoding the same) suppresses expression or activity of PTB by at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%of the endogenous or native level. As provided herein, a composition (e.g., Cas with nPTB-targeting gRNA or polynucleotide encoding the same) suppresses expression or activity of nPTB by about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%of the endogenous or native level.
In some embodiments, a composition as provided herein (e.g., Cas with PTB/nPTB-targeting gRNA or polynucleotide encoding the same) directly suppress the expression level of PTB/nPTB, e.g., suppressing the transcription, translation, or protein stability of PTB and/or nPTB.
In some embodiments, a composition as provided herein (e.g., Cas with PTB/nPTB-targeting gRNA or polynucleotide encoding the same) directly effects on the expression or activity of PTB/nPTB, without affecting other cellular signaling pathway.
In some embodiments, the composition comprising the composition comprises at least one gRNA that targets a primate PTB mRNA sequence or the at least one expression vector encodes at least one gRNA that targets a primate PTB mRNA sequence. In another embodiment, the composition comprising the composition comprises no more than one gRNA that targets a primate PTB mRNA sequence or the at least one expression vector encodes no more than one gRNA that targets a primate PTB mRNA sequence. In yet another embodiment, the composition comprising the composition comprises two, three, four five or six different gRNAs that target a primate PTB mRNA sequence or the at least one expression vector encodes two, three, four, five, or six different gRNAs that target a primate PTB mRNA sequence.
As provided herein, contacting the non-neuronal cell with a composition comprising a composition as provided herein, can be performed in any appropriate manner, depending on the type of non-neuronal cell to be reprogrammed, the environment in which the non-neuronal cell resides, and the desired cell reprogramming outcome.
In some embodiments, the non-neuronal cell is contacted with a composition comprising a composition as provided herein in the form of a polynucleotide encoding the encoding the Cas with PTB/nPTB-targeting gRNA. Thus, in one embodiment, the non-neuronal cell is contacted with a composition comprising a composition as provided herein in the form of at least one expression vector encoding a Cas effector protein and encoding a gRNA that targets a primate PTB mRNA sequence.
Promotors
In some embodiments, the at least one expression vector can comprise a nucleotide sequence encoding a Cas effector protein that is operably linked to a promoter that causes expression of the Cas effector protein in non-neuronal cell. In some embodiments, the promoter that causes expression in non-neuronal cell can be a glial cell-specific promoter such as a GFAP promoter, an ALDH1L1 promoter, an EAAT1/GLAST promoter, a glutamine synthetase promoter, a S100 beta promoter, or an EAAT2/GLT1 promoter. In some embodiments, the glial cell-specific promoter can be a Müller glia (MG) cell-specific promoter. In some embodiments, the MG cell-specific promoter is selected from the group consisting of: the GFAP promoter, the ALDH1L1 promoter, GLAST (also known as Slc1a3) promoter and the Rlbp1 promoter. In some embodiments, the glial cell-specific promoter or Müller glia (MG) cell-specific promoter can be a primate, human, or non-human primate promoter.
A GFAP is a glial fibrillary acid protein, which is an intermediate filament protein found almost exclusively in astrocytes. ALDH1L1, or aldehyde dehydrogenase 1 family member 1, is a protein which is highly expressed in CNS parenchymal astrocytes. EAAT1/GLAST/Slc1a3, also known as excitatory amino acid transporter 1, functions to terminate excitatory neurotransmission in the central nervous system. Glutamine synthetase, found in astrocytes, is the only enzyme capable of converting glutamate and ammonia to glutamine in the mammalian brain. S100 beta is a multifunctional protein found in large amounts in astrocytes. EAAT2/GLT1/Slc1a2 is a glutamate transporter. Rlbp1, also known as retinaldehyde-binding protein 1, plays a critical role as an 11-cis-retinal acceptor, which facilitates the enzyme isomerization of all 11-trans-retinal to 11-cis-retinal in the isomerization of rods and cones in the visual cycle.
In some embodiments, the at least one expression vector comprises at least one nucleotide sequence encoding a gRNA that targets a primate PTB mRNA sequence, which nucleotide sequence is operably linked to a promoter that causes expression of the gRNA in the non-neuronal cell. In some embodiments, the promoter that is operably linked to a gRNA coding sequence (and that causes expression of the gRNA in the non-neuronal cell) can be a promoter from a U6 snRNA gene, such as a primate, human, or non-human primate U6 promoter.
Codon optimization
In some embodiments, a nucleotide sequence encoding the Cas effector protein can be adapted to optimize its codon usage to that of the primate, human, or non-human primate non-neuronal host cell. The adaptiveness of a nucleotide sequence encoding a polypeptide to the codon usage of a host cell may be expressed as codon adaptation index (CAI) . The codon adaptation index is herein defined as a measurement of the relative adaptiveness of the codon usage of a gene towards the codon usage of highly expressed genes in a particular host cell or organism. The relative adaptiveness (w) of each codon is the ratio of the usage of each codon, to that of the most abundant codon for the same amino acid. The CAI index is defined as the geometric mean of these relative adaptiveness values. Non-synonymous codons and termination codons (dependent on genetic code) are excluded. CAI values range from 0 to 1, with higher values indicating a higher proportion of the most abundant codons (see Sharp and Li, 1987, Nucleic Acids Research 15: 1281-1295; also see: Jansen et al., 2003, Nucleic Acids Res. 31 (8) : 2242-51) . An adapted nucleotide sequence encoding the Cas effector protein preferably has a CAI of at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9. Codon optimization methods for optimum gene expression in heterologous organisms are known in the art and have been previously described (see e.g., Welch et al., 2009, PLoS One 4: e7002; Gustafsson et al., 2004, Trends Biotechnol. 22: 346-353; Wu et al., 2007, Nucl. Acids Res. 35: D76-79; Villalobos et al., 2006, BMC Bioinformatics 7: 285; U.S. Patent Publication 2011/0111413; and U.S. Patent Publication 2008/0292918) .
Additional factors
In other embodiments of the invention, a composition comprising the PTB-targeting composition as defined above, can comprise additional components, such as one or more non-PTB-targeting compositions, to further increase neuron conversion efficiency. The combination of the PTB-targeting compositions with one or more non-PTB-targeting compositions can act synergistically in increasing neuron conversion efficiency. Thus, in one embodiment, a composition comprising a PTB-targeting composition as defined above, can further comprise i) one or more dopamine neuron-associated factors, and/or ii) at least one expression vector for expression of one or more dopamine neuron-associated factors, preferably in a non-neuronal cell. The composition can be administered to a to a non-neuronal cell in the striatum for generating a functional dopaminergic neuron, whereby the composition can be administered to a glial cell in the striatum for generating a functional dopaminergic neuron.
In one embodiment, the one or more dopamine neuron-associated factors are selected from the group consisting of: Lmx1a, Lmx1b, FoxA2, Nurr1, Pitx3, Gata2, Gata3, FGF8, BMP, En1, En2, PET1, a Pax family protein, SHH, a Wnt family protein, and a TGF-β family protein. In one embodiment, the one or more dopamine neuron-associated factors are selected from the group consisting of: FoxA2, Lmx1a, and Nurr1. In a preferred embodiment, the one or more dopamine neuron-associated factors are FoxA2 alone, Lmx1a alone, Nurr1 alone, the combination of FoxA2 and Lmx1a, the combination of FoxA2 and Nurr1, the combination of Nurr1 and Lmx1a, or the combination of FoxA2, Lmx1a and Nurr1. Suitable primate and human amino acid and/or nucleotide sequences of these dopamine neuron-associated factors/genes can be accessed in publicly available databases. Construction and delivery of expression vectors for the expression of the one or more dopamine neuron-associated factors in the non-neuronal cell can be as described herein above for expression vectors of the PTB-targeting composition.
In another embodiment, a composition comprising a PTB-targeting composition as defined above, further comprises i) one or more factors selected from β-catenin, OSK factors (also known as Oct4, Sox2, Klf4, and other factors involved in the cell re-cycling or epigenetic remodeling) , Crx, Brn3a, Brn3b, Math5, Nr2e3, and Nrl, and/or ii) at least one expression vector for expression of one or more factors selected from β-catenin, OSK factors (also known as Oct4, Sox2, Klf4 and other factors involved in the cell re-cycling or epigenetic remodeling) , Crx, Brn3a, Brn3b, Math5, Nr2e3, and Nrl, preferably in a non-neuronal cell. In one embodiment, the one or more factors are selected from the group consisting of: β-catenin and the OSK factors, Oct4, Sox2, and Klf4. In a preferred embodiment, the one or more factors are a combination of β-catenin and Oct4, a combination of β-catenin and Sox2, a combination of β-catenin and Klf4, a combination of β-catenin and Oct4 with Sox2, a combination of β-catenin and Oct4 with Klf4, a combination of β-catenin and Sox2 with Klf4, a combination of β-catenin and all three OSK factors, Oct4, Sox2 and Klf4, or a combination of all three OSK factors, Oct4, Sox2 and Klf4 (without β-catenin) . Preferably, such a composition comprising both the PTB-targeting composition and the factor is used for local administration to a non-neuronal cell in a mature retina. More preferably, the composition is administered to a to a non-neuronal cell in the mature retina for generating a functional retinal ganglion cell (RGC) neuron and/or a functional retinal photoreceptor, whereby it is further preferred that the composition is administered to a glial cell or Müller glia (MG) cell in the mature retina for generating a functional retinal ganglion cell (RGC) neuron and/or a functional retinal photoreceptor. In a preferred embodiment, the factor is β-catenin. Suitable primate and human amino acid and/or nucleotide sequences of β-catenin, Oct4, Sox2, Klf4, Crx, Brn3a, Brn3b, Math5, Nr2e3, and Nrl or their genes are known to the skilled person and can be accessed in publicly available databases. Construction and delivery of expression vectors for the expression of the one or more factors selected from β-catenin, Oct4, Sox2, Klf4, Crx, Brn3a, Brn3b, Math5, Nr2e3, and Nrl in the non-neuronal cell can be as described herein above for expression vectors of the PTB-targeting composition. Alternatively, a composition can have no co-factors expressed.
Methods
In one aspect, the invention relates to a method of treating a neurological condition associated with degeneration of functional neurons in a region in the nervous system of a non-human primate or a human. The method can comprise the administration to a subject in need thereof an effective amount of a composition.
Specifically, one aspect of the invention provides methods of reprogramming a non-neuronal cell to a mature neuron. An exemplary method comprises providing a non-neuronal cell and contacting the non-neuronal cell with a composition comprising a composition (such as CRISPR/Cas effector with guide RNA (gRNA) or polynucleotide encoding the same) that suppresses expression and/or activity of PTB and/or nPTB in the non-neuronal cell, thereby reprogramming the non-neuronal cell to a mature neuron. The methods and compositions not only convert cells in vitro but also directly in vivo in nervous system (such as in striatum, retina, inner ear, and spinal cord) .
In some embodiments, the invention pertains to a method of treating a neurological condition associated with degeneration of functional neurons in a region in the nervous system of a non-human primate or a human, the method comprising the administration to a non-neuronal cell in the region in the nervous system of a subject in need thereof, an effective amount of a composition comprising i) an RNA-targeting Cas effector protein and a guide RNA (gRNA) that targets a primate PTB mRNA sequence or ii) at least one expression vector encoding an RNA-targeting Cas effector protein and encoding a gRNA that targets a primate PTB mRNA sequence, to suppress the expression or activity of PTB in the non-neuronal cell, thereby allowing the non-neural cell to reprogram into a functional neuron.
In another embodiment, the disclosure provides a method of reprogramming an astrocyte to a mature neuron. An exemplary method comprises providing the astrocyte to be reprogrammed and contacting the astrocyte with a composition comprising a composition (e.g., Cas with PTB/nPTB-targeting gRNA or polynucleotide encoding the same) that suppresses the expression or activity of PTB in the astrocyte for at least 1 day, thereby reprogramming the astrocyte to a mature neuron such as a dopamine neuron. In some embodiments, a single composition (e.g., Cas with PTB-targeting gRNA) that suppresses the expression or activity of PTB in an astrocyte can directly convert the astrocyte into a neuron such as a dopamine neuron. In some embodiments, the astrocyte is in striatum.
