Patent Application
20240366680
CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats) evolved in bacteria and archaea as an adaptive immune system to defend against viral attack. Upon exposure to a virus, short segments of viral DNA are integrated into the CRISPR locus. RNA is transcribed from a portion of the CRISPR locus that includes the viral sequence. That RNA, which contains sequence complementary to the viral genome, mediates targeting of a site-specific nuclease protein such as Cas9 or Cpf1 to a target sequence in the viral genome. The RNA-guided nuclease, in turn, cleaves and thereby silences the viral target.
CRISPR systems have been adapted for gene editing in eukaryotic cells. These systems generally include a protein component (the RNA-guided nuclease) and a nucleic acid component (generally referred to as a guide RNA or “gRNA”). These two components form a complex that interacts with specific target DNA sequences recognized by, or complementary to, the two components of the system and optionally edits or alters the target sequence, for example by means of site-specific DNA cleavage. The value of nucleases such as these as a tool for the treatment of many diseases is widely recognized.
In one aspect, the present disclosure provides a gRNA comprising a spacer comprising the nucleotide sequence of any one of SEQ ID NOs:1-310.
In one aspect, the present disclosure provides a gRNA, wherein the spacer consists of the nucleotide sequence of any one of SEQ ID NOs:1-310.
In one aspect, the present disclosure provides a composition comprising a gRNA comprising any one of SEQ ID NOs.: 1-310.
In one aspect, the present disclosure provides an AAV vector comprising the nucleotide sequence of any one of SEQ ID NOs:1-310.
In one aspect, the present disclosure provides an AAV vector comprising a nucleotide sequence encoding two or more gRNAs, wherein the two or more gRNAs comprise a spacer comprising the nucleotide sequence of any one of SEQ ID NOs:1-310.
In one aspect, the present disclosure provides an AAV vector comprising two or more nucleotide sequences, wherein the two or more nucleotide sequences encode two or more gRNAs comprising a spacer comprising the nucleotide sequence of any one of SEQ ID NOs:1-310.
In one aspect, the present disclosure provides an AAV vector comprising a first nucleotide sequence encoding a gRNA comprising a spacer comprising the nucleotide sequence of SEQ ID NO:5, and a second nucleotide sequence encoding a gRNA comprising a spacer comprising the nucleotide sequence of SEQ ID NO:189.
In one aspect, the present disclosure provides an AAV vector comprising a first nucleotide sequence encoding a gRNA comprising a spacer comprising the nucleotide sequence of SEQ ID NO:7, and a second nucleotide sequence encoding a gRNA comprising a spacer comprising the nucleotide sequence of SEQ ID NO:182.
In one aspect, the present disclosure provides an AAV vector comprising a first nucleotide sequence encoding a gRNA comprising a spacer comprising the nucleotide sequence of SEQ ID NO:7, and a second nucleotide sequence encoding a gRNA comprising a spacer comprising the nucleotide sequence of SEQ ID NO:189.
In one aspect, the present disclosure provides an AAV vector comprising a first nucleotide sequence encoding a gRNA comprising a spacer comprising the nucleotide sequence of SEQ ID NO:5, and a second nucleotide sequence encoding a gRNA comprising a spacer comprising the nucleotide sequence of SEQ ID NO:182.
In some embodiments, the AAV vector which further comprises a nucleotide sequence encoding an RNA-guided nuclease. In some such embodiments, the cell comprises the AAV vector.
In one aspect, the present disclosure provides a method comprising administering to a subject in need thereof (i) a first nucleotide sequence that encodes a guide RNA (gRNA) that targets a gene encoding a voltage-gated sodium channel subunit, and (ii) a second nucleotide sequence that encodes an RNA-guided nuclease, wherein the gRNA comprises a spacer comprising or consisting of the nucleotide sequence of any one of SEQ ID NOs:1-310.
In one aspect, the present disclosure provides a method comprising: administering to a subject in need thereof a composition comprising (i) a gRNA that targets a gene encoding a voltage-gated sodium channel subunit, wherein the gRNA comprises a spacer comprising or consisting of the nucleotide sequence of any one of SEQ ID NOs:1-310; and (ii) an RNA-guided nuclease.
In some embodiments, the subject has or is at risk of a pain disorder and/or a disorder associated with pain.
In some embodiments, the subject has a genetic mutation within the SCN9A and/or SCN10A locus.
In some embodiments, the pain disorder is nociceptive and/or neuropathic pain.
In one aspect, the present disclosure provides a method of treating a subject for one or more symptoms associated with a pain disorder, the method comprising administering to a subject in need thereof (i) a first nucleotide sequence that encodes a gRNA that targets a gene encoding a voltage-gated sodium channel subunit, and (ii) a second nucleotide sequence that encodes an RNA-guided nuclease, wherein the gRNA comprises a spacer comprising or consisting of the nucleotide sequence of one of SEQ ID NOs:1-310, e.g., wherein a level or measure of at least one symptom is reduced or ameliorated relative to control (e.g., a level or measure of the at least one symptom in the subject prior to the administering).
In one aspect, the present disclosure provides a method of treating a subject for one or more symptoms associated with a pain disorder, the method comprising: administering to a subject in need thereof a composition comprising (i) a gRNA that targets a gene encoding a voltage-gated sodium channel subunit, wherein the gRNA comprises a spacer comprising or consisting of the nucleotide sequence of any one of SEQ ID NOs:1-310; and (ii) an RNA-guided nuclease, wherein a level or measure of at least one symptom is reduced or ameliorated relative to control (e.g., a level or measure of the at least one symptom in the subject prior to the administering).
In some embodiments, the method comprises administering a complex of the gRNA and the RNA-guided nuclease. In some embodiments, the method comprises administering an RNP complex of the gRNA and the RNA-guided nuclease.
In some embodiments, the method comprises a subject, wherein the subject has a genetic mutation within the SCN9A and/or SCN10A locus.
In some embodiments, the method comprises a subject, wherein the subject has or is at risk of a pain disorder and/or disorder associated with pain.
In one aspect, the present disclosure provides a method of treating a subject, the method comprising administering to a neuronal cell of a subject in need thereof (i) a first nucleotide sequence that encodes a gRNA that targets a gene encoding a voltage-gated sodium channel subunit, and (ii) a second nucleotide sequence that encodes an RNA-guided nuclease, wherein the gRNA comprises a spacer comprising or consisting of the nucleotide sequence of one of SEQ ID NOs:1-310.
In some embodiments, the administering comprises contacting at least a portion of a neuronal cell with the first and/or second nucleotide sequences.
In some embodiments, the neuronal cell is a neuron or progenitor thereof. In some embodiments, the neuronal cell or progenitor is a dorsal root ganglion or dorsal root ganglion cell progenitor.
In one aspect, the present disclosure provides a method comprising administering to a neuronal cell of a subject in need thereof (i) a gRNA that targets a gene encoding a voltage-gated sodium channel subunit, and (ii) an RNA-guided nuclease, wherein the gRNA comprises a spacer comprising or consisting of the nucleotide sequence of one of SEQ ID NOs:1-310.
In some embodiments, the method comprises administering a complex of the gRNA and the RNA-guided nuclease.
In some embodiments, the method comprises administering an RNP complex of the gRNA and the RNA-guided nuclease.
In some embodiments, the administering comprises contacting at least a portion of the neuronal cell with the gRNA and the RNA-guided nuclease.
In some embodiments, the neuronal cell is a neuron or progenitor thereof. In some embodiments, the neuronal cell or progenitor is a dorsal root ganglion or dorsal root ganglion cell progenitor.
In some embodiments, the neuronal cell has a genetic mutation within the SCN9A or SCN10A locus.
In one aspect, the present disclosure provides a method of editing a neuronal cell in a subject in need thereof, the method comprising: administering to the subject (i) a first nucleotide sequence that encodes a gRNA that targets a gene encoding a voltage-gated sodium channel subunit, wherein the gRNA comprises a spacer comprising or consisting of the nucleotide sequence of any one of SEQ ID NOs:1-310; and (ii) a second nucleotide sequence that encodes an RNA-guided nuclease, wherein upon expression of the gRNA and the RNA-guided nuclease, the nuclease creates one or more single- or double-strand breaks within the gene or within a regulatory element of the gene.
In some embodiments, the one or more breaks results in one or more nucleotide changes in the gene or regulatory element of the gene.
In some embodiments, the level of expression of the gene is changed relative to control.
In some embodiments, the gene is SCN9A and/or SCN10A.
In some embodiments, the one or more changes is a permanent insertion, deletion, and/or substitution of at least one nucleotide within or near the SCN9A and/or SCN10A gene(s) or regulatory element(s). In some embodiments, the change in gene expression is a reduction in expression.
In some embodiments, the level of SCN9A and/or SCN10A gene expression is reduced as compared to control.
In one aspect, the present disclosure provides a method of editing a neuronal cell in a subject in need thereof comprising: administering to the subject (i) a first nucleotide sequence that encodes a gRNA that targets an SCN9A and/or SCN10A gene or regulatory element, wherein the gRNA comprises a spacer comprising or consisting of the nucleotide sequence of any one of SEQ ID NOs:1-310; and (ii) a second nucleotide sequence that encodes an RNA-guided nuclease, wherein upon expression of the gRNA and the RNA-guided nuclease, the nuclease creates one or more single- or double-strand breaks within the SCN9A or SCN10A genes or regulatory element.