In another embodiment, the invention provides a method of reprogramming an MG cell (e.g., one in mature retina) to an RGC neuron. An exemplary method comprises providing the MG cell to be reprogrammed and contacting the MG cell with a composition comprising a composition (e.g., Cas with PTB/nPTB-targeting gRNA or polynucleotide encoding the same) that suppresses the expression or activity of PTB and/or nPTB in the MG cell for at least 1 day, thereby reprogramming the MG cell to an RGC neuron. In some embodiments, a single composition (e.g., Cas with PTB-targeting gRNA) that suppresses the expression or activity of PTB in an MG cell can directly convert the MG cell into an RGC neuron. In some embodiments, the MG cell is in mature retina.
According to certain embodiments, an exemplary method comprises providing a human non-neuronal cell to be reprogrammed; and contacting the human non-neuronal cell with a composition comprising a single composition (e.g., a Cas with PTB/nPTB-targeting gRNA) that yields a decrease in expression or activity of PTB in the human non-neuronal cell, and a decrease of expression or activity of nPTB after the expression or activity of PTB is decreased. In some embodiments, the composition can lead to a sequential event as to the expression or activity levels of PTB and nPTB in a certain type of non-neuronal cell, e.g., human non-neuronal cell, e.g., human glial cell. In some embodiments, the direct effect of contacting with the composition (e.g., Cas with PTB-targeting gRNA) is a decrease of expression or activity of PTB in the non-neuronal cell. In some embodiments, in the non-neuronal cell, the decrease of expression or activity of PTB in the non-neuronal cell accompanies an initial increase of nPTB expression level in the non-neuronal cell. In some embodiments, an initial nPTB expression level increases to a high nPTB expression level as expression or activity of PTB is suppressed. In some embodiments, following the initial increase, nPTB expression decreases from the high nPTB expression level to a low nPTB expression level. In some embodiments, the low nPTB expression level is still higher than the initial nPTB expression level after expression or activity of PTB is suppressed. In some embodiments, the nPTB expression level decreases after the initial increase spontaneously without external intervention other than the composition that suppresses the expression or activity of PTB. Without being bound to a certain theory, the subsequent decrease of nPTB expression level in the non-neuronal cell after PTB expression or activity can be decreased by the composition can be correlated with the direct conversion of the non-neuronal cell to a mature neuron by the composition. According to some embodiments, a single composition (e.g., a Cas with PTB-targeting gRNA) that suppresses the expression or activity of PTB does not induce the sequential event as described above in a human adult fibroblast cell, e.g., nPTB can experience the initial rise in expression level, but no subsequent decrease to a certain low level.
Conversion Efficiency
According to some embodiments of the present disclosure, the methods provided herein comprise reprogramming a plurality of non-neuronal cells into mature neurons at a high efficiency.
In some embodiments, at least 40%of the astrocytes are converted to mature neurons that are Map2 positive. In some embodiments, at least about 20%, 25%, 30%, 35%, 38%, 40%, 42%, 44%, 46%, 48%, 50%, 52%, 54%, 56%, 58%, 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, or 100%of the MG cells and/or astrocytes are converted to mature neurons that are positive for NeuN or Map2.
In some embodiments, at least 10 MG cells are converted to RGCs which express Brn3a or Rbpms in retinal ganglion cell layer (GCL) per 10 mm X 50 μm. In some embodiments, at least about 1, 2, 4, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, or 60 MG cells are converted to RGCs which express Brn3a or Rbpms in retinal ganglion cell layer (GCL) per 10 mm X 50 μm.
In some embodiments, the methods comprise reprogramming astrocytes into mature neurons, and at least 40%, at least 60%, or at least 80%of the astrocytes are converted to mature neurons that are Map2 or NeuN positive. In some embodiments, at least 20%, at least 40%or at least 60%of the human astrocytes are converted to mature neurons that are Map2 or NeuN positive. In some embodiments, at least about 20%, 25%, 30%, 35%, 38%, 40%, 42%, 44%, 46%, 48%, 50%, 52%, 54%, 56%, 58%, 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, or 100%of the astrocytes are converted to mature neurons that are positive for Map2 or NeuN.
In some embodiments, the methods as provided herein comprise reprogramming a plurality of non-neuronal cells, e.g., human non-neuronal cells, e.g., human glial cells, or astrocytes, and at least about 20%, 25%, 30%, 35%, 38%, 40%, 42%, 44%, 46%, 48%, 50%, 52%, 54%, 56%, 58%, 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, or 99%of the non-neuronal cells, e.g., human non-neuronal cells, e.g., human glial cells, or astrocytes are reprogrammed to mature neurons. In some embodiments, the methods as provided herein reprogram about 20%, 25%, 30%, 35%, 38%, 40%, 42%, 44%, 46%, 48%, 50%, 52%, 54%, 56%, 58%, 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 92%94%, 96%98%99%, or 100%of the non-neuronal cells, e.g., human non-neuronal cells, e.g., human glial cells, MG cells, or astrocytes are reprogrammed to mature neurons.
In some embodiments, a mature neuron is characterized by its expression of one or more neuronal markers selected from the group consisting of NeuN (neuronal nuclei antigen) , Map2 (microtubule-associated protein 2) , NSE (neuron specific enolase) , 160 kDa neurofilament medium, 200 kDa neurofilament heavy, PDS-95 (postsynaptic density protein 95) , Synapsin I, Synaptophysin, GAD67 (glutamate decarboxylase 67) , GAD65 (glutamate decarboxylase 67) , parvalbumin, DARPP32 (dopamine-and cAMP-regulated neuronal phosphoprotein 32) , vGLUT1 (vesicular glutamate transporter 1) , vGLUT2 (vesicular glutamate transporter 2) , acetylcholine, vesicular GABA transporter (VGAT) , and gamma-aminobutyric acid (GABA) , and TH (tyrosine hydroxylase) . In some embodiments, at least 40%of the non-neuronal cells, e.g., human non-neuronal cells, human glial cells, or astrocytes are reprogrammed to mature neurons.
In some embodiments, MG cells are converted into retinal photoreceptors characterized by the expression of one or more rod cell markers selected from rhodopsin and GNAT1, and/or characterized by expression of one or more cone cell markers selected from S-opsin, M-opsin, and mCAR.
The expression of the markers mentioned above can be assessed. For example, immunostaining using antibodies against specific cell type markers as described herein can reveal whether or not the cell of interest expresses the corresponding cell type marker. Immunostaining under certain conditions can also uncover the subcellular distribution of the cell type marker, which can also be important for determining the developmental stage of the cell of interest. For instance, expression of Map2 can be found in various neurites (e.g., dendrites) in a post-mitotic mature neuron, but which absent in axon of the neuron. Expression of voltage-gated sodium channels (e.g., a subunits Navi. 1-1.9 and b subunits) can be another example because they can be clustered in a mature neuron at axon initial segment, where an action potential can be initiated, and/or in the Node of Ranvier. In some embodiments, other techniques such as, but not limited to, flow cytometry, mass spectrometry, in situ hybridization, RT-PCR, and microarrays, can also be used for assessing expression of specific cell type markers.
Certain aspects of the present disclosure provide methods that comprise reprogramming a plurality of non-neuronal cells, and at least about 20%, 25%, 30%, 35%, 38%, 40%, 42%, 44%, 46%, 48%, 50%, 52%, 54%, 56%, 58%, 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, or 99%of the non-neuronal cells, e.g., glial cells, MG cells, or astrocytes are reprogrammed to functional neurons.
In some embodiments, the methods provided herein reprogram at least 20%of the non-neuronal cells, e.g., glial cells, MG cells, or astrocytes, are reprogrammed to functional neurons. In some embodiments, the methods provided herein reprogram about 20%, 25%, 30%, 35%, 38%, 40%, 42%, 44%, 46%, 48%, 50%, 52%, 54%, 56%, 58%, 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%92%94%, 96%, 98%, 99%, or 100%of the non-neuronal cells, e.g., glial cells, MG cells, or astrocytes, are reprogrammed to functional neurons.
In some embodiments, wherein the methods provided herein comprise the use of a combination of i) a PTB-targeting composition as defined herein, and ii) one or more non-PTB-targeting compositions as defined herein, the combination increases the neuron conversion efficiency by at least a factor 1.1, 1.2, 1.5, 2.0, 3.0, 4.0, 5.0, 8.0, 10, 15, or 20.
Functional Assessment
In some embodiments, functional neurons are characterized by their ability to form a neuronal network, to send and receive neuronal signals, or both. In some embodiments, functional neurons fire action potentials. In some embodiments, functional neurons establish synaptic connections with other neurons. For instance, a functional neuron can be a postsynaptic neuron in a synapse, e.g., having its dendritic termini (dendritic spines) forming postsynaptic compartments in synapses with another neuron. For instance, a functional neuron can be a presynaptic neuron in a synapse, e.g., having axonal terminal forming presynaptic terminal in synapses with another neuron.
A functional neuron can form synapses with another neuron which can include, but are not limited to, axoaxonic, axodendritic, and axosomatic synapses. A functional neuron can form synapses with another neuron which can be excitatory (e.g., glutamatergic) , inhibitory (e.g., GABAergic) , modulatory, or any combination thereof. In some embodiments, a functional neuron forms synapses with another neuron which are glutamatergic, GABAergic, cholinergic, adrenergic, dopaminergic, or any other appropriate type.
As a presynaptic neuron, a functional neuron can release neurotransmitters such as, but not limited to, glutamate, GABA, acetylcholine, aspartate, D-serine, glycine, nitric oxide (NO) , carbon monoxide (CO) , hydrogen sulfide (H2S) , dopamine, norepinephrine (also known as noradrenaline) , epinephrine (adrenaline) , histamine, serotonin, phenethylamine, N-methylphenethylamine, tyramine, 3-iodothyronamine, octopamine, tryptamine, somatostatin, substance P, opioid peptides, adenosine triphosphate (ATP) , adenosine, and anandamide. As a postsynaptic neuron, a functional neuron can elicit a postsynaptic response to a neurotransmitter released by a presynaptic neuron into the synaptic cleft. The postsynaptic response of a functional neuron as generated in the method provided herein can be either excitatory, inhibitory, or any combination thereof, depending on the type of neurotransmitter receptor the functional neuron expresses. In some embodiments, the functional neuron expresses ionic neurotransmitter receptors, e.g., ionic glutamate receptors and ionic GABA receptors. Ionic glutamate receptors can include, but not limited to, a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) -type glutamate receptors (e.g., GluAl/GRIAl; GluA2/GRIA2; GluA3/GRIA3; GluA4 /GRIA4 ) , delta receptors (e.g., GluDl/GRIDl; GluD2/GRID2) , kainate receptors (e.g., GluKl/GRIKl; GluK2/GRIK2; GluK3/GRIK3 ; GluK4/GRIK4; GluK5/GRIK5) and iV-methyl-D-aspartate (NMDA) receptors (e.g., GluNl/GRINl; GluN2A/GRIN2A; GluN2B/GRIN2B; GluN2C/GRIN2C; GluN2D/GRIN2D; GluN3A/GRIN3A; GluN3B/GRIN3B) . Ionic GABA receptors can include, but are not limited to, GABAA receptor. In some embodiments, the functional neuron expresses metabolic neurotransmitter receptors, e.g., metabolic glutamate receptors (e.g., mGluRi, mGluRs, mGluR , mGluR , mGluRi, mGluRe, mGluR , mGluRs) , and metabolic GABA receptors (e.g., GABAB receptor) . In some embodiments, the functional neuron expresses a type of dopamine receptor, either Dl-like family dopamine receptor, e.g., D1 and D5 receptor (DIR and D5R) , or D2-like family dopamine receptor, e.g., D2, D3, and D4 receptors (D2R, D3R, and D4R) . In some embodiments, a functional neuron as provided herein forms electrical synapse with another neuron (e.g., a gap junction) . In some embodiments, a function neuron as provided herein forms either chemical or electrical synapse (s) with itself, as known as an autapse.
The characteristics of a functional neuron can be assessed. For example, the electrical properties of a functional neuron, such as firing of action potential and postsynaptic response to neurotransmitter release can be examined by techniques such as patch clamp recording (e.g., current clamp and voltage clamp recordings) , intracellular recording, and extracellular recording (e.g., tetrode recording, single-wire recording, and filed potential recording) . Specific properties of a functional neuron (e.g., expression of ion channels and resting membrane potential) can also be examined by patch clamp recording, where different variants of patch clamp recording can be applied for different purposes, such as cell-attached patch, inside-out patch, outside-out patch, whole-cell recording, perforated patch, or loose patch. Assessment of postsynaptic response by electrical methods can be coupled with either electrical stimulation of presynaptic neurons, application of neurotransmitters, or receptor agonists or antagonists. In some cases, AMPA-type glutamate receptor-mediated postsynaptic current can be assessed by AMPA receptor agonists, e.g., AMPA, or antagonists, e.g., 2, 3-dihydroxy-6-nitro-7-sulfamoyl-benzoquinoxaline (NBQX) or 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX) . In some cases, NMDA-type glutamate receptor-mediated postsynaptic current can be assessed by NMDA receptor agonists, e.g., NMDA and glycine, or antagonists, e.g., AP5 and ketamine.
In some embodiments, functional neurons are examined by techniques other than electrical approaches. For example, various fluorescent dyes or genetically encoded fluorescent proteins and imaging techniques can be utilized for monitoring electrical signals conveyed or transmitted by a functional neuron. In this context, calcium-dependent fluorescent dyes (e.g., calcium indicators) , such as, but not limited to, fura-2, indo-1, fluo-3, fluo-4, and Calcium Green-1, and calcium-dependent fluorescent proteins, such as, but not limited to, Cameleons, FIP-CBSM, Pericams, GCaMP, TN-L15, TN-humTnC, TN-XL, TNXXL, and Twitchs, can be used to trace calcium influx and efflux as an indicator of neuronal membrane potential. Alternatively or additionally, voltage-sensitive dyes that can change their spectral properties in response to voltage changes can also be used for monitoring neuronal activities.
Neurotransmitter release can be an important aspect of a functional neuron. The methods provided herein can comprise reprograming of a non-neuronal cell to a functional neuron that releases a certain type of neurotransmitter. In some embodiments, the functional neuron releases neurotransmitters such as, but not limited to, glutamate, GABA, acetylcholine, aspartate, D-serine, glycine, nitric oxide (NO) , carbon monoxide (CO) , hydrogen sulfide (H2S) , dopamine, norepinephrine (also known as noradrenaline) , epinephrine (adrenaline) , histamine, serotonin, phenethylamine, N-methylphenethylamine, tyramine, 3-iodothyronamine, octopamine, tryptamine, somatostatin, substance P, opioid peptides, adenosine triphosphate (ATP) , adenosine, and anandamide.
In some embodiments, the functional neuron releases dopamine as a major neurotransmitter. In some embodiments, the functional neuron releases more than one type of neurotransmitter. In some embodiments, the functional neuron releases neurotransmitters in response to an action potential. In some embodiments, the functional neuron releases neurotransmitters in response to graded electrical potential (e.g., membrane potential changes that do not exceed a threshold for eliciting an action potential) . In some embodiments, the functional neuron exhibits neurotransmitter release at a basal level (e.g., spontaneous neurotransmitter release) .
Neurotransmitter release as described herein from a functional neuron can be assessed by various techniques. In some embodiments, imaging approaches can be used for characterizing a functional neuron′s neurotransmitter release, for instance, by imaging a genetically encoded fluorescent fusion molecule comprising a vesicular protein, one can monitor the process of synaptic vesicles being fused to presynaptic membrane.
Alternatively or additionally, other methods can be applied to directly monitor the level of a specific neurotransmitter. For example, a high-performance liquid chromatography (HPLC) probe can be used to measure the amount of dopamine in a culture dish or in a brain region where a functional neuron projects its axon. The level of dopamine as detected by HPLC can indicate the presynaptic activity of a functional neuron. In some embodiments, such an assessment can be coupled with stimulation of the functional neuron in order to change its membrane potential, e.g., to make it elicit action potential.
In an aspect, the present disclosure provides a method of generating a functional neuron in vivo. An exemplary method comprises administering to a region in the nervous system, e.g., mature retina, inner ear, or a region in the brain or spinal cord (e.g., striatum) , of a subject a composition comprising a composition (e.g., a Cas effector protein and a gRNA targeting or complementing to PTB and/or nPTB, or a polynucleotide encoding the same) in a non-neuronal cell (e.g., a glial cell or astrocyte) in the region in the nervous system, and allowing the non-neuronal cell to reprogram into the functional neuron. In some embodiments, the composition suppresses the expression or activity of PTB and/or nPTB.
Administration Routes
According to some embodiments of the present disclosure, the methods provided herein comprise direct administration of a composition (e.g., a Cas effector protein and a gRNA targeting /complementing to PTB and/or nPTB or polynucleotide encoding the same) into a region in the nervous system (e.g., mature retina, inner ear, or a region in the brain or spinal cord (e.g., striatum) ) of a subject. In some embodiments, the composition (e.g., a Cas effector protein and a gRNA targeting or complementing PTB and/or nPTB or a polynucleotide encoding the same) is delivered locally to a region in the nervous system (e.g., mature retina or a region in the brain or spinal cord (e.g., striatum) ) . In one embodiment, a composition comprising a composition, such as a viral vector (e.g. AAV vector) , is administered to the subject or organism by stereotaxic or convection enhanced delivery to a brain region (e.g., striatum) .
Using a stereotaxic positioning system, a specific brain region (e.g., striatum) can be located that is to be administered with the composition comprising the composition. In another embodiment, a composition as provided herein is delivered systemically to a subject or to a region in nervous system, e.g., brain (e.g., striatum) or spinal cord, of a subject, e.g., delivered to cerebrospinal fluid or cerebral ventricles, and the composition comprises one or more agents that are configured to relocate the composition to a particular region in the nervous system (e.g., striatum) or a particular type of cells in the nervous system of the subject.
In some embodiments, the composition used in the methods provided herein comprises a virus that expresses a Cas effector and an anti-PTB or anti-nPTB gRNA, and the methods comprise injection of the virus in a desired brain region stereotaxically. In some embodiments, the virus comprises adenovirus, lentivirus, adeno-associated virus (AAV) , poliovirus, herpes simplex virus (HSV) , or murine Maloney-based virus. The AAV that can be used in the methods provided herein can be any appropriate serotype of AAV, such as, but not limited to, AAV2, AAV5, AAV6, AAV7, AAV8, and AAV9. In some embodiments, the methods comprise delivering an AAV2-or AAV9-based viral vector that expresses an agent that suppresses expression or activity of PTB and/or nPTB in a non-neuronal cell in a region in nervous system, e.g., brain (e.g., striatum) or spinal cord.
In some embodiments, the methods provided herein comprise reprogramming a variety of non-neuronal cells to mature neurons. In some embodiments, the methods provided herein comprise administering to a region in the nervous system, e.g., the brain (e.g., striatum) or spinal cord, of a subject a composition comprising a composition that suppresses the expression or activity of PTB and/or nPTB in a variety of non-neuronal cells (e.g., glial cells) , allowing the non-neuronal cells to reprogram into functional neurons. Types of glial cells can include, but are not limited to, astrocytes, oligodendrocytes, NG2 cells, satellite cells, microglial cells, radial glial cells, Schwann cells, precentral gyrus cells, or ependymal cells. In some embodiments, the methods provided herein comprise reprogramming astrocyte in a region in the nervous system, e.g., brain (e.g., striatum) or spinal cord, of a subject into a functional neuron.
Methods provided herein can comprise reprogramming a non-neuronal cell in a specific brain region (e.g., striatum) into a functional neuron. Exemplary brain regions include the hindbrain, midbrain, and forebrain. In some embodiments, the methods provided herein comprise administering to a midbrain, striatum, or cortex of a subject a composition comprising a composition that suppresses the expression or activity of PTB in a non-neuronal cell in mature retina or in the striatum and allowing the non-neuronal cell to reprogram into the functional neuron. In some embodiments, the methods provided herein comprise administering to mature retina or in the striatum of a subject a composition comprising a composition that suppresses the expression or activity of PTB/nPTB in a non-neuronal cell in the mature retina or in the striatum and allowing the non-neuronal cell to reprogram into the functional neuron.
In some embodiments, the methods provided herein comprise reprogramming a non-neuronal cell into a functional neuron, wherein the non-neuronal cell is located in a brain region, such as, but not limited to, medulla oblongata, medullary pyramids, olivary body, inferior olivary nucleus, rostral ventrolateral medulla, caudal ventrolateral medulla, solitary nucleus, respiratory center-respiratory groups, dorsal respiratory group, ventral respiratory group or apneustic centre, pre-botzinger complex, botzinger complex, retrotrapezoid nucleus, nucleus retrofacialis, nucleus retroambiguus, nucleus para-ambiguus, paramedian reticular nucleus, gigantocellular reticular nucleus, parafacial zone, cuneate nucleus, gracile nucleus, perihypoglossal nuclei, intercalated nucleus, prepositus nucleus, sublingual nucleus, area postrema, medullary cranial nerve nuclei, inferior salivatory nucleus, nucleus ambiguous, dorsal nucleus of vagus nerve, hypoglossal nucleus, metencephalon, pons, pontine nuclei, pontine cranial nerve nuclei, chief or pontine nucleus of the trigeminal nerve sensory nucleus, motor nucleus for the trigeminal nerve (v) , abducens nucleus (vi) , facial nerve nucleus (vii) , vestibulocochlear nuclei (vestibular nuclei and cochlear nuclei) (viii) , superior salivatory nucleus, pontine tegmentum, pontine micturition center (barrington′s nucleus) , locus coeruleus, pedunculopontine nucleus, laterodorsal tegmental nucleus, tegmental pontine reticular nucleus, parabrachial area, medial parabrachial nucleus, lateral parabrachial nucleus, subparabrachial nucleus (Kolliker-Fuse nucleus) , pontine respiratory group, superior olivary complex, medial superior olive, lateral superior olive, medial nucleus of the trapezoid body, paramedian pontine reticular formation, parvocellular reticular nucleus, caudal pontine reticular nucleus, cerebellar peduncles, superior cerebellar peduncle, middle cerebellar peduncle, inferior cerebellar peduncle, fourth ventricle, cerebellum, cerebellar vermis, cerebellar hemispheres, anterior lobe, posterior lobe, flocculonodular lobe, cerebellar nuclei, fastigial nucleus, interposed nucleus, globose nucleus, emboliform nucleus, dentate nucleus, midbrain (mesencephalon) , tectum, corpora quadrigemina, inferior colliculi, superior colliculi, pretectum, tegmentum, periaqueductal gray, rostral interstitial nucleus of medial longitudinal fasciculus, midbrain reticular formation, dorsal raphe nucleus, red nucleus, ventral tegmental area, parabrachial pigmented nucleus, paranigral nucleus, rostromedial tegmental nucleus, caudal linear nucleus, rostral linear nucleus of the raphe, interfascicular nucleus, substantia nigra, pars compacta, pars reticulata, interpeduncular nucleus, cerebral peduncle, crus cerebri, mesencephalic cranial nerve nuclei, oculomotor nucleus (iii) , edinger-westphal nucleus, trochlear nucleus (iv) , mesencephalic duct (cerebral aqueduct, aqueduct of sylvius) , forebrain (prosencephalon) , diencephalon, epithalamus, pineal body, habenular nuclei, stria medullaris, taenia thalami, third ventricle, subcommissural organ, thalamus, anterior nuclear group, anteroventral nucleus (a.k.a. ventral anterior nucleus) , anterodorsal nucleus, anteromedial nucleus, medial nuclear group, medial dorsal nucleus, midline nuclear group, paratenial nucleus, reuniens nucleus, rhomboidal nucleus, intralaminar nuclear group, centromedian nucleus, parafascicular nucleus, paracentral nucleus, central lateral nucleus, lateral nuclear group, lateral dorsal nucleus, lateral posterior nucleus, pulvinar, ventral nuclear group, ventral anterior nucleus, ventral lateral nucleus, ventral posterior nucleus, ventral posterior lateral nucleus, ventral posterior medial nucleus, metathalamus, medial geniculate body, lateral geniculate body, thalamic reticular nucleus, hypothalamus (limbic system) (hpa axis) , anterior, medial area, parts of preoptic area, medial preoptic nucleus, suprachiasmatic nucleus, paraventricular nucleus, supraoptic nucleus (mainly) , anterior hypothalamic nucleus, lateral area, parts of preoptic area, lateral preoptic nucleus, anterior part of lateral nucleus, part of supraoptic nucleus, other nuclei of preoptic area, median preoptic nucleus, periventricular preoptic nucleus, tuberal, medial area, dorsomedial hypothalamic nucleus, ventromedial nucleus, arcuate nucleus, lateral area, tuberal part of lateral nucleus, lateral tuberal nuclei, posterior, medial area, mammillary nuclei, posterior nucleus, lateral area, posterior part of lateral nucleus, optic chiasm, subfornical organ, periventricular nucleus, pituitary stalk, tuber cinereum, tuberal nucleus, tuberomammillary nucleus, tuberal region, mammillary bodies, mammillary nucleus, subthalamus, subthalamic nucleus, zona incerta, pituitary gland, neurohypophysis, pars intermedia (intermediate lobe) , adenohypophysis, frontal lobe, parietal lobe, occipital lobe, temporal lobe, cerebellum, brainstem, centrum semiovale, corona radiata, internal capsule, external capsule, extreme capsule, subcortical, hippocampus, dentate gyrus, cornu ammonis (CA fields) , cornu ammonis area 1 (CA1) , cornu ammonis area 2 (CA2) , cornu ammonis area 3 (CA3) , cornu ammonis area 4 (CA4) , amygdala, central nucleus of amygdala, medial nucleus of amygdala, cortical and basomedial nuclei of amygdala, lateral and basolateral nuclei of amygdala, extended amygdala, stria terminalis, bed nucleus of the stria terminalis, claustrum, basal ganglia, striatum, dorsal striatum, putamen, caudate nucleus, ventral striatum, nucleus accumbens, olfactory tubercle, globus pallidus, ventral pallidum, subthalamic nucleus, basal forebrain, anterior perforated substance, substantia innominata, nucleus basalis, diagonal band of broca, septal nuclei, medial septal nuclei, lamina terminalis, vascular organ of lamina terminalis, rhinencephalon (paleopallium) , olfactory bulb, olfactory tract, anterior olfactory nucleus, piriform cortex, anterior commissure, uncus, periamygdaloid cortex, cerebral cortex, frontal lobe, cortex, motor cortex, primary motor cortex (precentral gyrus, Ml) , supplementary motor cortex, premotor cortex, prefrontal cortex, orbitofrontal cortex, dorsolateral prefrontal cortex, gyri, superior frontal gyrus, middle frontal gyrus, inferior frontal gyrus, Brodmann areas 4, 6, 8, 9, 10, 11, 12, 24, 25, 32, 33, 44, 45, 46, and 47, parietal lobe, cortex, primary somatosensory cortex (SI) , secondary somatosensory cortex (S2) , posterior parietal cortex, gyri, postcentral gyrus (primary somesthetic area) , precuneus, Brodmann areas 1, 2, 3, 5, 7, 23, 26, 29, 31, 39, and 40, occipital lobe, cortex, primary visual cortex (VI) , v2, v3, v4, v5/mt, gyri, lateral occipital gyrus, cuneus, Brodmann areas 17 (VI, primary visual cortex) ; 18, and 19, temporal lobe, cortex, primary auditory cortex (Al) , secondary auditory cortex (A2) , inferior temporal cortex, posterior inferior temporal cortex, gyri, superior temporal gyrus, middle temporal gyrus, inferior temporal gyrus, entorhinal cortex, perirhinal cortex, parahippocampal gyrus, fusiform gyrus, Brodmann areas 20, 21, 22, 27, 34, 35, 36, 37, 38, 41, and 42, medial superior temporal area (MST) , insular cortex, cingulate cortex, anterior cingulate, posterior cingulate, retrosplenial cortex, indusium griseum, subgenual area 25, and Brodmann areas 23, 24; 26, 29, 30 (retrosplenial areas) ; 31, and 32. Alternatively, the non-neuronal cell may be located in the mature retina, the inner ear, or the spinal cord. In some embodiments, a non-neuronal cell which is located in the striatum is located in the putamen.