In one aspect, the present disclosure provides a method of treating a subject, the method comprising: (i) administering to an autologous iPSC cell (a) a first nucleotide sequence that encodes a gRNA that targets a gene encoding a voltage-gated sodium channel subunit, wherein the gRNA comprises a spacer comprising or consisting of the nucleotide sequence of any one of SEQ ID NOs:1-310; and (b) a second nucleotide sequence that encodes an RNA-guided nuclease; (ii) differentiating the iPSC cell into a neuron or neuronal progenitor; and (iii) transplanting the differentiated cell into a subject.
In some embodiments, the present disclosure provides a gRNA that targets a PAM that is targeted by a gRNA comprising a spacer comprising the nucleotide sequence of any one of SEQ ID NOs:1-310.
In one aspect, the present disclosure provides a gRNA that targets (i) a target sequence of a gene encoding a voltage-gated sodium channel subunit or (ii) a target sequence of a regulatory element of the gene, wherein the target sequence is targeted by a gRNA comprising a spacer comprising the nucleotide sequence of any one of SEQ ID NOs:1-310.
In some embodiments, the method comprises administering a vector comprising the first nucleotide sequence and/or the second nucleotide sequence.
In some embodiments, the vector is an AAV vector. In some embodiments, the AAV vector comprises components from one serotype of AAV. In some embodiments, the AAV vector comprises components from more than one serotype of AAV. In some embodiments, the serotype is 1, 2, 3, 4, 5, 6, 7, 8, or 9.
In some embodiments, a cell is a neuronal cell or precursor thereof. In some embodiments the neuronal cell is a peripheral neuronal cell or precursor thereof. In some embodiments, the peripheral neuronal cell is a dorsal root ganglion cell.
In some embodiments, the composition further comprises an RNA-guided nuclease. In some embodiments, the composition further comprises a complex of a gRNA and an RNA-guided nuclease. In some embodiments, the composition comprises an RNP complex of the gRNA and the RNA-guided nuclease.
In some embodiments, the composition comprises an AAV vector. In some embodiments, the composition further comprises a second AAV vector comprising a nucleotide sequence encoding an RNA-guided nuclease.
Throughout the specification, several terms are employed that are defined in the following paragraphs. Other definitions are also found within the body of the specification.
As used herein, the terms “about” and “approximately,” in reference to a number, is used herein to include numbers that fall within a range of 20%, 10%, 5%, or 1% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).
As used herein, the term “cleavage” refers to the breakage of the covalent backbone of a DNA molecule. In some embodiments, cleavage can be initiated by a variety of methods including, but not limited to, enzymatic or chemical hydrolysis of a phosphodiester bond. In some embodiments, both single-stranded cleavage and double-stranded cleavage are possible, and double-stranded cleavage can occur as a result of two distinct single-stranded cleavage events. In some embodiments, DNA cleavage can result in the production of either blunt ends or cohesive ends.
As used herein, a “conservative substitution” refers to a substitution of an amino acid made among amino acids within the following groups: i) methionine, isoleucine, leucine, valine, ii) phenylalanine, tyrosine, tryptophan, iii) lysine, arginine, histidine, iv) alanine, glycine, v) serine, threonine, vi) glutamine, asparagine and vii) glutamic acid, aspartic acid. In some embodiments, a conservative amino acid substitution refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein in which the amino acid substitution was made.
As used herein, the term “heterologous,” in reference to polypeptide domains, refers to the fact that the polypeptide domains do not naturally occur together (e.g., in the same polypeptide). For example, in fusion proteins generated by the hand of man, a polypeptide domain from one polypeptide may be fused to a polypeptide domain from a different polypeptide. The two polypeptide domains would be considered “heterologous” with respect to each other, as they do not naturally occur together.
As used herein, the term “host cell” is a cell that is manipulated according to the present disclosure, e.g., into which nucleic acids are introduced. A “transformed host cell” is a cell that has undergone transformation such that it has taken up exogenous material such as exogenous genetic material, e.g., exogenous nucleic acids.
As used herein, the term “identity” refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules. In some embodiments, polymeric molecules are considered to be “substantially identical” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. Calculation of the percent identity of two nucleic acid or polypeptide sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In certain embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of the length of a reference sequence. The nucleotides at corresponding positions are then compared. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. As is well known in the art, amino acid or nucleic acid sequences may be compared using any of a variety of algorithms, including those available in commercial computer programs such as BLASTN for nucleotide sequences and BLASTP, gapped BLAST, and PSI-BLAST for amino acid sequences. Exemplary such programs are described in Altschul, et al., Basic local alignment search tool, J. Mol. Biol., 215(3): 403-410, 1990; Altschul, et al., Methods in Enzymology; Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997; Baxevanis et al., Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins, Wiley, 1998; and Misener, et al., (eds.), Bioinformatics Methods and Protocols (Methods in Molecular Biology, Vol. 132), Humana Press, 1999.
As used herein, the term “operably linked” refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. In some embodiments, a regulatory element is “operably linked” to a functional element. In some such embodiments, an operably linked regulatory element is associated in such a way that expression and/or activity of the functional element is achieved under conditions compatible with the regulatory element. In some embodiments, “operably linked” regulatory elements are contiguous (e.g., covalently linked) with the coding elements of interest; in some embodiments, regulatory elements act in trans to or otherwise at a from the functional element of interest.
As used herein, the term “nuclease” refers to a polypeptide capable of cleaving the phosphodiester bonds between the nucleotide subunits of nucleic acids; the term “endonuclease” refers to a polypeptide capable of cleaving the phosphodiester bond within a polynucleotide chain.
As used herein, the terms “nucleic acid”, “nucleic acid molecule” or “polynucleotide” are used herein interchangeably. They refer to a polymer of deoxyribonucleotides or ribonucleotides in either single- or double-stranded form, and unless otherwise stated, encompass known analogs of natural nucleotides that can function in a similar manner as naturally occurring nucleotides. The terms encompass nucleic acid-like structures with synthetic backbones, as well as amplification products. DNAs and RNAs are both polynucleotides. The polymer may include natural nucleosides (i.e., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine), nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, C5-propynylcytidine, C5-propynyluridine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-methylcytidine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine, and 2-thiocytidine), chemically modified bases, biologically modified bases (e.g., methylated bases), intercalated bases, modified sugars (e.g., 2′-fluororibose, ribose, 2′-deoxyribose, arabinose, and hexose), or modified phosphate groups (e.g., phosphorothioates and 5′-N-phosphoramidite linkages).
As used herein, the term “oligonucleotide” refers to a string of nucleotides or analogues thereof. In some embodiments, oligonucleotides may be obtained by a number of methods including, for example, chemical synthesis, restriction enzyme digestion or PCR. As will be appreciated by one skilled in the art, the length of an oligonucleotide (i.e., the number of nucleotides) can vary widely, often depending on the intended function or use of the oligonucleotide. Throughout the specification, whenever an oligonucleotide is represented by a sequence of letters (chosen from the four base letters: A, C, G, and T, which denote adenosine, cytidine, guanosine, and thymidine, respectively), the nucleotides are presented in the 5′ to 3′ order from the left to the right. In certain embodiments, the sequence of an oligonucleotide includes one or more degenerate residues described herein.
As used herein, the term “off-target” refers to binding, cleavage and/or editing of an unintended or unexpected region of DNA by an RNA guided nuclease. In some embodiments, a region of DNA is an off-target region when it differs from the region of DNA intended or expected to be bound, cleaved and/or edited by 1, 2, 3, 4, 5, 6, 7 or more nucleotides.
As used herein, the term “on-target” refers to binding, cleavage and/or editing of an intended or expected region of DNA by an RNA guided nuclease.
As used herein, the term “polynucleotide” generally has its art-recognized meaning of a polymer of nucleic acids. The term is also used to refer to specific functional classes of polynucleotides, such as, for example, guide RNA molecules.
As used herein, the term “polypeptide” generally has its art-recognized meaning of a polymer of amino acids. The term is also used to refer to specific functional classes of polypeptides such as for example nucleases antibodies etc
As used herein, the term “regulatory element” refers to a DNA sequence that controls or impacts one or more aspects of gene expression. In some embodiments, a regulatory element is or includes a promoter, an enhancer, a silencer, and/or a termination signal. In some embodiments, a regulatory element controls or impacts inducible expression.
As used herein, the term “target site” refers to a portion of a double-stranded nucleic acid to which a binding molecule, e.g., a guide RNA or a guide RNA:Cas complex, will bind, provided sufficient conditions for binding exist. Typically, the target site comprises a nucleic acid sequence to which a binding molecule, e.g., a guide RNA or a guide RNA:Cas complex described herein, binds and/or that is cleaved as a result of such binding. In some embodiments, a target site comprises a nucleic acid sequence (also referred to herein as a target sequence or protospacer) that is complementary to a DNA sequence to which the targeting sequence (also referred to herein as the spacer) of a guide RNA described herein binds. In the context of RNA-guided nucleases, e.g., CRISPR/Cas nucleases, a target site typically comprises a nucleotide sequence (also referred to herein as a target sequence or a protospacer) that is complementary to a sequence comprised in a guide RNA (also referred to herein as the targeting sequence or the spacer) of the RNA-programmable nuclease. The target site further comprises a protospacer adjacent motif (PAM) at the 3′ end or 5′ end adjacent to the guide RNA-complementary sequence. For the RNA-guided nuclease Cas9, the target sequence may be, in some embodiments, 16-24 base pairs plus a 3-6 base pair PAM (e.g., NNN, wherein N represents any nucleotide). Exemplary PAM sequences for RNA-guided nucleases, such as Cas9, are known to those of skill in the art and include, without limitation, NNG, NGN, NAG, NGA, NGG, NGAG and NGCG wherein N represents any nucleotide. In addition, Cas9 nucleases from different species have been described, e.g., S. thermophilus recognizes a PAM that comprises the sequence NGGNG, and Cas9 from S. aureus recognizes a PAM that comprises the sequence NNGRRT. Additional PAM sequences are known in the art, including, but not limited to NNAGAAW and NAAR (see, e.g., Esvelt and Wang, Molecular Systems Biology, 9:641 (2013), the entire contents of which are incorporated herein by reference). For example, the target site of an RNA-guided nuclease, such as, e.g., Cas9, may comprise the structure [Nz]-[PAM], where each N is, independently, any nucleotide, and z is an integer between 1 and 50. In some embodiments, z is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50. In some embodiments, z is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. In some embodiments, Z is 20.
The present disclosure encompasses, in part, the discovery of RNA-guided nucleases that can target one or more DNA sequences in order to modify the one or more sequences in a way that impacts one or more symptoms of a pain disorder and/or a disorder associated with pain in a subject. Accordingly, in some embodiments, the present disclosure provides one or more technologies (e.g., compositions, systems, methods, etc.) related to genomic editing, e.g., a gene of interest. In some embodiments, a gene of interest is SCN9A and/or SCN10A. In some embodiments, a protein of interest is NaV1.7 and/or NaV1.8.
In some embodiments, the present disclosure provides technologies for delivery of one or more functional effector proteins, e.g., nucleases, e.g., site-specific nucleases, e.g. Cas9 nucleases (including variants and fusions thereof), and/or nucleic acids (e.g., guide RNAs (gRNAs)) to a cell (e.g., in-vitro, ex-vivo, in-vivo). In some embodiments, the gene editing proteins include CRISPR/Cas proteins.
In some embodiments, technologies provided by the present disclosure may be useful for delivery of such functional effector proteins into a cell, e.g., in the context of manipulating the cell for a research or therapeutic purpose, e.g., via gene editing. For example, delivery of site-specific proteins, such as Cas proteins (or variants or fusions thereof) using the technologies provided herein allows for the targeted manipulation/modification of the genome of a host cell, e.g., in or around SCN9A and/or SCN10A, in-vitro, ex-vivo, or in-vivo.
In some embodiments, gene editing refers to adding, disrupting and/or changing a sequence of specific genes and/or regulatory elements thereof by insertion, removal and/or substitution or mutation of DNA from a genome using artificially engineered proteins and related molecules. For example, gene editing proteins, such as Cas nucleases, may be delivered to a cell using a method described herein. The gene editing protein can introduce double stranded breaks at a target locus in the host cell genome, resulting in altered gene function and/or expression. The activity of gene editing proteins can also promote DNA repair (e.g., non-homologous end joining or homology-directed repair), which is useful for rescue construct-mediated stable integration of foreign genetic material into the genome of a host cell.
In some embodiments, a CRISPR/Cas9-based gene editing approach can be used. A CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 gene editing system can efficiently disrupt genes at desired loci, enabling either complete gene knockout or homology directed repair. For example, in some embodiments, a guide RNA (gRNA) directs a site-directed polypeptide (e.g., an RNA-guided nuclease, e.g., a Cas9 endonuclease) to specific sites in the genome proximal to a protospacer adjacent motif (PAM), causing a double-strand break (DSB). Host cell machinery efficiently repairs the break via a non-homologous end joining (NHEJ) pathway, and during this process also introduces one or more changes (e.g., insertions or deletions (“indels”)) at the target site. Accordingly, gene disruption (e.g., frameshifts) can be achieved if the one or more changes (e.g., indels) in a target site is/are within the coding sequence of a given gene.
In some embodiments, a site-directed polypeptide is or comprises a Casendonuclease. In some embodiments, a Cas endonuclease is or comprises, for example, S. pyogenes Cas9, S. aureus Cas9, N meningitides Cas9, S. thermophilus CRISPRI Cas9, S. thermophilus CRISPR3 Cas9, T denticola Cas9, and Acidaminococcus sp. BV3L6 Cpf1, and variants thereof. Additional suitable Cas nucleases are known in the art. In some preferred embodiments, an endonuclease is a Cas9 endonuclease. In some such embodiments, a Cas9 endonuclease is an S. aureus endonuclease.
In some embodiments, a genome editing system uses a guide RNA (gRNA) comprising a spacer that comprises or consists of the nucleotide sequence of any one of SEQ ID Nos. 1-310. In some embodiments, a gRNA targets a target sequence. In some such embodiments, a target sequence is a gene or regulatory region of SCN9A and/or SCN10A.
In some embodiments, a gRNA that targets SCN9A comprises a spacer that comprises or consists of the nucleotide sequence of any one of SEQ ID Nos. 1-176.
In some embodiments, a gRNA that targets SCN10A comprises a spacer that comprises or consists of the nucleotide sequence of any one of SEQ ID Nos. 177-310.
In some embodiments, a system comprises at least one gRNA of SEQ ID Nos. 1-310 and a site-directed polypeptide (e.g., an RNA-guided nuclease described herein).
In some embodiments, a gRNA is or comprises an sgRNA (single-guide RNA), which sgRNA combines tracrRNA and crRNA (the two, separate molecules in the native CRISPR/Cas9 system in S. pyogenes) into a single RNA construct. In some such embodiments, such an sgRNA may be used for plasmid or in-vitro translation (IVT) expression.
In some embodiments, a gene editing system of the disclosure can be adapted for treating one or more symptoms of a pain disorder and/or disorder associated with pain, which one or more symptoms result from dysfunction/dysregulation in/of a voltage gated sodium channel, e.g., NaV1.7 and/or NaV1.8, or its subunits (e.g., SCN9A and/or SCN10A). For example, CRISPR/Cas9 components for targeting a gene encoding a subunit of NaV1.7 (i.e., SCN9A) and/or NaV1.8 (i.e., SCN10A) can be delivered to a subject (e.g., a human subject), having one or more mutant alleles, for example a dominant allele, of a gene encoding a subunit of NaV1.7 (i.e., SCN9A) and/or NaV1.8 (i.e., SCN10A).
SCN9A has a cytogenetic location of 2q24.3 and the genomic coordinate are on Chromosome 2 on the forward strand at position 166, 195, 185-166,375,993. A nucleotide sequence of SCN9A is shown as SEQ ID NO: 311. SCN7A is the gene upstream of SCN9A on the reverse strand and RN7SKP152 is the gene downstream of SCN9A on the reverse strand. AC010127.3 is a gene located on the forward strand opposite of SCN9A. SCN9A has a NCBI gene ID of 6335, Uniprot ID of Q15858 and Ensembl Gene ID of ENSG00000169432. SCN9A has 3906 SNPs, 39 introns and 38 exons. Sequences for the SCN9A gene, and SNPs, among other things, are provided for in International Publication No. WO/2018/007980.
In some embodiments, SCN9A may also be referred to as Sodium Channel, Voltage Gated, Type IX Alpha Subunit, Sodium Voltage-Gated Channel Alpha Subunit 9, Sodium Channel, Voltage-Gated, Type IX, Alpha Polypeptide, Voltage-Gated Sodium Channel Subunit Alpha Navl. 7, Sodium Channel Protein Type IX Subunit Alpha, Neuroendocrine Sodium Channel, Peripheral Sodium Channel 1, HNE-Na, NENA, PN1, GEFSP7, HSAN2D, Navl 0.7, FEB3B, ETHA, and SFNP.
SCN10A has a cytogenetic location of 3p22.2 and the genomic coordinate are on Chromosome 3 on the reserve strand at position 38,696,802-38,794,010. A nucleotide sequence of human SCN10A is shown as SEQ ID NO: 312. AC116038.1 is the gene upstream of SCN10A on the reverse strand and SCN5A is the gene downstream of SCN10A on the reverse strand. SCN10A has a NCBI gene ID of 6336, Uniprot ID of Q9Y5Y9 and Ensembl Gene ID of ENSG00000185313. SCNIOA has 6380 SNPs, 26 introns and 27 exons. Sequences for the SCN10A gene, and SNPs, among other things, are provided for in International Publication No. WO/2018/007976.
In some embodiments, SCN10A may also be referred to Sodium Channel, Voltage Gated, Type X Alpha Subunit, Sodium Voltage-Gated Channel Alpha Subunit 10, Sodium Channel Voltage-Gated Type X Alpha Polypeptide, Voltage-Gated Sodium Channel Subunit Alpha Nav1.8, Sodium Channel Protein Type X Subunit Alpha, Peripheral Nerve Sodium Channel 3, HPN3, PN3, Navl 0.8, FEPS2, and SNS.