In one aspect, the invention provides a method of generating a dopaminergic neuron in vivo. In an embodiment, the method can comprise administering to the striatum in the brain of a subject a composition comprising a composition (e.g., a Cas effector protein and a coding sequence for gRNA against PTB and/or nPTB) that suppresses expression or activity of PTB and/or nPTB in a non-neuronal cell in the brain (e.g., a glial cell or astrocyte) , and allowing the non-neuronal cell to reprogram into the dopaminergic neuron. In some embodiments, the method comprises administering to the putamen of the subject a composition comprising a composition (e.g., a Cas effector protein and a coding sequence for gRNA against PTB and/or nPTB) that suppresses expression or activity of PTB and/or nPTB in a non-neuronal cell in the brain (e.g., a glial cell or astrocyte) , and allowing the non-neuronal cell to reprogram into the dopaminergic neuron. In some embodiments, the method comprises administering exclusively to the putamen of the subject (e.g. avoiding administration to the caudate nucleus and/or other parts of the striatum) and allowing the non-neuronal cell to reprogram into the dopaminergic neuron.
In another aspect, the invention provides a method of generating a retinal ganglion cell (RGC) neuron or a functional retinal photoreceptor in vivo. An exemplary method comprises administering to the mature retina of a subject a composition comprising a composition (e.g., a Cas effector protein and a coding sequence for gRNA against PTB and/or nPTB) that suppresses expression or activity of PTB and/or nPTB in a non-neuronal cell in the mature retina (e.g., a glial cell or MG cell) , and allowing the non-neuronal cell to reprogram into the RGC neuron or the functional retinal photoreceptor.
In some embodiments, the methods provided herein comprise administering to a region in the nervous system, e.g., brain or spinal cord, of a subject a composition comprising a cell programming agent (e.g., a Cas effector protein and a coding sequence for gRNA against PTB and/or nPTB) that suppresses the expression or activity of PTB and/or nPTB in a non-neuronal cell in the region, and allowing the non-neuronal cell to reprogram into a functional neuron of a subtype that is predominant in the region.
Without being bound to a particular theory, the methods provided herein can take advantage of local induction signals in a region, e.g., a specific brain region, when reprogramming a non-neuronal cell into a functional neuron in vivo. For example, local signals in the striatum may induce the conversion of non-neuronal cells with PTB/nPTB suppressed into dopamine neurons. Local neurons, non-neuronal cells, e.g., astrocytes, microglia, and/or other local constituents of the striatum can contribute to the subtype specification of the neuron that is generated from the non-neuronal cell under the induction of the composition.
In some embodiments, the methods provided herein comprise administering to a brain region (e.g., striatum) of a subject a composition comprising a composition (e.g., a Cas effector protein and a coding sequence for gRNA against PTB and/or nPTB) that suppresses the expression or activity of PTB/nPTB in a plurality of non-neuronal cell in the brain region, and the methods further comprise reprogramming at least about 5%, 10%, 20%, 25%, 30%, 35%, 38%, 40%, 42%, 44%, 46%, 48%, 50%, 52%, 54%, 56%, 58%, 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, or 99%of the non-neuronal cells to dopaminergic neurons. Expression of PTB/nPTB can be suppressed by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800-fold, at least about 900-fold, at least about 1000-fold, at least about 5000-fold, at least about 10,000-fold, at least about 100,000-fold, or at least about 1,000,000-fold. Expression of PTB/nPTB can be suppressed by at least about 5%, 10%, 20%, 25%, 30%, 35%, 38%, 40%, 42%, 44%, 46%, 48%, 50%, 52%, 54%, 56%, 58%, 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, or 99%. Expression can be suppressed by 100%.
In some embodiments, the methods provided herein comprise administering to the mature retina or a brain region (e.g., striatum) of a subject a composition comprising a composition (e.g., a Cas effector protein and a coding sequence for gRNA against PTB and/or nPTB) that suppresses the expression or activity of PTB/nPTB in a plurality of non-neuronal cell in the brain region, so at least about 5%, 10%, 20%, 25%, 30%, 35%, 38%, 40%, 42%, 44%, 46%, 48%, 50%, 52%, 54%, 56%, 58%, 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, or 99%of the functional neurons generated by the methods are RGC or dopaminergic, respectively.
In some embodiments, the dopaminergic neuron generated in the methods provided herein expresses one or more markers of dopaminergic neurons, including, but not limited to, dopamine, tyrosine hydroxylase (TH) , dopamine transporter (DAT) , vesicular monoamine transporter 2 (VMAT2) , engrailed homeobox 1 (Enl) , Nuclear receptor related-1 (Nurrl) , G protein-regulated inward-rectifier potassium channel 2 (Girk2) , forkhead box A2 (FoxA2) , orthodenticle homeobox 2 (OTX2) , and/or LIM homeobox transcription factor 1 alpha (Lmx1a) .
In some embodiments, the dopamine neuron generated in the methods provided herein exhibit Ih current, which can be mediated by Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. Ih current can be characterized as a slowly activating, inward current, which can be activated by hyperpolarizing steps. For instance, under voltage clamp and the holding potential Vh is -40 mV, an inward slowly activating current can be triggered in a dopamine neuron, with a reversal potential close to -30 mV. The activation curve of Ih current characteristic of a dopamine neuron generated in the methods provided herein can range from -50 to -120 mV with a mid-activation point of -84-1 mV.
In some embodiments, the dopaminergic neurons generated in the methods provided herein have gene expression profile similar to a native dopaminergic neuron. In some embodiments, the dopaminergic neurons generated in the methods provided herein release dopamine as a neurotransmitter.
A dopaminergic neuron generated in the methods provided herein can be of any subtype of dopaminergic neuron, including, but not limited to, A9 (e.g., immunopositive for Girk2) , A10 (e.g., immunopositive for calbindin-D28 k) , A11, A12, A13, A16, Aaq, and telencephalic dopamine neurons.
According to some embodiments of the present disclosure, the methods provided herein comprise reprogramming a non-neuronal cell in a region in the nervous system, e.g., mature retina or a region of the brain or spinal cord (e.g., striatum) , of a subject to a functional neuron. In some embodiments, the functional neuron as discussed here is integrated into the neural network in the nervous system. As described herein, the reprogrammed functional neuron can form synaptic connections with local neurons, e.g., neurons that are adjacent to the reprogrammed functional neurons. For example, synaptic connections between the reprogrammed neuron and neighboring primary neuron (e.g., glutamatergic neurons) , GABAergic interneurons, or other neighboring neurons (e.g., dopaminergic neuron, adrenergic neurons, or cholinergic neurons) can form as the reprogrammed neuron matures in vivo. Among these synaptic connections with local neurons, the reprogrammed functional neuron can be a presynaptic neuron, a postsynaptic neuron, or both. In some embodiments, the reprogrammed functional neuron sends axonal projections to remote brain regions.
In some embodiments, a reprogrammed functional neuron can integrate itself into one or more existing neural pathways in the brain or spinal cord, for instance, but not limited to, superior longitudinal fasciculus, arcuate fasciculus, uncinate fasciculus, perforant pathway, thalamocortical radiations, corpus callosum, anterior commissure, amygdalofugal pathway, interthalamic adhesion, posterior commissure, habenular commissure, fornix, mammillotegmental fasciculus, incertohypothalamic pathway, cerebral peduncle, medial forebrain bundle, medial longitudinal fasciculus, myoclonic triangle, mesocortical pathway, mesolimbic pathway, nigrostriatal pathway, tuberoinfundibular pathway, extrapyramidal system, pyramidal tract, corticospinal tract or cerebrospinal fibers, lateral corticospinal tract, anterior corticospinal tract, corticopontine fibers, frontopontine fibers, temporopontine fibers, corticobulbar tract, corticomesencephalic tract, tectospinal tract, interstitiospinal tract, rubrospinal tract, rubro-olivary tract, olivocerebellar tract, olivospinal tract, vestibulospinal tract, lateral vestibulospinal tract, medial vestibulospinal tract, reticulospinal tract, lateral raphespinal tract, posterior column-medial lemniscus pathway, gracile fasciculus, cuneate fasciculus, medial lemniscus, spinothalamic tract, lateral spinothalamic tract, anterior spinothalamic tract, spinomesencephalic tract, spinocerebellar tract, spino-olivary tract, and spinoreticular tract.
Without being bound to a certain theory, the local cellular environment can be correlated with the projections of a functional neuron generated according to some embodiments of the present disclosure. For instance, a functional neuron generated in striatum according to some embodiments of the methods provided herein can be affected by other cells in the local environment of striatum.
Conditions and Diseases
In an aspect, the present disclosure provides a method of treating a neurological condition or disease. Non-limiting examples of neurological conditions and diseases include: Alzheimer’s disease, amyotrophic lateral sclerosis, ataxia, Bell’s palsy, brain tumors, spinal cord injury, epilepsy, seizures, cerebral aneurysms, Guillain-Barre Syndrome, multiple sclerosis, Parkinson’s disease, stroke, Friedreich ataxia, Huntington’s disease, schizophrenia, depression, drug addition, blindness, deafness, Lewy body disease, motor neuron disease, movement disorder, choreoathetosis, dyskinesias, bipolar disorder, Autism spectrum disorder, spinal muscular atrophy, or head injury. In some embodiments, the neurological condition or disease can be associated with degeneration of functional neurons in a region in the nervous system.
The methods provided herein can also find use in treating or ameliorating one or more symptoms of neurodegenerative diseases including, but not limited to, autosomal dominant cerebellar ataxia, autosomal recessive spastic ataxia of Charlevoix-Saguenay, Corticobasal degeneration, Corticobasal syndrome, Creutzfeldt-Jakob disease, fragile X-associated tremor/ataxia syndrome, frontotemporal dementia and parkinsonism linked to chromosome 17, Kufor-Rakeb syndrome, Lyme disease, Machado-Joseph disease, Niemann-Pick disease, pontocerebellar hypoplasia, Refsum disease, pyruvate dehydrogenase complex deficiency, Sandhoff disease, Shy-Drager syndrome, Tay-Sachs disease, and Wobbly hedgehog syndrome.
In some embodiments, the neurological condition is associated with degeneration of functional neurons in the mature retina of a subject. Non-limiting examples of neurological conditions associated with the retina include, but are not limited to, glaucoma, age-related RGC loss, optic nerve injury, retinal ischemia, Leber’s congenital amaurosis (LCA) , retinitis pigmentosa (RP) , and Leber’s hereditary optic neuropathy.
As provided herein, ″neurodegeneration″ or its grammatical equivalents, can refer to the progressive loss of structure, function, or both of neurons, including death of neuron. Neurodegeneration can be due to any type of mechanisms. A neurological condition of the methods provided herein are applicable to can be of any etiology. A neurological condition can be inherited or sporadic, can be due to genetic mutations, protein misfolding, oxidative stress, or environment exposures (e.g., toxins or drugs of abuse) .
In some embodiments, the methods provided herein treat a neurological condition associated with degeneration of dopaminergic neurons in a brain region. In some embodiments, the methods provided herein treat a neurological condition associated with degeneration of RGC neurons in the mature retina. In other embodiments, the methods provided herein treat a neurological condition associated with degeneration of any type of neurons, such as, but not limited to, glutamatergic neurons, GABAergic neurons, cholinergic neurons, adrenergic neurons, dopaminergic neurons, or any other appropriate type neurons that release neurotransmitter aspartate, D-serine, glycine, nitric oxide (NO) , carbon monoxide (CO) , hydrogen sulfide (H2S) , norepinephrine (also known as noradrenaline) , histamine, serotonin, phenethylamine, N-methylphenethylamine, tyramine, 3-iodothyronamine, octopamine, tryptamine, somatostatin, substance P, opioid peptides, adenosine triphosphate (ATP) , adenosine, or anandamide. The methods provided herein can find use in treating a neurological condition associated with neuronal degeneration in any region, such as, but limited to, midbrain regions (e.g., substantial nigra or ventral tegmental area) , forebrain regions, hindbrain regions, or spinal cord. The methods provided herein can comprise reprogramming non-neuronal cells to functional neurons in any appropriate region (s) in the nervous system in order to treat a neurological condition associated with neuronal degeneration.
Methods provided herein can find use in treating or ameliorating one or more symptoms associate with Parkinson′s disease. Parkinson′s disease is a neuro-degenerative disease with early prominent functional impairment or death of dopaminergic neurons in the substantia nigra pars compacta (SNpc) . The resultant dopamine deficiency within the basal ganglia can lead to a movement disorder characterized by classical parkinsonian motor symptoms. Parkinson′s disease can also be associated with numerous non-motor symptoms. One standard for diagnosis of Parkinson′s disease can be the presence of SNpc degeneration and Lewy pathology at post-mortem pathological examination. Lewy pathology can include abnormal aggregates of asynuclein protein, called Lewy bodies and Lewy neurites. Patients with Parkinson′s disease can exhibit a number of symptoms, including motor symptoms and non-motor symptoms. Methods provided herein can treat or ameliorate one or more of these motor or non-motor symptoms associated with Parkinson′s disease. Motor symptoms of Parkinson′s disease (Parkinsonism symptoms) can include bradykinesia (slowness) , stiffness, impaired balance, shuffling gait, and postural instability. Motor features in patients with Parkinson′s disease can be heterogeneous, which has prompted attempts to classify subtypes of the disease, for instance, tremor-dominant Parkinson′s disease (with a relative absence of other motor symptoms) , non-tremor-dominant Parkinson′s disease (which can include phenotypes described as akinetic-rigid syndrome and postural instability gait disorder) , and an additional subgroup with a mixed or indeterminate phenotype with several motor symptoms of comparable severity. Non-motor symptoms of Parkinson′s disease can include olfactory dysfunction, cognitive impairment, psychiatric symptoms (e.g., depression) , sleep disorders, autonomic dysfunction, pain, and fatigue. These symptoms can be common in early Parkinson′s disease. Non-motor features can also be frequently present in Parkinson′s disease before the onset of the classical motor symptoms. This premotor or prodromal phase of the disease can be characterized by impaired olfaction, constipation, depression, excessive daytime sleepiness, or rapid eye movement sleep behavior disorder.
In some embodiments, methods provided herein mitigate or slow the progression of Parkinson′s disease. Progression of Parkinson′s disease can be characterized by worsening of motor features. As the disease advances, there can be an emergence of complications related to long-term symptomatic treatment, including motor and non-motor fluctuations, dyskinesia, and psychosis.
One pathological feature of Parkinson′s disease can be loss of dopaminergic neurons within the substantial nigra, e.g., substantial nigra pars compacta (SNpc) . According to some embodiments, methods provided herein replenish dopamine (secreted from converted dopamine neuron in the striatum) diminished due to loss of dopamine neuron in substantial nigra (e.g., SNpc) of a patient. Neuronal loss in Parkinson′s disease can also occur in many other brain regions, including the locus ceruleus, nucleus basalis of Meynert, pedunculopontine nucleus, raphe nucleus, dorsal motor nucleus of the vagus, amygdala, and hypothalamus. In some embodiments, methods of treating or ameliorating one or more symptoms of Parkinson′s disease in a subject as provided herein include reprogramming non-neuronal cells to functional neurons in brain regions experiencing neuronal loss in a patient with Parkinson′s disease.
Methods provided herein can find use in treating Parkinson′s disease of different etiology. For example, there can be Parkinson′s disease as a result of one or more genetic mutations, such as, but not limited to, mutations in genes SNCA, LRRK2 , VPS35 , EIF4G1, DNAJC13, CHCHD2 , Parkin, PINK1, DJ-1, ATP13A2, C9ORF72, FBX07, PLA2G6, POLG1, SCA2, SCA3, SYNJ1, RAB39B, and possibly one or more genes affected in 22qll. 2 microdeletion syndrome. Alternatively, there can be Parkinson′s disease with no known genetic traits.
As provided herein, the one or more symptoms of Parkinson′s disease the methods provided herein can ameliorate can include not only the motor symptoms and non-symptoms as described above, but also pathological features at other levels. For example, reduction in dopamine signaling in the brain of a patient with Parkinson′s disease can be reversed or mitigated by methods provided herein by replenishing functional dopamine neurons, which can be integrated into the neural circuitry and reconstruct the dopamine neuron projections to appropriate brain regions.
In an aspect, the present disclosure also provides methods of restoring dopamine release in subject with a decreased amount of dopamine biogenesis compared to a normal level. An exemplary method comprises reprogramming a non-neuronal cell in a brain region of the subject (e.g., striatum) , and allowing the non-neuronal cell to reprogram into a dopaminergic neuron, thereby restoring at least 50%of the decreased amount of dopamine. In some embodiments, the reprogramming is performed by administering to the brain region of the subject (e.g., striatum) a composition comprising a composition that suppresses the expression or activity of PTB/nPTB in a non-neuronal cell (e.g., an astrocyte) in the brain region. In some embodiments, the methods provided herein restore at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%of the decreased amount of dopamine. In some embodiments, the methods provided herein restore about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100%of the decreased amount of dopamine. In some embodiments, the methods provided herein restore at least about 50%of the decreased amount of dopamine.
Pharmaceutical Compositions, Dosing, and Administration
Methods provided herein can comprise suppressing the expression or activity of PTB and/or nPTB in a non-neuronal cell for a certain period of time sufficient for reprogramming the non-neuronal cell to a mature neuron.
In some embodiments, exemplary methods comprise contacting the non-neuronal cell with a composition that suppresses the expression or activity of PTB and/or nPTB in the non-neuronal cell for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 2 months, at least 3 months, at least 4 months, at least 5 months, or at least 1 year, thereby reprogramming the non-neuronal cell to a mature neuron.
In certain embodiments, suppression of PTB or nPTB expression or activity is sequential. For example, the expression or activity of PTB is first suppressed for any one of the above-mentioned time periods before the expression or activity of nPTB is suppressed. In certain embodiments, suppression of PTB and nPTB expression or activity is concurrent.
In some embodiments, exemplary methods comprise contacting the non-neuronal cell with a composition that suppresses the expression or activity of PTB in the non-neuronal cell for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 3 weeks, 4 weeks, 5 weeks, 2 months, 3 months, 4 months, or 5 months, before contacting the non-neuronal cell with a composition that suppresses the expression or activity of nPTB in the non-neuronal cell, thereby reprogramming the non-neuronal cell to a mature neuron.
In some configurations, the methods provided herein comprise administering the composition only once, e.g., adding the composition to a cell culture comprising the non-neuronal cell, or delivering the composition to a brain region in a subject comprising the non-neuronal cell (e.g., the striatum) , once and the composition can remain active, suppressing expression or activity of PTB and/or nPTB in the non-neuronal cell for a desirable amount of time. A desirable amount of time can be for at least 1 day, at least 2 days, at least 4 days, or at least 10 days. For instance, when the composition comprises an AAV vector expressing a Cas effector and a coding sequence for an anti-PTB gRNA, the design of the AAV vector can enable it to remain transcriptionally active for an extended period of time.
In some embodiments, the methods provided herein comprise administering the composition more than once, e.g., at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 12 times, at least 15 times, at least 20 times or more.
Immunosuppressants
In some embodiments, the methods provided herein further comprise administering at least one immunosuppressant. An immunosuppressant is a drug that inhibits or prevents activity of the immune system. The immunosuppressant can be administered prior to, simultaneously with, or after the administration of the composition, for example, when the composition comprises an AAV vector expressing a Cas effector and a coding sequence for an anti-PTB gRNA. Suitable immunosuppressants include, but are not limited to, corticosteroids (e.g. prednisone, prednisolone, dexamethasone, etc. ) , calcineurin inhibitors (e.g. cyclosporine, tacrolimus, etc. ) , mTOR inhibitors (sirolimus, everolimus, etc. ) , IMDH inhibitors (azathioprine, mycophenolate, etc. ) , antibodies (e.g. basiliximab, rituximab, alemtuzumab, etc. ) , interferons (e.g. IFN-β, IFN-γ, etc. ) , Janus kinase inhibitors (e.g. tofacitinib, etc. ) , and biologics (anakinra, etc. ) .
In one embodiment, the immunosuppressant is administered about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 3 weeks, 4 weeks, 5 weeks, 2 months, 3 months, 4 months, or 5 months, before and/or after administration of the composition. In one embodiment, the immunosuppressant is administered about simultaneously with the administration of the composition.
In one aspect, the present disclosure provides pharmaceutical compositions comprising a composition in an amount effective to reprogram a mammalian non-neuronal cell to a mature neuron by suppressing the expression or activity of PTB/nPTB in the non-neuronal cell. An exemplary pharmaceutical composition can further comprise another component, such as a pharmaceutically acceptable carrier, stabilizer, dilutent, dispersing agent, suspending agent, thickening agent or excipient. As described above, a composition as provided herein can be a Cas effector protein and a coding sequence for a gRNA against PTB/nPTB.