In some embodiments, a gene editing system of the disclosure can be used to modify one or more wild-type or non-wild-type alleles at one or more loci of a targeted gene (e.g., SCN9A and/or SCN10A). In some embodiments, a replacement copy of a gene encoding a non-wild-type alpha subunit of NaV1.7 and/or NaV1.8 protein can be provided. In some embodiments, a replacement copy of a gene is not provided. In some such embodiments, a targeted gene may be modified in a way that produces a change such that protein is no longer produced from that gene (e.g., a frameshift mutation that results in no functional protein produced from the gene).
In some embodiments, technologies (e.g., methods) of the present disclosure can involve editing one or both alleles of a particular gene. Gene editing to modify the allele(s) has the advantage of permanently altering the target gene or gene products in a particular cell or cells derived therefrom. In some embodiments, any CRISPR endonuclease may be used in the methods of the present disclosure. As is known to those of skill in the art, any given CRISPR endonuclease has its own associated PAM, which may or may not be disease, condition, and/or disorder specific. For example, in some embodiments,
In some embodiments, methods of the present disclosure are performed in-vitro, ex-vivo, and/or in-vivo.
In some embodiments, ex-vivo methods of the present disclosure can comprise editing cells of or derived from a peripheral nervous system (e.g., a neuron or a glial cell such as Schwann cell in nerves or satellite glial cell in ganglia, a neuronal progenitor, or neural precursor cell). In some such embodiments, one or more cells is isolated from a subject (e.g., patient in need of treatment, a control subject, etc.) using one or more aspects of gene editing as described herein. In some embodiments, ex-vivo methods in accordance with the present disclosure can comprise editing a patient-specific cell (e.g., iPSC or mesenchymal stem cell, neural stem cell, neuronal progenitor cell, neuronal cell, etc.).
In some embodiments, methods of the present disclosure are performed in-vitro. In some such embodiments, in-vitro methods of the present disclosure can comprise editing cells in culture, wherein such cells have been obtained from, e.g., a cell line and have been or will be differentiated into a committed progenitor cell, e.g., a neuronal progenitor cell.
In some embodiments, methods of the present disclosure are performed in-vivo. In some such embodiments, in-vivo methods of the present disclosure can comprise editing cells in patients with or at risk of one or more symptoms of a pain disorder and/or a disorder associated with pain using one or more aspects of gene editing as described herein.
In some embodiments, whether ex-vivo, in-vitro, or in-vivo, the present disclosure provides technologies that comprise editing the SCN9A and/or SCN10A gene(s) in a human cell using methods and approaches comprising gene editing as described herein.
In some embodiments, patients experiencing or at risk of one or more symptoms of a pain disorder or a disorder associated with pain may exhibit a wide range of changes (e.g., insertions, deletions, substitutions, etc.) in the SCN9A and/or SCN10A gene(s) relative to wild type SCN9A and/or SCN10A gene(s). In some embodiments, patients experiencing or at risk of one or more symptoms of a pain disorder or a disorder associated with pain may exhibit no of changes (e.g., insertions, deletions, substitutions, etc.) in the SCN9A and/or SCN10A gene(s). Accordingly, in some embodiments, different patients may require different editing strategies.
For example, in some embodiments, expression of the SCN9A and/or SCN10A gene(s) may be disrupted or eliminated by introducing random insertions or deletions (indels) that arise due to the imprecise NHEJ repair pathway. In some embodiments, target regions may be the coding sequences of the SCN9A and/or SCN10A gene(s) (i.e., exons). In some embodiments, inserting or deleting nucleotides into the coding sequence of a gene may cause a “frame shift” where the normal 3-letter codon pattern is disturbed. In some such embodiments, gene expression and therefore protein production can be reduced or eliminated. Similar approaches may also be used to target any intron, intron:exon junction, or regulatory DNA element of the SCN9A and/or SCN10A gene(s) where sequence alteration may interfere with the expression of the SCN9A and/or SCN10A gene(s) and any resulting gene product(s).
In some embodiments, NHEJ can also be used to delete segments of a target gene (e.g., SCN9A and/or SCN10A), either directly or by altering splice donor or acceptor sites through cleavage by one gRNA targeting several locations, or several gRNAs. In some embodiments, such an approach can be useful if small random indels are inefficient to knock-out the target gene or genes. In some embodiments, pairs of gRNAs (e.g., targeting different locations within a gene or regulatory element and/or targeting different genes) have been used for this type of deletions. Without a donor present, the ends from a DNA break or ends from different breaks can be joined using the several non-homologous repair pathways in which the DNA ends are joined with little or no base-pairing at the junction. In addition to canonical NHEJ, there are similar repair mechanisms, such as alt-NHEJ. If there are two breaks, the intervening segment can be deleted or inverted. In some embodiments, NHEJ repair pathways can lead to insertions, deletions or mutations at the joints.
In some embodiments, NHEJ can also lead to homology-independent target integration. For example, inclusion of a nuclease target site on a donor plasmid can promote integration of a transgene into the chromosomal double-strand break following in-vivo nuclease cleavage of both the donor and the chromosome (Cristea, Biotechnol Bioeng. 2013 March; 1 10(3):871-80). NHEJ was used to insert a 15-kb inducible gene expression cassette into a defined locus in human cell lines after nuclease cleavage. (See e.g., Maresca, M., Lin, V. G., Guo, N. & Yang, Y., Genome Res 23, 539-546 (2013); Suzuki et al. Nature, 540, 144-149 (2016)). In some such embodiments, the integrated sequence may disrupt the reading frame of the SCN9A and/or SCN10A gene(s) or alter the structure (e.g., primary structure, secondary structure, etc.) of the gene(s).
In some embodiments, homology directed repair (HDR) can also be used to knock-out a gene or alter the gene function. For example, an HDR knock-out strategy can comprise disrupting the structure or function of the SCN9A and/or SCN10A gene(s) by inserting into the SCN9A and/or SCN10A gene(s) a nonfunctional or irrelevant sequence. In some such embodiments, this can be achieved by inducing one single stranded break or double stranded break in the gene of interest with one or more CRISPR endonucleases and a gRNA {e.g., crRNA +tracrRNA, or sgRNA), or two or more single stranded breaks or double stranded breaks in the gene of interest with one or more CRISPR endonucleases and two or more gRNAs, in the presence of a donor DNA template introduced exogenously to direct the cellular DSB response to Homology-Directed Repair (the donor DNA template can be a short single stranded oligonucleotide, a short double stranded oligonucleotide, a long single or double stranded DNA molecule). In some instances, this approach can require development and optimization of gRNAs and donor DNA molecules for the SCN9A and/or SCN10A gene(s).
Generally, homology directed repair (HDR) is considered an error-free mechanism that uses a supplied homologous DNA sequence as a template during DSB repair. The rate of HDR is a function of the distance between the mutation and the cut site so choosing overlapping or nearest target sites is important. Templates can include extra sequences flanked by the homologous regions or can contain a sequence that differs from the genomic sequence, thus allowing sequence editing. The most common form of HDR is homologous recombination. There are additional pathways for HDR, including single-strand annealing and alternative-HDR. Genome engineering tools allow researchers to manipulate the cellular homologous recombination pathways to create site-specific modifications to the genome.
In some embodiments, cells can repair a double-stranded break using a synthetic donor molecule provided in trans. Therefore, in some embodiments, by introducing a double-stranded break near a specific mutation and providing a suitable donor, targeted changes can be made in the genome. In some embodiments, specific cleavage increases the rate of HDR more than 1,000 fold above the rate of 1 in 10<6>cells receiving a homologous donor alone. The rate of homology directed repair (HDR) at a particular nucleotide is a function of the distance to the cut site, so choosing overlapping or nearest target sites is important. Accordingly, in some embodiments, the present disclosure appreciates that technologies comprising gene editing offer advantages over, e.g., gene addition, as, in some embodiments, editing in situ can leave the rest of the genome essentially unperturbed.
In some embodiments, supplied donors for editing by HDR vary markedly but can contain a particular portion of a target sequence with small or large flanking homology arms to allow annealing to the genomic DNA. In some embodiments, homology regions flanking the introduced genetic changes can be 30 bp or smaller, or as large as a multi-kilobase cassette that can contain promoters, cDNAs, etc. In some embodiments, single-stranded and/or double-stranded oligonucleotide donors can be used. In some such embodiments, oligonucleotides range in size from less than 100 nt to over many kb, though longer ssDNA can also be generated and used. In embodiments comprising double-stranded donors, such donors may be or include, without limitation, PCR amplicons, plasmids, and mini-circles.
In some embodiments, an AAV vector can be a very effective means of delivery of a donor template, though the packaging limits for individual donors is <5kb. In some embodiments, active transcription of a donor can increase HDR three-fold, indicating the inclusion of promoter may increase conversion. In some embodiments, CpG methylation of a donor can decrease gene expression and HDR.
In addition to wild-type endonucleases, such as Cas9, nickase variants exist that have one or the other nuclease domain inactivated resulting in cutting of only one DNA strand. HDR can be directed from individual Cas nickases or using pairs of nickases that flank the target area. Donors can be single-stranded, nicked, or dsDNA.