Excipients
A pharmaceutical composition provided herein can comprise one or more carriers or excipients, including but not limited to: buffers, carbohydrates, mannitol, proteins, peptides or amino acids such as glycine, antioxidants, bacteriostats, chelating agents, suspending agents, thickening agents and/or preservatives. A pharmaceutical composition can comprise water, oils (e.g., petroleum oil, animal oil, vegetable oil, synthetic oil , peanut oil, soybean oil, mineral oil, or sesame oil) , saline solutions, aqueous dextrose and glycerol solutions, flavoring agents, coloring agents, detackifiers and other acceptable additives, binders, or other pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH buffering agents, tonicity adjusting agents, emulsifying agents, or wetting agents, etc.. Examples of excipients can include, but are not limited to, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, and ethanol. In another instance, the composition is substantially free of preservatives. In other embodiments, the composition contains at least one preservative.
A pharmaceutically-acceptable carrier can be present at about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.1%, about 99.2%, about 99.3%, about 99.4%, about 99.5%, about 99.6%, about 99.7%, about 99.8%, or about 99.9%by mass of the total composition.
A pharmaceutically-acceptable excipient can be present at about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.1%, about 99.2%, about 99.3%, about 99.4%, about 99.5%, about 99.6%, about 99.7%, about 99.8%, or about 99.9%by mass of the total composition.
Dosage forms can include, but are not limited to, feed, food, pellets, lozenges, liquids, elixirs, aerosols, inhalants, sprays, powders, tablets, pills, capsules, gels, nanoparticles, microgels, suppositories, aqueous suspensions, oily suspensions, ointments, patches, lotions, emulsions, creams, drops, dispersible powders or granules, emulsions in hard or soft capsules, syrups, phytoceuticals, nutraceuticals, or any combination thereof. General methodology on pharmaceutical dosage forms can be found in Ansel et ah, Pharmaceutical Dosage Forms and Drug Delivery Systems (Lippencott Williams & Wilkins, Baltimore Md. (1999) ) . It will be recognized that, while any suitable carrier known to those of ordinary skill in the art can be employed to administer the pharmaceutical compositions described herein, the type of carrier can vary depending on the mode of administration. Suitable formulations and additional carriers are described in Remington ″The Science and Practice of Pharmacy″ (20th Ed., Lippincott Williams & Wilkins, Baltimore Md. ) , the teachings of which are incorporated by reference in their entirety herein.
A pharmaceutical composition can be formulated for injection, inhalation, parenteral administration, intravenous administration, subcutaneous administration, intramuscular administration, intradermal administration, topical administration, opthalamic administration, nasal administration, or oral administration. A pharmaceutical composition can be administered in a local or systemic manner (e.g., via injection of the compound directly into an organ or in the form of an intermediate release formulation) . In some embodiments, a pharmaceutical composition can be administered to the nervous system. In some embodiments, a pharmaceutical composition can be administered to a region of the nervous system (e.g., the mature retina, the inner ear, or the stratium of the brain) . In certain embodiments, the pharmaceutical composition comprising an AAV vector encoding a Cas effector and a coding sequence for a gRNA against PTB/nPTB can be injected into the mature retina, or the striatum of a subject′s brain. In some embodiments, the composition is administered and directly affects functional neurons. In some embodiments, the composition is administered to non-functional neurons and reprograms them into functional neurons, thereby replenishing the degenerated neurons in the region.
In some cases a subject can be an animal. An animal can be a mammal. An animal can be a human. An animal can be a non-human primate, such as, but not limited to, rhesus macaques, crab-eating macaques, stump-tailed macaques, pig-tailed macaques, squirrel monkeys, owl monkeys, baboons, chimpanzees, marmosets and spider monkeys. An animal can be a bovine, canine, caprine, cervine, cricetine, feline, equine, lapine, murine, musteline, or ovine. An animal can be a research animal, a genetically modified animal, or any other appropriate type of animal.
As one of ordinary skills in the art will appreciate, pharmaceutical compositions can comprise any appropriate carrier or excipient, depending on the type of composition and the administration route the composition is designed for. For example, a composition comprising a composition as provided herein can be formulated for parenteral administration and can be presented in unit dose form in ampoules, pre-filled syringes, small volume infusion or in multi dose containers with an added preservative. The composition can take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, for example solutions in aqueous polyethylene glycol. For example, for injectable formulations, a vehicle can be chosen from those known in the art to be suitable, including aqueous solutions or oil suspensions; emulsions with sesame oil, corn oil, cottonseed oil, or peanut oil; elixirs; mannitol; dextrose; a sterile aqueous solution; or similar pharmaceutical vehicles.
The formulation can also comprise polymer compositions which are biocompatible, biodegradable, such as poly (lactic-co-glycolic) acid. These materials can be made into micro or nanospheres, loaded with a drug and further coated or derivatized to provide superior sustained release performance.
Vehicles suitable for periocular or intraocular injection include, for example, suspensions of active agent in injection grade water, liposomes, and vehicles suitable for lipophilic substances. A composition as provided herein can further comprise additional agents besides a composition and a pharmaceutically acceptable carrier or excipient. For example, additional agents can be provided for promoting neuronal survival. Alternatively in addition to, additional agents can be provided for monitoring pharmacodynamics purpose. In some embodiments, a composition comprises additional agents as a penetration enhancer or for sustained or controlled release of the active ingredient, e.g., a composition.
A composition provided herein can be administered in a therapeutically effective amount. A ″therapeutically effective amount″ of a composition of the disclosure will vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the composition to elicit a desired response in the individual. A therapeutically effective amount can also be one in which any toxic or detrimental effects of the composition are outweighed by the therapeutically beneficial effects. Without wishing to be bound by a particular theory, it is contemplated that, in some cases, a therapeutically effective amount of composition as provided herein can be an amount of composition that converts a certain proportion of astrocytes in a brain region that experiences neuronal loss, conversion of such proportion of astrocytes to functional neurons in the brain region is sufficient to ameliorate or treating the disease or condition associated with the neuronal loss in the brain region, and meanwhile, such proportion of astrocytes does not exceed a threshold level that can lead to aversive effects that can overweigh the beneficial effects brought by the neuronal conversion, for instance, due to excessive reduction in the number of astrocytes in the brain region as a direct consequence of the neuronal conversion.
A composition provided herein can be administered to a subject in a dosage volume of about 0.0005, 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.7, 0.8, 0.9, 1.0 mL, or more. The composition can be administered as a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more dose course regimen. Sometimes, the composition can be administered as a 2, 3, or 4 dose course regimen. Sometimes the composition can be administered as a 1 dose course regimen.
The administration of the first dose (e.g., an AAV vector encoding a Cas effector and a gRNA against PTB) and second dose (e.g., an AAV vector encoding a Cas effector and a gRNA against nPTB) of a multi dose course regimen can be separated by about 0 days, 1 day, 2 days, 5 days, 7 days, 14 days, 21 days, 30 days, 2 months, 4 months, 6 months, 9 months, 1 year, 1.5 years, 2 years, 3 years, 4 years, 5 years, 10 years, 20 years, or more. A composition described herein can be administered to a subject once a day, once a week, once two weeks, once a month, a year, twice a year, three times a year, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more years. Sometimes, the composition can be administered to a subject every 2, 3, 4, 5, 6, 7, or more years. Sometimes, the composition can be administered to a subject once.
Tissue Transplantation
Some embodiments of the disclosure provide methods and compositions for cell or tissue transplantation. An exemplary method can comprise reprogramming a non-neuronal cell to a neuron in vitro and transplanting the reprogrammed neuron into the brain region in a subject. In some embodiments, in vitro reprogramming can be performed according to the methods provided herein. An exemplary composition can comprise a neuron reprogrammed according to any embodiment of the methods provided herein.
In other embodiments, a method provided herein comprises reprogramming a non-neuronal cell to a neuron in vivo and explanting the reprogrammed neuron. In some embodiments, the explant comprises brain tissue comprising the reprogramed neuron. In some embodiments, the explant is transplanted into the brain region of a subject. As provided herein, the transplantation of neurons reprogrammed according to the methods provided herein can be used to replenish degenerated neurons in a subject suffering a condition associated with neuronal loss.
Also provided herein is a brain tissue (e.g., explant) of an animal that comprises one or more neurons reprogrammed according to any embodiment of the present disclosure. Such brain tissue can be alive. In some embodiments, brain tissue can be fixed by any appropriate fixative. Brain tissue can be used for transplantation, medical research, basic research, or any type of purpose.
The disclosure demonstrates that the method is applicable to disease models of neurodegeneration. For example, the disclosure shows that astrocyte-to-neuron conversion strategy can work in a chemical-induced Parkinson′s disease model. The methods and compositions can convert astrocytes to neurons including dopaminergic, glutamatergic, and GABAergic neurons, these neurons are able to form synapses in the brain, and remarkably, the converted neurons can efficiently reconstruct the lesioned nigrostriatal pathway to correct measurable Parkinson′s phenotypes. The effectiveness of this method was demonstrated both in astrocytes in culture (human and mouse) as well as in vivo in a mouse Parkinson′s disease model. Therefore, this strategy has the potential to cure Parkinson′s disease, which can also be applied to a wide range of neurodegenerative diseases (e.g., other neurological diseases associated with neuronal dysfunction) .
In some embodiments, the approach of the disclosure exploits the genetic foundation of a neuronal maturation program already present, but latent, in both mammalian astrocytes that progressively produce mature neurons once they are reprogrammed by PTB suppression. These findings provide a clinically feasible approach to generate neurons from local astrocytes in mammalian brain using a single dose of a vector comprising coding sequence for a Cas effector and a gRNA against PTB/nPTB. The phenotypes of PTB/nPTB knockdown-induced neurons can be a function of the context in which they are produced and/or the astrocytes from which they are derived.
The disclosure demonstrates the potent conversion of astrocytes to neurons (e.g., dopamine neurons in the striatum) . More particularly, the disclosure shows that in a primate model, the strategy efficiently can convert astrocytes to neurons, thus satisfying all five factors for in vivo reprogramming. The data provided herein show that PTB reduction in the primate brain can convert astrocytes to dopamine neurons (e.g., dopaminergic neurons) .
EXAMPLES
The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion. The present examples, along with the methods described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses which are encompassed within the spirit of the invention as defined by the scope of the claims will occur to those skilled in the art.
Example 1: Materials and methods
Mice and cell lines
C57BL/6 mice were purchased from Shanghai SLAC Laboratory. The mice were housed in a light/dark cycle room with water and food. All animal experiments were performed and approved by the Animal Care and Use Committee of the CEBSIT, Chinese Academy of Sciences, Shanghai, China. The Cos7, 293T and N2a cell lines were obtained from Cell bank of Shanghai Institute of Biochemistry and Cell Biology (SIBCB) , Chinese Academy of Sciences (CAS) , and cultured in DMEM with 10%FBS and 1%penicillin/streptomycin in a 37℃ incubator under 5%CO
2.
Transfection, qPCR and RNA-seq
Transient transfection was conducted with 4 μg vectors expressing CAG-CasRx-P2A-GFP/CAG-Cas13X-P2A-GFP + 2 μg U6-gRNA-CMV-mCherry plasmid using Lipofectamine 3000. CAG-CasRx-P2A-GFP/CAG-Cas13X-P2A-GFP plasmid was used as a control. Two days after transient transfection, around 30K GFP and mCherry double-positive (GFP top 20%) cells were collected by fluorescence activated cell sorting (FACS) and lysed for qPCR analysis. RNA was first extracted using Trizol (Ambion) and then converted to cDNA using a reverse transcription kit (HiScript Q RT SuperMix for qPCR, Vazyme, Biotech) . The amplification was tracked by AceQ qPCR SYBR Green Master Mix (Vazyme, Biotech) . For the RNA-seq, Cos7 cells were cultured in 10-cm dishes. Around 100k positive cells (GFP top 20%) were isolated by FACS, RNA was extracted and reverse transcribed to cDNA, which was then used for RNA-seq. Analysis of the RNA-seq data was performed and presented as the mean of all repeats.
The DNA sequence of CasRx is:
Guide RNA sequences
The guide sequences (i.e. the sequence that is complementary to the PTB mRNA sequence to be targeted) in the guide RNAs as used are shown in Table 1. Guide sequences of guide RNAs (Cas13d) and the relative expression level (vs control without gRNA) achieved by the respective guide RNAs. High efficiency gRNAs (bold, < 0.12) , almost all located in the key region (positions 951-1487 of SEQ ID NO: 87, which is SEQ ID NO: 88; see Table 2 and Figure 1) , are indicated in bold print.
Table 1: Guide Sequences
Table 2: PTB Sequences
As shown in Table 3, Cas13X guide sequences of guide RNAs and the relative expression level (vs control without gRNA) achieved by the respective guide RNAs. High efficiency gRNAs (bold) , all located in the key region (positions 951-1487 of SEQ ID NO: 87, which is SEQ ID NO: 88; see also Figure 2) , are indicated in bold print. The sequence of Cas13X is:
Table 3: Cas13X guide sequences
NHP MPTP model
All subjects in this experiment were adult (7-13 years old) male cynomolgus monkeys (Macaca fascicularis) , which were cared and handled according to the Guidelines for Animal Experiments of WuXi AppTec and the Guide for the Care and Use of Laboratory Animals of GuochenBiotek (Suzhou, Jiangsu province, China) . To induce bilateral parkinsonism in these monkeys, MPTP were intravenously injected once daily until the motor symptoms, including bradykinesia, tremor and rigid, were observed. Once the administration of MPTP were stopped, these animals were turned to observation period for at least 4 weeks to ensure the stability of symptoms. PET-CT was used to determine the elimination of dopamine signals in the striatum.
Stereotactic injection and immunofluorescence staining
AAV8 was used in this study. Stereotactic injections in the mice were performed as previously described (Zhou et al., 2014, Elife 3, e02536, doi: 10.7554/eLife. 02536) . The mice were placed in a stereotactic frame. Next, the skin over the skull was shaven and opened using a razor. A craniotomy with coordinates (AP +0.8 mm, ML ± 1.6 mm) was made over the boundary of frontal and parietal bones, allowing the placement of an injection micropipette (~20 μm outside diameter at the tip) . The viral solution containing either AAV-GFAP-CasRx-Ptbp1+AAV-GFAP-mCherry or AAV-GFAP-CasRx+AAV-GFAP-mCherry was injected slowly (~0.3 μl/min) . Mice were injected in the striatum (AP +0.8 mm, ML ± 1.6 mm and DV 2.6 mm) with AAVs (> 1 X 1012 vg/ml, 1 μL, mice aged 8-10 weeks) .
For Macaca fascicularis, the enrolled animals were rated into three stages (severe, moderate, and mild) based on their motor symptoms. In each stage, the assignment into treatment and control groups was blindly. Around 14 hours before AAV injection, the immunosuppressant dexamethasone or Tacrolimus was administrated via intramuscular injection to reduce immunological reaction. For AAV injection, both sides of the putamen were injected with 60-80 μL (around 1 X 1013 vg/ml) AAV-GFAP-CasRx-Ptbp1+AAV-GFAP-mCherry and AAV-GFAP-CasRx+AAV-GFAP-mCherry, respectively. The volume ratio between GFAP-mCherry and GFAP-CasRx or GFAP-CasRx-Ptbp1 were 1: 20. The viral solution was injected into putamen of both hemispheres using a 100ul syringe and a syringe pump. After the surgery, animals were treated with Ceftriaxone sodium and Meloxicam to avoid infection and painfulness. Daily administration of Dexamethasone or Tacrolimus was performed for about 1 week after surgery.
For immunofluorescence staining, the brains were perfused and fixed with 4%paraformaldehyde (PFA) overnight, and kept in 30%sucrose for at least 12 hours for mice and 2 weeks for Macaca fascicularis. Brains were sectioned after embedding and freezing, and slices with the thickness of 30 μm for mice and 30 μm for Macaca fascicularis were used for immunofluorescence staining. Brain sections were rinsed thoroughly with 0.1 M phosphate buffer (PB) . Primary antibodies: Rabbit-anti-NeuN (1: 500, 24307S, Cell Signaling Technology) , Guinea Pig anti-NeuN antibody (1: 500, ABN90, Millipore) , Mouse anti-Flag (1: 2000, F3165, Sigma) , Rabbit anti-TH anti-body (1: 500, AB152, Millipore) , Rat anti-DAT (1: 100, MAB369, Millipore) and Rabbit anti-RBPMS (Proteintech, Cat# 15187-1-AP) . Secondary antibodies: Alexa Fluora 488 AffiniPure Donkey Anti-Rabbit IgG (H+L) (1: 500, 711-545-152, Jackson ImmunoResearch) Alexa Fluora 488 AffiniPure Donkey Anti-Mouse IgG (H+L) (1: 500, 715-545-150, Jackson ImmunoResearch) Cy5-AffiniPure Donkey Anti-Guinea Pig IgG (H+L) (1: 500, 706-175-148, Jackson ImmunoResearch) Cy5 AffiniPure Donkey Anti-Rabbit IgG (H+L) (1: 500, 711-175-152, Jackson ImmunoResearch) were used in this study. After antibody incubation, slices were washed and then covered with mountant (Life Technology) . Images were collected under an Olympus FV3000 microscope.
Subretinal injection and immunofluorescence staining
For Mus musculus, NMDA and AAV8 were introduced via subretinal injection. Mice were anaesthetized and AAVs were slowly injected into the subretinal space. To determine the conversion in intact retinas, in total 1 μl AAV-GFAP-GFP-Cre (0.2 μl) and AAV-GFAP-CasRx-Ptbp1 (0.4 μl) , or AAV-GFAP-GFP-Cre (0.2 μl) and AAV-GFAP-CasRx (0.4 ml) and PBS (0.4 μl) or AAV-GFAP-GFP-Cre (0.2 μl) and AAV-GFAP-CasRx-Ptbp1 (0.4 μl) and AAV-GFAP-β-catenin (0.4 μl) were delivered to the retina via subretinal injection (Ai9 and C57BL/6 mice, aged 4 months) . Two-three weeks after AAV injection, the eyes and optic nerves were collected.
For Macaca fascicularis, animals (2.8-year-old) were anaesthetized, and the pupil size was dilated, then AAVs were slowly injected into the subretinal space. The needle was removed, and the eye ointment was administrated after injection. To determine the conversion, AAV-GFAP-tdTomato + AAV-GFAP-CasRx-Ptbp1 or AAV-GFAP-tdTomato + AAV-GFAP-CasRx were delivered. Around one-to-two months after AAV injection, the eyes, optic nerves, and brain tissue were collected.
Tissue samples were then fixed with 4%paraformaldehyde (PFA) , and then maintained in the solution containing 30%sucrose. After embedding, the eyes were sectioned, and slices were washed and covered with mountant (Life Technology) . The images were collected using Olympus FV3000 microscope.
Video recording and analysis
To analyze the behavioral changes of these animals, 2 paradigms (home cage and open field) were used in these experiment and videos were recorded in a series of time points including healthy baseline (before MPTP administration) , T0 (after MPTP administration but before virus injection) and once per month after virus injection. In the paradigm of home cage, animals were filmed in their own cages for 1 hour without any disturbance and then follow with 3 times of feeding of peanuts (small piece of food to test finger flexibility) and 20 minutes of feeding of apple (big food to test hand coordination and eating speed) .
In the paradigm of open field, each animal was moved to a transparent cage (1.2m X 1.2m X 1.2m) with foods (apple and peanuts to induce animal movement) in each corner and recorded by 5 camaras (4 on the floor and 1 on the top) for 1.5h without human interference. After that, the animal keeper walked around the cage for 3 times and then 3 times of pole provoking (sticked a pole into the cage and swung it several times) toward to animal.
The behavioral changes of these animals were quantified with a rating score based on previous works. The movement of animal in open field was analyzed by Ethovision XT.
PET-CT scanning
To monitor the therapeutic efficiency in these monkey, PET-CT was typically performed before and after virus injection. For some animals, we also performed PET-CT scanning before MPTP administration. Monkeys were fasted for 12 hours and then habituated for more than 1 hour before imaging. Intramuscular injection of propofol or Zoletil50 were used to induce stable anesthesia before and during scanning. After anesthesia, each animal was intravenously injected with a PET tracer, [18F] DOPA, 30 minutes before emission scanning. The animals were placed on the PET scanner (Discovery IQ Gen 2, GE healthcare) and CT scan was performed for attenuation correction, followed by a 10-minute emission scan. The emission data were reconstructed using Ultra-TOF algorithm with an attenuation correction and no scatter correction. The regions of interest (ROIs) were manually drawn on the bilateral putamenand cerebellum. The analysis of PET data was performed using RadiAnt DICOM Viewer (Medixant, Poland) . The standardized uptake values (SUVs) of the left putamen, right putamen and cerebellum were quantified following the formular: SUV= cumulative radioactivity in ROIs / (injection dose/body weight) . Data was quantified as SUV ratio (SUVR) using cerebellum as internal control following the formular: SUVR= (SUV
putamen -SUV
cerebellum) / SUV
cerebellum and the average of SUVRs of both hemispheres were then used as the final SUVR of the monkey.
MRI scanning
Animals were anaesthetized with propofol, and then the T1-and T2-weighted MR images were obtained using a 3-tesla MRI scanner (Discovery
TM MR750w GEM 3.0T, GE Healthcare) . For the first scanning, a high-resolution 3D T1-weighted image was obtained for each animal by an Ax 3D T1BRAVO sequence (TR= 6.7ms, TE= 2.5ms, FA= 15., matrix= 192×192, Freq·FOV= 18.0mm, Phase FOV= 1.0mm, slice thickness= 1.0mm) to identify specific brain regions including hippocampus, cerebellum and striatum. A high-resolution 3D T2-weighted image was then obtained with T2 CUBE sequence (TR= 2500ms, TE= 292ms, matrix= 320×320, Freq·FOV= 17.6mm, Phase FOV= 0.90mm, slice thickness= 0.6mm) . The analysis of MRI data was performed using RadiAnt DICOM Viewer (Medixant, Poland) .
HPLC for dopamine concentration determination
To quantify the extracellular dopamine level in monkeys’ central nerve system, we collected cerebrospinal fluid (CSF) samples from monkeys in three time points (before MPTP administration, after MPTP but before AAV injection and after AAV injection) and analyzed them using high precision liquid chromatography (HPLC) . CSF samples were obtained via lumbar puncture during monkey anesthesia in the same time period (9 to 12 am) and then frozen in -80℃ refrigerator. All three samples of each monkey were examined at the same batch to avoid reading variability. To perform the HPLC (UPLC (Shimadzu) and MS (Triple Quad6500+) (Model No: LCMS021) ) , the samples were completely thaw at room temperature under yellow light and vortex individually. Then the samples, internal standard (2.00ng/mL Dopamine-D4 in de-ionized water) and blank sample (de-ionized water) were added in 96-well plate in turn. Borate Buffer and AQC (1.00mg/mL) were added into each sample immediately and stood for 1 min at room temperature. An aliquot of 10.0 μL of the mixture was injected into the LC-MS/MS system. The internal standard was used to quantify and identify the peak on the chromatograph and the level of dopamine were quantified and expressed as pg·mL
-1.
Example 2: Ptbp1 in M. Fascicularis
In this study, a system was designed that could specifically downregulate Ptbp1 expression in both mice, Macaca Fascicularis and human cells, and further showed that CasRx-mediated downregulation of Ptbp1 could directly convert astrocytes into dopamine neurons with high efficiency in the striatum of Macaca Fascicularis. Taken together, our study provides the pre-clinical evidence that Ptbp1-mediated dopamine neuron conversion is also feasible in primates, i.e. in both human and non-human primates, paving the road for the clinical trials.
To knock down the expression of Ptbp1, we used the RNA-guided and RNA-targeting CRISPR protein CasRx, which is an efficient and specific approach for downregulation of RNAs. To examine the efficiency of CasRx-mediated knockdown of Ptbp1, we designed 86 gRNAs targeting the human Ptbp1 gene. We observed that co-transfection of a vector containing CasRx gene with gRNAs that target Ptbp1, resulted in reduction of Ptbp1 mRNA in human 293T cells, 2 days after transfection (Figure 1A; for gRNA sequences, see Table 1) . Specifically, targeting of a key region in human Ptbp1 gene (positions 951-1487 of SEQ ID NO: 87, which correspond to position 1-536 of SEQ ID NO: 88) frequently induced potent downregulation (Figure 1B) . In this experiment, gRNAs targeting this key region achieved a knock-down to 40%or lower, and potent knock-down to 10%or lower is only observed in this key region. Besides CasRx, the key region was further confirmed by another RNA-targeting Cas13 protein Cas13X (Figure 2A and 2B) .