In some embodiments, donor DNA can be supplied with a nuclease or independently by a variety of different methods, for example by transfection, nano-particle, micro-injection, or viral transduction. A range of tethering options has been proposed to increase the availability of the donors for HDR. For example, in some embodiments, tethering is or comprises attaching the donor to the nuclease, attaching to DNA binding proteins that bind nearby, or attaching to proteins that are involved in DNA end binding or repair.
One of skill in the art will appreciate that choice of repair pathway can be guided by a number of culture conditions, such as those that influence cell cycling, or by targeting of DNA repair and associated proteins. For example, in some embodiments, key NHEJ molecules (e.g., KU70, KU80 or DNA ligase IV) can be suppressed to, e.g., increase HDR.
In addition to gene editing by NHEJ or HDR, in some embodiments, site-specific gene insertions that use both the NHEJ pathway and HDR may be used. In some embodiments, a combination approach may be applicable in certain settings. In some such embodiments, such an approach may include intron/exon borders. In some embodiments, NHEJ may prove effective for ligation in the intron, while the error-free HDR may be better suited in the coding region.
The SCN9A and/or SCN10A gene(s) contain(s) a number of exons which are known in the art. Any one or more of these exons or nearby introns can be targeted in order to create one or more changes (e.g., indels) that modify (e.g., disrupt) the reading frame and eventually eliminate the SCN9A and/or SCN10A protein presence and/or function/activity.
In some embodiments, technologies of the present disclosure provide gRNA pairs that make a deletion by cutting the gene twice at locations flanking an unwanted sequence. This sequence may include one or more exons, introns, intron:exon junctions, other DNA sequences encoding regulatory elements of the SCN9A and/or SCN10A gene(s) or combinations thereof. The cutting can be accomplished by a pair of DNA endonucleases that each makes a DSB in the genome, or by multiple nickases that together make a DSB in the genome.
In some embodiments, the present disclosure can provide one gRNA to make one double-strand cut within a coding or splicing sequence. The double-strand cut can be made by a single DNA endonuclease or multiple nickases that together make a DSB in the genome.
In some embodiments, splicing donor and acceptors are generally within 100 base pairs of the neighboring intron. In some such embodiments, the present disclosure provides technologies that can provide gRNAs that cut approximately +/−100-3100 bp with respect to each exon/intron junction of interest.
Any of the gene editing strategies described or derived from the present disclosure, gene editing can be confirmed by sequencing or PCR analysis.
In some embodiments, shifts in the location of the 5′ boundary and/or the 3′ boundary relative to particular reference loci can be used to facilitate or enhance particular applications of gene editing, which depend in part on the endonuclease system selected for the editing, as further described herein.
For example, many endonuclease systems have guidelines or criteria surrounding initial selection of potential target sites for cleavage, such as the requirement of a PAM sequence motif in a particular position adjacent to the DNA cleavage sites in the case of CRISPR Type II or Type V endonucleases.
In some embodiments, frequency of off-target activity for a particular combination of target sequence and gene editing endonuclease (i.e. the frequency of DSBs occurring at sites other than the selected target sequence) can be assessed relative to the frequency of on-target activity. In some embodiments, cells that have been correctly edited at a desired locus can have a selective advantage relative to other cells. By way of non-limiting example, a selective advantage may be or comprise acquisition of attributes such as enhanced rates of replication, persistence, resistance to certain conditions, enhanced rates of successful engraftment or persistence in-vivo following introduction into a patient, and other attributes associated with the maintenance or increased numbers or viability of such cells.
In some embodiments, cells that have been correctly edited at a desired locus can be positively selected for by one or more screening methods used to identify, sort or otherwise select for cells that have been correctly edited. Both selective advantage and directed selection methods can take advantage of the phenotype associated with the alteration.
In some embodiments, cells can be edited two or more times in order to create a second modification that creates a new phenotype that is used to select or purify the intended population of cells. In some such embodiments, a second modification could be created by adding an additional (e.g., second, third, fourth) gRNA enabling expression of a selectable or screenable marker. In some embodiments, cells can be correctly edited at a desired locus using a DNA fragment that contains the cDNA and also a selectable marker.
In some embodiments, target sequence selection can be guided by consideration of off-target frequencies in order to enhance the effectiveness of a given gene editing application and/or reduce the potential for undesired alterations at sites other than the desired target. In some embodiments, occurrence of off-target activity can be influenced by a number of factors including similarities and dissimilarities between the target site and various off-target sites, as well as the particular endonuclease used. Bioinformatics tools are available that assist in prediction of off-target activity, and frequently such tools can also be used to identify the most likely sites of off-target activity. In some such embodiments, assessments of off-target activity can then be confirmed or analyzed in experimental settings to evaluate relative frequencies of off-target to on-target activity. In some such embodiments, selection of sequences that have higher relative on-target activities can be achieved.
In some embodiments, target sequence selection can relate to homologous recombination events. Sequences sharing regions of homology can serve as focal points for homologous recombination events that result in deletion of intervening sequences. Such recombination events occur during the normal course of replication of chromosomes and other DNA sequences, and also at other times when DNA sequences are being synthesized, such as in the case of repairs of double-strand breaks (DSBs), which occur on a regular basis during the normal cell replication cycle but can also be enhanced by the occurrence of various events (such as UV light and other inducers of DNA breakage) or the presence of certain agents (such as various chemical inducers). In some embodiments, many such inducers cause DSBs to occur indiscriminately in the genome, and DSBs can be regularly induced and repaired in normal cells. During repair, the original sequence can often be reconstructed with complete fidelity, however, in some embodiments, small insertions or deletions (referred to as “indels”) are introduced at the DSB site.
In some embodiments, DSBs can be specifically induced at particular locations, as in the case of certain endonucleases systems described herein. In some such embodiments, such systems can be used to cause directed or preferential gene modification events at selected chromosomal locations. The tendency for homologous sequences to be subject to recombination in the context of DNA repair (as well as replication) can be taken advantage of in a number of circumstances, and is the basis for one application of gene editing systems, such as CRISPR, in which homology directed repair is used to insert a sequence of interest, provided through use of a donor polynucleotide, into a desired chromosomal location.
In some embodiments, regions of homology between particular sequences, which can be or comprise small regions of microhomology (comprising ten base pairs or fewer), can also be used to bring about desired deletions. For example, in some embodiments, a single DSB can be introduced at a site that exhibits microhomology with a nearby sequence. In some such embodiments, during the normal course of repair of such DSB, a result that occurs with high frequency is the deletion of the intervening sequence as a result of recombination being facilitated by the DSB and concomitant cellular repair process.
In some embodiments, selecting target sequences within regions of homology can also give rise to much larger deletions, including gene fusions (when deletions are in coding regions), which may or may not be desired given the particular circumstances.
The present disclosure provides and describes herein methods of selection of various target regions for the creation of DSBs designed to induce insertions, deletions or mutations that result in reduction or elimination of SCN9A and/or SCN10A gene(s) and resultant gene products (e.g., proteins) and activity, as well as the selection of specific target sequences within such regions that are designed to minimize off-target events relative to on-target events.
In some embodiments, one or more components of a gene editing system described herein is expressed using an expression vector. In some embodiments, one or more components of a gene editing system described herein is administered to a subject using a viral vector.
Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into a host cell, and thereby are replicated along with the host genome. In some embodiments, vectors can be capable of directing expression of nucleic acids to which they are operatively linked (e.g., often referred to as recombinant expression vectors or expression vectors).
In some embodiments, a regulatory sequence includes, for example, promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are well known in the art and are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences can include sequences that direct constitutive expression of a nucleotide sequence in many types of host cells, as well as those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend factors such as choice of host cell, level of expression desired or required, etc.
In some embodiments, a vector can comprise one or more transcription and/or translation control elements. Depending on the host/vector system utilized, any of a number of suitable transcription and translation control elements, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. can be used in the expression vector. In some embodiments, a vector can be a self-inactivating vector that either inactivates the viral sequences or the components of the CRISPR machinery or other elements. In some embodiments, a vector includes a promoter. In some embodiments, suitable eukaryotic promoters (i.e., promoters functional in a eukaryotic cell) include but are not limited to those from cytomegalovirus (CMV) immediate early, minimal CMV, herpes simplex virus (HSV) thymidine kinase, early and late SV40, long terminal repeats (LTRs) from retrovirus, human elongation factor-1 promoter (EF1), a hybrid construct comprising the cytomegalovirus (CMV) enhancer fused to the chicken beta-actin promoter (CAG), murine stem cell virus promoter (MSCV), phosphogly cerate kinase-1 locus promoter (PGK), and mouse metallothionein-I.
In some embodiments, a promoter can be a neuron specific promoter. Neuron specific promoters are known in the art and include, e.g., those derived from synapsin I (SYN), calcium/calmodulin-dependent protein kinase II Beta (CAMK2B), calcium/calmodulin-dependent protein kinase II alpha (CAMK2A), Methyl-CpG Binding Protein 2 (MECP2), Somatostat, tubulin alpha I (TUBA1A), neuron-specific enolase (ENO2), eSYN (hybrid promoter consisting of a 0.4Kb CMV enhancer (E) and the 0.45Kb human Synapsin I promoter fragment) and/or platelet-derived growth factor beta (PDGFB) promoters. In some embodiments, alternative neuronal specific promoters as known in the art may be utilized in constructs described herein.
In some embodiments, a promoter can be a constitutive promoter (e.g., CMV promoter, UBC promoter). In some embodiments, a promoter can be an inducible promoter (e.g., a heat shock promoter, tetracycline-regulated promoter, steroid-regulated promoter, metal-regulated promoter, estrogen receptor-regulated promoter, etc.). In some embodiments, a promoter can be a spatially restricted and/or temporally restricted promoter (e.g., a tissue specific promoter, a cell-type-specific promoter, etc.).
For expressing small RNAs, including gRNAs (e.g., such as any of those of SEQ ID NOs: 1-310) as described herein and optionally used in connection with a site-directed polypeptide (e.g., an RNA-guided nuclease, e.g., a Cas endonuclease), various promoters such as RNA polymerase III promoters, (e.g., U6 and/or HI) may be suitable or, in some embodiments, can be advantageous. Descriptions of and parameters for selecting and/or enhancing use of such promoters are known in art (see, e.g., Ma, H. et al, Molecular Therapy—Nucleic Acids 3, el 61 (2014) doi: 10.1038/mtna.2014.12.
In some embodiments, an expression vector can also contain a ribosome binding site for translation initiation and/or a transcription terminator. In some embodiments, an expression vector can also comprise appropriate sequences for amplifying expression (e.g., of a given sequence contained therein). In some embodiments, an expression vector can include nucleotide sequences encoding non-native tags (e.g., histidine tag, hemagglutinin tag, green fluorescent protein, etc.). In some such embodiments, the non-native tag is fused to a site-directed polypeptide resulting in a fusion protein.
In some embodiments, a nucleic acid encoding a genome-targeting nucleic acid of the disclosure and/or a site-directed polypeptide can be packaged into or on the surface of delivery vehicles for delivery to cells. Delivery vehicles contemplated include, but are not limited to, nanospheres, liposomes, quantum dots, nanoparticles, polyethylene glycol particles, hydrogels, and micelles. A variety of targeting moieties are known in the art and can be used to facilitate, improve and/or enhance interaction of such vehicles with desired cell types or locations.
In some embodiments, introduction of one or more complexes, polypeptides, and/or nucleic acids of the present disclosure into one or more cells can occur by viral or bacteriophage infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, nucleofection, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct micro-injection, nanoparticle-mediated nucleic acid delivery, etc.
By way of non-limiting example, in some embodiments, viral vectors or components thereof based on vaccinia virus, poliovirus, adenovirus, adeno-associated virus, SV40, herpes simplex virus, human immunodeficiency virus, retrovirus (e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus), lentivirus, alphavirus, enterovirus, pestivirus, baculovirus, herpesvirus, Epstein Barr virus, papovavirus, poxvirus, and other recombinant vectors. In some embodiments a vector may be or comprise pXT1, pSG5, pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia). In some preferred embodiments, one or more vectors comprises adeno-associated virus or components thereof. Other vectors can be used so long as they are compatible with the host cell to which a given vector is provided.
In certain embodiments of the disclosure, a viral vector described herein is an adeno-associated viral (AAV) vector. AAV systems are generally well known in the art (see, e.g., Kelleher and Vos, Biotechniques, 17(6):1110-17 (1994); Cotten et al., P.N.A.S. U.S.A., 89(13):6094-98 (1992); Curiel, Nat Immun, 13(2-3):141-64 (1994); Muzyczka, Curr Top Microbiol Immunol, 158:97-129 (1992); and Asokan A, et al., Mol. Ther., 20(4):699-708 (2012)). Methods for generating and using AAV vectors are described, for example, in U.S. Pat. Nos. 5,139,941 and 4,797,368.
Several AAV serotypes have been characterized, including AAV1, AAV2 (e.g., AAV2g9, AAV2.5, or AAV2i8), AAV3 (e.g., AAV3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV 11, as well as variants and/or hybrids thereof. For example, in some embodiments, an AAV vector is an AAVrh10, AAVhu68, AAV2/5, AAV2/6, AAV2/8 or AAV2/9 vector (e.g., AAV6, AAV8 or AAV9 serotype having AAV2 ITR). Other AAV vectors are described in, e.g., Sharma et al., Brain Res Bull. 2010 Feb. 15; 81(2-3): 273. Generally, any AAV serotype may be used to deliver a transgene described herein. However, in some embodiments, serotypes have different tropisms, e.g., they preferentially infect different tissues, thus, in some embodiments, a serotype may be chosen based upon tropism for a particular tissue and/or cell type. In some embodiments, one or more AAV serotypes can be matched to one or more target cell types.
A recombinant adeno-associated virus (rAAV) vector can be used for delivery. Techniques to produce rAAV particles, in which an AAV genome to be packaged that includes the polynucleotide to be delivered, rep and cap genes, and helper virus functions are provided to a cell are standard in the art. Production of rAAV typically requires that the following components are present within a single cell (denoted herein as a packaging cell): a rAAV genome, AAV rep and cap genes separate from (i.e., not in) the rAAV genome, and helper virus functions. The AAV rep and cap genes may be from any AAV serotype for which recombinant virus can be derived, and may be from a different AAV serotype than the rAAV genome ITRs, including, but not limited to, AAV serotypes described herein. Production of pseudotyped rAAV is disclosed in, for example, international patent application publication number WO 01/83692.
The AAV sequences of an AAV vector typically comprise the cis-acting 5′ and 3′ inverted terminal repeat sequences (See, e.g., B. J. Carter, in “Handbook of Parvoviruses”, ed., P. Tijsser, CRC Press, pp. 155 168 (1990)). The ITR sequences are about 145 bp in length. In some embodiments, substantially the entire sequences encoding the ITRs are used in an AAV vector, although some degree of minor modification of these sequences can be permissible. Ability to modify these ITR sequences is known to those of skill in the art. (See, e.g., texts such as Sambrook et al, “Molecular Cloning. A Laboratory Manual”, 2d ed., Cold Spring Harbor Laboratory, New York (1989); and K. Fisher et al., J Virol, 70:520 532 (1996)). For example, in some embodiments, an AAV vector of the present disclosure is a “cis-acting” plasmid containing a transgene, in which the selected transgene sequence and associated regulatory elements are flanked by the 5′ and 3′ AAV ITR sequences. The AAV ITR sequences may be obtained from any known AAV, including known mammalian AAV types and/or those described herein.
In some embodiments, an AAV vector may be a dual or triple AAV vector, e.g., for the delivery of large transgenes (e.g., transgenes of greater than approximately 5kb). In some embodiments, a dual AAV vector may include two separate AAV vectors, each including a fragment of the full sequence of the large transgene of interest, and when recombined, the fragments form the full sequence of the large transgene of interest, or a functional portion thereof.
In some embodiments, a triple AAV vector may include three separate AAV vectors, each including a fragment of the sequence of the large transgene of interest, and when recombined, the fragments form the full sequence of the large transgene of interest, or a functional portion thereof.
The multiple AAV vectors of the dual or triple AAV vectors can be delivered to and co-transduced into the same cell, where the two or three fragments of transgene recombine together and generate a single mRNA transcript of the entire large transgene of interest. In some embodiments, the fragmented transgenes include a non-overlapping sequences. In some embodiments, the fragmented transgenes include a specified overlapping sequences.
In some embodiments, the multiple AAV vectors of the dual or triple may be the same type of AAV vector (e.g., the same serotype, construct, etc.). In some embodiments, the multiple AAV vectors of the dual or triple may be different types of AAV vector (e.g., same serotype, construct, etc.).
Exemplary rAAV nucleic acid vectors useful in accordance with the present disclosure include single-stranded (ss) or self-complementary (sc) AAV nucleic acid vectors.
In some embodiments, an rAAV particle comprises a nucleic acid vector, such as a single-stranded (ss) or self-complementary (sc) AAV nucleic acid vector. In some embodiments, the nucleic acid vector contains an expression construct as described herein and one or more regions comprising inverted terminal repeat (ITR) sequences (e.g., wild-type ITR sequences or engineered ITR sequences) flanking the expression construct. In some embodiments, the nucleic acid is encapsidated by a viral capsid.
Accordingly, in some embodiments, a rAAV particle, vector and/or nucleic acid comprises a viral capsid and a nucleic acid vector as described herein, which is encapsidated by the viral capsid. In some embodiments, the viral capsid comprises 60 capsid protein subunits comprising VP1, VP2 and VP3. In some embodiments, the VP1, VP2, and VP3 subunits are present in the capsid at a ratio of approximately 1:1:10, respectively.
In some embodiments, ITR sequences of a nucleic acid or nucleic acid vector of the present disclosure can be derived from any AAV serotype (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or others as described herein) or can be derived from more than one serotype. In some embodiments, ITR sequences are derived from AAV2. In some embodiments, the ITR sequences are derived from one or more other serotypes. ITR sequences and plasmids containing ITR sequences are known in the art and are commercially available (see, e.g., products and services available from Vector Biolabs Philadelphia PA: Cellbiolabs San Diego CA: Agilent Technologies, Santa Clara, Ca; and Addgene, Cambridge, MA; and Gene delivery to skeletal muscle results in sustained expression and systemic delivery of a therapeutic protein. Kessler P D, Podsakoff G M, Chen X, McQuiston S A, Colosi P C, Matelis L A, Kurtzman G J, Byrne B J. Proc Natl Acad Sci USA. 1996 Nov. 26; 93(24): 14082-7; and Curtis A. Machida. Methods in Molecular Medicine™. Viral Vectors for Gene Therapy Methods and Protocols. 10.1385/1-59259-304-6:201 © Humana Press Inc. 2003. Chapter 10. Targeted Integration by Adeno-Associated Virus. Matthew D. Weitzman, Samuel M. Young Jr., Toni Cathomen and Richard Jude Samulski; U.S. Pat. Nos. 5,139,941 and 5,962,313).