The targeting site of gRNA 60 is conserved in the Macaca Fascicularis, human Ptbp1 and mouse gene and enabled the potent downregulation of Ptbp1 gene in human 293T, Monkey Cos7 cells and mouse N2a cells, thus it was used in the following experiments (Figures 3) . To determine the targeting specificity of this strategy, we performed RNA-seq and found that Ptbp1 was specifically downregulated in Cos7 cells (Figure 4) . A recent study showed that Ptbp1 knockdown could convert striatal astrocytes into dopamine neurons in mice, we next examined whether Ptbp1 knockdown in the nonhuman primate striatal astrocytes could locally convert astrocytes into dopamine neurons in vivo. To validate the AAV vectors in vivo, we first injected wild type mice with AAV-GFAP-CasRx-Ptbp1 expressing CasRx and gRNA 60, together with AAV-GFAP-mCherry that fluorescently labeled astrocytes. We also constructed the control vector AAV-GFAP-CasRx that does not express Ptbp1 gRNA (Figures 5A) . One week after injection, we found that both mCherry and CasRx were specifically expressed in astrocytes and showed a high co-localization efficiency in the striatum (Figure 5B) . In addition, mCherry+ cells expressed mature neuron markers NeuN were observed after injected with AAV-GFAP-mCherry and AAV-GFAP-CasRx-Ptbp1 but not in the control striatum injected with AAV-GFAP-CasRx (Figure 5C) . Moreover, a large fraction of mCherry+ cells expressed dopamine neuron marker DAT at one month after AAV injection (Figure 5D) .
Besides astrocyte-to-dopamine neuron conversion, our previous results showed that knockdown of Ptbp1 could convert Muller Glia (MG) into retinal ganglion cells (RGCs) in the mature retinas aged 4-8 weeks. However, it is unknown whether this strategy is applicable to middle-aged and old mice. We thus introduced AAV-GFAP-CasRx-Ptbp1 and AAV-GFAP-GFP-Cre into the eyes of ai9 mice (CAG-LSL-tdTomato) aged 4-5 months via subretina injection, and observed a small number of tdTomato+ axons in the optic nerve, 3 weeks after injection. To explore whether stimulation of MG re-cycling could increase the MG-to-RGC conversion, we co-injected AAV-GFAP-CasRx-Ptbp1, AAV-GFAP-GFP-Cre with AAV-GFAP-β-catenin into the retinas aged 4-5 months. Around 3 weeks after injection, we found that injection AAV-GFAP-CasRx-Ptbp1 + AAV-GFAP-β-catenin + AAV-GFAP-GFP-Cre enhanced the number of converted RGCs compared to that of AAV-GFAP-CasRx-Ptbp1 and AAV-GFAP-GFP-Cre (see Figure 6) . Taken together, we provide a new strategy to enhance the RGC conversion in the middle-aged or old mammalian.
Example 3: Alleviation of symptoms in PD model monkeys
To explore whether knockdown of Ptbp1 in Macaca Fascicularis striatum could converted astrocytes into dopamine neuron, we injected AAV-GFAP-CasRx-Ptbp1 together with AAV-GFAP-mCherry into the striatum of the right hemisphere of a healthy Macaca Fascicularis aged 8 years, and AAV-GFAP-CasRx together with AAV-GFAP-mCherry into the left striatum. One month after AAV injection, we observed that the expression of mCherry colocalized with GFAP in the striatum injected with control AAVs, suggesting the high astrocyte-targeting specificity of GFAP promoter in the monkey striatum (Figure 7A) . In addition, we found mCherry+TH+ cells in the right striatum but not in the control striatum (Figure 7B) , demonstrating that knockdown of Ptbp1 could also convert astrocytes into dopamine neurons in nonhuman primates.
To determine whether astrocyte-to-dopamine neuron conversion could be achieved in the putamen of PD monkey, we performed immunofluorescence staining to observe a large fraction mCherry+ cells expressed the dopamine neuron marker tyrosine hydroxylase (TH) in the putamen injected with AAV-GFAP-mCherry and AAV-GFAP-CasRx-Ptbp1, but not in the putamen injected with control AAVs (AAV-GFAP-mCherry and AAV-GFAP-CasRx) (Figure 8A) . We further revealed that a high percentage of mCherry+TH+ cells also express mature dopaminergic neuron marker dopamine transporter (DAT) and nigra A9-type dopaminergic neuron marker GIRK2 (Figure 8A and 8B) . Together, these results indicated successful induction of A9-specific dopamine neurons via downregulating Ptbp1 expression in putamen astrocytes. To explore whether induced dopamine neurons have the ability to release dopamine, we performed immunostaining and observed that most of mCherry+TH+ cells expressed vesicular monoamine transporter 2 (VMAT2) (Figure 8C) , which plays an essential role in the packaging, storage, and release of dopamine. Next, to evaluate whether induced dopamine neurons could alleviate the symptoms of Parkinson′s disease, we used a video-based analysis of monkey movements. Our results show that the PD monkeys injected with AAV-GFAP-mCherry + AAV-GFAP-CasRx-Ptbp1 show significant alleviation of PD symptoms (an alleviation of around 40%) nine months after AAV injection (Figure 9) . However, we did not observe obvious alleviation of PD symptoms in the control monkey injected with AAV-GFAP-mCherry + AAV-GFAP-CasRx that does not encode gRNA targeting Ptbp1. To determine whether the induced dopamine neurons functioned as dopaminergic neurons in vivo, we performed 18F-Dopa PET to detect presynaptic dopaminergic function in the putamen. We find the enhancement of these signals in the putamen injected with AAV-GFAP-mCherry and AAV-GFAP-CasRx-Ptbp1, but not for control AAVs (Figure 10A, 10B and 10C) . Furthermore, to examine whether induced dopaminergic neurons could increase dopamine level, we performed HPLC to detect dopamine concentration in CSF. We find the improvement of dopamine level in the PD monkey injected with AAV-GFAP-mCherry and AAV-GFAP-CasRx-Ptbp1, but not in the one injected with control AAVs (Figure 11) . These results showed the functionality of induced dopamine neurons in the putamen of PD monkey.
While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed in practicing the disclosure. It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.
Claims (40)
- A composition for regulating a primate PTBP gene expression comprising:(1) a Cas effector protein, or a nucleic acid encoding the same,(2) a guide RNA (gRNA) that targets a target sequence, or its complement, of the primate PTBP gene or a transcript thereof, or a nucleic acid encoding the same,wherein the composition is capable of suppressing the expression of the primate PTBP gene.
- The composition of claim 1, wherein the target sequence is located within a key region of the primate PTBP gene.
- The composition of claim 2, wherein the key region is SEQ ID NO: 88.
- The composition of any one of claims 1-3, wherein the Cas effector protein is an RNA-targeting Cas effector protein.
- The composition of claim 4, wherein the RNA-targeting Cas effector protein is selected from the group consisting of Cas13d, CasRx, Cas13X, Cas13a, Cas13b, Cas13c, Cas13Y, and a functional domain thereof.
- The composition of claim 5, wherein the Cas effector protein is CasRx.
- The composition of claim 5, wherein the Cas effector protein is Cas13X.
- The composition of any one of claims 1-7, wherein the gRNA sequence has at least 90%sequence identity to SEQ ID NOs: 1-86.
- The composition of any one of claims 1-7, wherein the gRNA sequence has at least 90%sequence identity to SEQ ID NOs: 89-112.
- The composition of any one of claims 1-9, wherein the nucleic acid encoding the Cas effector protein is located on an expression vector.
- The composition of claim 10, wherein the expression vector is a gene therapy vector.
- The composition of claim 11, wherein the gene therapy vector is a viral gene therapy vector.
- The composition of claim 12, wherein the viral gene therapy vector is selected from the group consisting of an adeno-associated virus (AAV) vector, an adenovirus vector, a lentivirus vector, a retrovirus vector, a herpes virus, a SV40 vector, a poxvirus vector, and a combination thereof.
- The composition of any one of claims 10-13, wherein the expression vector is comprised in a nanoparticle.
- The composition of any one of claims 10-14, wherein the expression vector further comprises a glial cell-specific promoter that causes expression of the Cas effector protein in a non-neuronal cell.
- The composition of claim 15, wherein the glial cell-specific promoter is selected from the group consisting of a GFAP promoter, a ALDH1L1 promoter, a EAAT1/GLAST promoter, a glutamine synthetase promoter, a S100 beta promoter and a EAAT2/GLT-1 promoter.
- The composition of claim 15, wherein the glial cell-specific promoter is a Müller glia (MG) cell-specific promoter.
- The composition of claim 17, wherein the MG cell-specific promoter is selected from the group consisting of a GFAP promoter, a ALDH1L1 promoter, a Glast (also known as Slc1a3) promoter and a Rlbp1 promoter.
- The composition of any one of claims 1-18, wherein the nucleic acid encoding the gRNA is located on an expression vector.
- The composition of claim 19, wherein the expression vector further comprises a promoter that causes expression of the gRNA in a non-neuronal cell.
- The composition of claim 20, wherein the promoter that causes expression of the gRNA in the non-neuronal cell is a U6 promoter.
- The composition of any one of claims 1-21, wherein the primate PTBP gene is located in a non-neuronal cell.
- The composition of claim 22, wherein the non-neuronal cell is located in mature retina, striatum, substantia nigra, inner ear, spinal cord, prefrontal cortex, motor cortex, or ventral tegmental area (VTA) .
- The composition of claim 23, wherein the non-neuronal cell located in the striatum is in putamen.
- The composition of claim 24, wherein the non-neuronal cell is a glial cell.
- The composition of claim 25, wherein the glial cell is an astrocyte.
- The composition of any one of claims 1-26 further comprising one or more dopamine neuron-associated factors, or an expression vector for expression of the one or more dopamine neuron-associated factors thereof.
- The composition of claim 27, wherein the one or more dopamine neuron-associated factors are selected from the group consisting of: Lmx1a, Lmx1b, FoxA2, Nurr1, Pitx3, Gata2, Gata3, FGF8, BMP, En1, En2, PET1, a Pax family protein, SHH, a Wnt family protein, and a TGF-β family protein.
- The composition of any one of claims 1-28 further comprising one or more factors selected from β-catenin, Oct4, Sox2, Klf4, Crx, Brn3a, Brn3b, Math5, Nr2e3 and Nrl, or an expression vector for expression of the one or more factors thereof.
- The composition of any one of claims 1-29 further comprising an immunosuppressant.
- The composition of claim 30, wherein the immunosuppressant is selected from the group consisting of a corticosteroid, a calcineurin inhibitor, an mTOR inhibitor, an IMDH inhibitor, an immune-suppressive antibody, an interferon, a Janus kinase inhibitor and an anakinra.
- A method of treating a disease comprising administering an effective amount of the composition of any one of claims 1-31 to a subject in need thereof.
- The method of claim 32, wherein the composition suppresses the expression or activity of the primate PTBP protein in a non-neuronal cell, thereby allowing the non-neuronal cell to reprogram into a functional neuron.
- The method of claim 32 or 33, wherein the disease is a neurological condition associated with degeneration of functional neurons.
- The method of claim 34, wherein the neurological condition is a neurological condition associated with degeneration of functional neurons in the mature retina which is selected from the group consisting of: glaucoma, age-related RGC loss, optic nerve injury, retinal ischemia, LCA (Leber′s congenital amaurosis) , RP (Retinitis pigmentosa) disease and Leber′s hereditary optic neuropathy.
- The method of claim 34, wherein the condition associated with degeneration of functional neurons is selected from the group consisting of Parkinson′s disease, Alzheimer′s disease, Huntington′s disease, Schizophrenia, depression, drug addiction, stroke, movement disorder, spinal cord injury, choreoathetosis, dyskinesias, bipolar disorder, and Autism spectrum disorder (ASD) .
- The method of any one of claims 32-36, wherein the composition is administered to a glial cell or a MG cell in a mature retina to generate a functional retinal ganglion cell (RGC) neuron or a functional retinal photoreceptor.
- The method of any one of claims 32-37, wherein an expression level of PTBP mRNA is less than 40%compared to a corresponding control.
- The method of any one of claims 32-37, wherein an expression level of PTBP mRNA is less than 50%compared to a corresponding control.
- The method of any one of claims 32-37, wherein there are no co-factors expressed.
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