In some embodiments, an expression construct is at least 2, 3, 4, 5, 6, 7, 8, 9, 10 kilobases in size. In some embodiments, an expression construct is no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or less kilobases in size. In some embodiments, the expression construct is between 4 and 7 kilobases in size.
An rAAV vector, particle, and/or nucleic acid may be of any AAV serotype (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10), including any derivative (including non-naturally occurring variants of a serotype) or pseudotype.
In some embodiments, the rAAV serotype is rAAV2. In some embodiments, the rAAV serotype is rAAV5. In some embodiments, the rAAV serotype is rAAV8. In some embodiments, the rAAV serotype is rAAV9. In some embodiments, the rAAV serotype is rAAV5. Such AAV serotypes and derivatives/pseudotypes, and methods of producing such derivatives/pseudotypes are known in the art (see, e.g., Mol Ther. 2012 April; 20(4):699-708. doi: 10.1038/mt.2011.287. Epub 2012 Jan. 24. The AAV vector toolkit: poised at the clinical crossroads. Asokan Al, Schaffer D V, Samulski R J.).
In some embodiments, an AAV vector may utilize or be based on a serotype selected from any of the following serotypes, and variants thereof including but not limited to AAV1, AAV10, AAV106.1/hu.37, AAV11, AAV114.3/hu.40, AAV 12, AAV127.2/hu.41, AAV127.5/hu.42, AAV128.1/hu.43, AAV128.3/hu.44, AAV130.4/hu.48, AAV145.1/hu.53, AAV145.5/hu.54, AAV145.6/hu.55, AAV16.12/hu.11, AAV16.3, AAV16.8/hu.10, AAV161.10/hu.60, AAV161.6/hu.61, AAV1-7/rh.48, AAV1-8/rh.49, AAV2, AAV2.5, AAV2i8, AAV2.5T, AAV2-15/rh.62, AAV223.1, AAV223.2, AAV223.4, AAV223.5, AAV223.6, AAV223.7, AAV2-3/rh.61, AAV24.1, AAV2-4/rh.50, AAV2-5/rh.51, AAV27.3, AAV29.3/bb. 1, AAV29.5/bb.2, AAV2G9, AAV-2-pre-miRNA-101, AAV3, AAV3.1/hu.6, AAV3.1/hu.9, AAV3-11/rh.53, AAV3-3, AAV33.12/hu.17, AAV33.4/hu.15, AAV33.8/hu.16, AAV3-9/rh.52, AAV3a, AAV3b, AAV4, AAV4-19/rh.55, AAV42.12, AAV42-10, AAV42-11, AAV42-12, AAV42-13, AAV42-15, AAV42-1b, AAV42-2, AAV42-3a, AAV42-3b, AAV42-4, AAV42-5a, AAV42-5b, AAV42-6b, AAV42-8, AAV42-aa, AAV43-1, AAV43-12, AAV43-20, AAV43-21, AAV43-23, AAV43-25, AAV43-5, AAV4-4, AAV44.1, AAV44.2, AAV44.5, AAV46.2/hu.28, AAV46.6/hu.29, AAV4-8/r 11.64, AAV4-8/rh.64, AAV4-9/rh.54, AAV5, AAV52.1/hu.20, AAV52/hu.19, AAV5-22/rh.58, AAV5-3/rh.57, AAV54.1/hu.21, AAV54.2/hu.22, AAV54.4R/hu.27, AAV54.5/hu.23, AAV54.7/hu.24, AAV58.2/hu.25, AAV6, AAV6.1, AAV6.1.2, AAV6.2, AAV7, AAV7.2, AAV7.3/hu.7, AAV8, AAV-8b, AAV-8h, AAV9, AAV9.11, AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV9.84, AAV9.9, AAVA3.3, AAVA3.4, AAVA3.5, AAV A3.7, AAV-b, AAVCI, AAVC2, AAVC5, AAVCh.5, AAVCh.5R1, AAVcy.2, AAVcy.3, AAVcy.4, AAVcy.5, AAVCy.5R1, AAVCy.5R2, AAVCy.5R3, AAVCy.5R4, AAVcy.6, AAV-DJ, AAV-DJ8, AAVF3, AAVF5, AAV-h, AAVH-1/hu. 1, AAVH2, AAVH-5/hu.3, AAVH6, AAVhE1.1, AAVhER1.14, AAVhEr1.16, AAVhEr1.18, AAVhER1.23, AAVhEr1.35, AAVhEr1.36, AAVhEr1.5, AAVhEr1.7, AAVhEr1.8, AAVhEr2.16, AAVhEr2.29, AAVhEr2.30, AAVhEr2.31, AAVhEr2.36, AAVhEr2.4, AAVhEr3.1, AAVhu.1, AAVhu.10, AAVhu.11, AAVhu.12, AAVhu.13, AAVhu.14/9, AAVhu.15, AAVhu.16, AAVhu.17, AAVhu.18, AAVhu.19, AAVhu.2, AAVhu.20, AAVhu.21, AAVhu.22, AAVhu.23.2, AAVhu.24, AAVhu.25, AAVhu.27, AAVhu.28, AAVhu.29, AAVhu.29R, AAVhu.3, AAVhu.31, AAVhu.32, AAVhu.34, AAVhu.35, AAVhu.37, AAVhu.39, AAVhu.4, AAVhu.40, AAVhu.41, AAVhu.42, AAVhu.43, AAVhu.44, AAVhu.44R1, AAVhu.44R2, AAVhu.44R3, AAVhu.45, AAVhu.46, AAVhu.47, AAVhu.48, AAVhu.48R1, AAVhu.48R2, AAVhu.48R3, AAVhu.49, AAVhu.5, AAVhu.51, AAVhu.52, AAVhu.53, AAVhu.54, AAVhu.55, AAVhu.56, AAVhu.57, AAVhu.58, AAVhu.6, AAVhu.60, AAVhu.61, AAVhu.63, AAVhu.64, AAVhu.66, AAVhu.67, AAVhu.7, AAVhu.8, AAVhu.9, AAVhu.t 19, AAVLG-10/rh.40, AAVLG-4/rh.38, AAVLG-9/hu.39, AAVLG-9/hu.39, AAV-LK01, AAV-LK02, AAVLK03, AAV-LK03, AAV-LK04, AAV-LK05, AAV-LK06, AAV-LK07, AAV-LK08, AAV-LK09, AAV-LK10, AAV-LK 11, AAV-LK12, AAV-LK13, AAV-LK14, AAV-LK15, AAV-LK17, AAV-LK18, AAV-LK19, AAVN721-8/rh.43, AAV-PAEC, AAV-PAEC11, AAV-PAEC12, AAV-PAEC2, AAV-PAEC4, AAV-PAEC6, AAV-PAEC7, AAV-PAEC 8, AAVpi. 1, AAVpi.2, AAVpi.3, AAVrh.10, AAVrh.12, AAVrh.13, AAVrh.13R, AAVrh.14, AAVrh.17, AAVrh.18, AAVrh.19, AAVrh.2, AAVrh.20, AAVrh.21, AAVrh.22, AAVrh.23, AAVrh.24, AAVrh.25, AAVrh.2R, AAVrh.31, AAVrh.32, AAVrh.33, AAVrh.34, AAVrh.35, AAVrh.36, AAVrh.37, AAVrh.37R2, AAVrh.38, AAVrh.39, AAVrh.40, AAVrh.43, AAVrh.44, AAVrh.45, AAVrh.46, AAVrh.47, AAVrh.48, AAVrh.48, AAVrh.48.1, AAVrh.48.1.2, AAVrh.48.2, AAVrh.49, AAVrh.50, AAVrh.51, AAVrh.52, AAVrh.53, AAVrh.54, AAVrh.55, AAVrh.56, AAVrh.57, AAVrh.58, AAVrh.59, AAVrh.60, AAVrh.61, AAVrh.62, AAVrh.64, AAVrh.64R1, AAVrh.64R2, AAVrh.65, AAVrh.67, AAVrh.68, AAVrh.69, AAVrh.70, AAVrh.72, AAVrh.73, AAVrh.74, AAVrh.8, AAVrh.8R, AAVrh8R, AAVrh8R A586R mutant, AAVrh8R R533A mutant, BAAV, B P61 AAV, B P62 AAV, B P63 AAV, bovine AAV, caprine AAV, Japanese AAV10, true type AAV (ttAAV), UPENN AAV 10, AAV-LK 16, AAAV, AAV Shuffle 100-1, AAV Shuffle 100-2, AAV Shuffle 100-3, AAV Shuffle 100-7, AAV Shuffle 10-2, AAV Shuffle 10-6, AAV Shuffle 10-8, AAV SM 100-10, AAV SM 100-3, AAV SM 10-1, AAV SM 10-2, and/or AAV SM 10-8. In some embodiments, the AAV serotype may be, or have, a mutation in the AAV9 sequence as described by N Pulicherla et al. (Molecular Therapy 19(6): 1070-1078 (2011)), such as but not limited to, AAV9.9, AAV9.11, AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV9.84.
In some embodiments, a serotype may be AAVDJ or a variant thereof, such as AAVDJ8 (or AAV-DJ8), as described by Grimm et al. (Journal of Virology 82(12): 5887-5911 (2008)). The amino acid sequence of AAVDJ8 may comprise two or more mutations in order to remove the heparin binding domain (HBD). By way of non-limiting example, an AAV-DJ sequence of SEQ ID NO: 1 in U.S. Pat. No. 7,588,772, may comprise two mutations: (1) R587Q where arginine (R; Arg) at amino acid 587 is changed to glutamine (Q; Gin) and (2) R590T where arginine (R; Arg) at amino acid 590 is changed to threonine (T; Thr). As another non-limiting example, may comprise three mutations: (1) K406R where lysine (K; Lys) at amino acid 406 is changed to arginine (R; Arg), (2) R587Q where arginine (R; Arg) at amino acid 587 is changed to glutamine (Q; Gin) and (3) R590T where arginine (R; Arg) at amino acid 590 is changed to threonine (T; Thr).
In some embodiments, an AAV serotype may comprise or have a sequence as described in, e.g., U.S. Pat. No. 6,156,303, or derivatives thereof. In some embodiments, an AAV serotype may be or comprise a sequence as described in International Application Publication No. WO2015121501, such as, but not limited to, true type AAV (ttAAV) (SEQ ID NO: 2 of WO2015121501), “UPenn AAV10” (SEQ ID NO: 8 of WO2015121501), “Japanese AAV10” (SEQ ID NO: 9 of WO2015121501), or variants thereof.
In some embodiments, an AAV serotype may be from any number of species. For example, in some embodiments, an AAV may be an avian AAV (AAAV). In some embodiments, an rAAV serotype may be or comprise a sequence as described in U.S. Pat. No. 9,238,800, such as, but not limited to, AAAV (SEQ ID NO: 1, 2, 4, 6, 8, 10, 12, and 14 of U.S. Pat. No. 9,238,800), or variants thereof. In some embodiments, an AAV may be a bovine AAV (BAAV). The BAAV serotype may be or comprise a sequence as described in U.S. Pat. No. 9,193,769, such as, but not limited to, BAAV (SEQ ID NO: 1 and 6 of U.S. Pat. No. 9,193,769), or variants thereof. The BAAV serotype may be or have a sequence as described in U.S. Pat. No. 7,427,396, such as, but not limited to, BAAV (SEQ ID NO: 5 and 6 of U.S. Pat. No. 7,427,396), or variants thereof. In some embodiments, an AAV may be a caprine AAV. The caprine AAV serotype may be or comprise a sequence as described in U.S. Pat. No. 7,427,396, such as, but not limited to, caprine AAV (SEQ ID NO: 3 of U.S. Pat. No. 7,427,396), or variants thereof.
In some embodiments, an AAV may be engineered as a hybrid AAV from two or more parental serotypes. In one example, the AAV may be AAV2G9 which comprises sequences from AAV2 and AAV9. The AAV2G9 AAV serotype may be, or have, a sequence as described in U.S. Patent Publication No. 20160017005. For example, in some embodiments, an AAV vector is an AAV2/5, AAV2/6, AAV2/8 or AAV2/9 vector (e.g., AAV6, AAV8 or AAV9 serotype having AAV2 ITR). Other AAV vectors are described in, e.g., Sharma et al., Brain Res Bull. 2010 Feb. 15; 81(2-3): 273.
In some embodiments, an AAV may be a serotype generated by the AAV9 capsid library with mutations in amino acids 390-627 (VP1 numbering) as described by Pulicherla et al. (Molecular Therapy 19(6): 1070-1078 (2011). In some such embodiments, a serotype and corresponding nucleotide and amino acid substitutions may be, but is not limited to, AAV9.1 (G1594C; D532H), AAV6.2 (T1418A and T1436X; V473D and I479K), AAV9.3 (T1238A; F413Y), AAV9.4 (T1250C and A1617T; F417S), AAV9.5 (A1235G, A1314T, A1642G, C1760T; Q412R, T548A, A587V), AAV9.6 (T1231A; F4111), AAV9.9 (G1203A, G1785T; W595C), AAV9.10 (A1500G, T1676C; M559T), AAV9.11 (A1425T, A1702C, A1769T; T568P, Q590L), AAV9.13 (A1369C, A1720T; N457H, T574S), AAV9.14 (T1340A, T1362C, T1560C, G1713A; L447H), AAV9.16 (A1775T; Q592L), AAV9.24 (T1507C, T1521G; W503R), AAV9.26 (A1337G, A1769C; Y446C, Q590P), AAV9.33 (A1667C; D556A), AAV9.34 (A1534G, C1794T; N512D), AAV9.35 (A1289T, T1450A, C1494T, A1515T, C1794A, G1816A; Q430L, Y484N, N98K, V606I), AAV9.40 (A1694T, E565V), AAV9.41 (A1348T, T1362C; T450S), AAV9.44 (A1684C, A1701T, A1737G; N562H, K567N), AAV9.45 (A1492T, C1804T; N498Y, L602F), AAV9.46 (G1441C, T1525C, T1549G; G481R, W509R, L517V), 9.47 (G1241A, G1358A, A1669G, C1745T; S414N, G453D, K557E, T5821), AAV9.48 (C1445T, A1736T; P482L, Q579L), AAV9.50 (A1638T, C1683T, T1805A; Q546H, L602H), AAV9.53 (G1301A, A1405C, C1664T, G1811T; R134Q, S469R, A555V, G604V), AAV9.54 (CI 531 A, T1609A; L511I, L537M), AAV9.55 (T1605A; F535L), AAV9.58 (C1475T, C1579A; T492I, H527N), AAV.59 (T1336C; Y446H), AAV9.61 (A1493T; N498I), AAV9.64 (C1531A, A1617T; L511I), AAV9.65 (C1335T, T1530C, C1568A; A523D), AAV9.68 (C1510A; P504T), AAV9.80 (G1441A,; G481R), AAV9.83 (C1402A, A1500T; P468T, E500D), AAV9.87 (T1464C, T1468C; S490P), AAV9.90 (A1196T; Y399F), AAV9.91 (T1316G, A1583T, C1782G, T1806C; L439R, K5281), AAV9.93 (A1273G, A1421G, A1638C, C1712T, G1732A, A1744T, A1832T; S425G, Q474R, Q546H, P571L, G578R, T582S, D611V), AAV9.94 (A1675T; M559L) and AAV9.95 (T1605A; F535L).
In some embodiments, the AAV may be a variant, such as PUP. A or PHP.B as described in Deverman. 2016. Nature Biotechnology. 34(2): 204-209.
In some embodiments, an rAAV vector, particle, and/or nucleic acid comprise(s) capsid proteins having one or more amino acid substitutions (e.g., AAV2(tripleY-F) (Y444F+Y500F+Y730F), see Petrs-Silva et al., Novel Properties of Tyrosine-mutant AAV2 Vectors in the Mouse Retina, Mol. Ther. 19(2): 293-301 (2011); AAV2(quadY-F+T-V) (Y272F+Y444F+Y500F+Y730F+T491V), see Kay et al., Targeting Photoreceptors via Intravitreal Delivery Using Novel, Capsid-Mutated AAV Vectors, PLoS One, 8(4): e62097 (2013); or AAV2(MAX)deltaHS, also known as AV2(4pMut)deltaHS (Y444F+Y500F+Y730F+T491V+R585S+R588T+R487G), see Boye et al., Impact of Heparan Sulfate Binding on Transduction of Retina by Recombinant Adeno-Associated Virus Vectors, J. Virol. 90(8): 4215-4231 (2016)).
In some embodiments, an rAAV vector, particle, and/or nucleic acid comprises a capsid that includes modified capsid proteins (e.g., capsid proteins comprising a modified VP3 region). Methods of producing modified capsid proteins are known in the art (see, e.g., US20130310443). In some embodiments, the rAAV vector, particle, and/or nucleic acid comprises a modified capsid protein comprising at least one non-native amino acid substitution at a position that corresponds to a surface-exposed amino acid (e.g., a surface exposed Tyrosine) in a wild-type capsid protein. In some embodiments, the rAAV vector, particle, and/or nucleic acid comprises a modified capsid protein comprising a non-tyrosine amino acid (e.g., a phenylalanine) at a position that corresponds to a surface-exposed tyrosine amino acid in a wild-type capsid protein, a non-threonine amino acid (e.g., a valine) at a position that corresponds to a surface-exposed threonine amino acid in the wild-type capsid protein, a non-lysine amino acid (e.g., a glutamic acid) at a position that corresponds to a surface-exposed lysine amino acid in the wild-type capsid protein, a non-serine amino acid (e.g., a valine) at a position that corresponds to a surface-exposed serine amino acid in the wild-type capsid protein, or a combination thereof.
In some embodiments, a rAAV vector, particle, and/or nucleic acid (e.g., a rAAV2 or other rAAV serotype particle) comprises a capsid that includes modified capsid proteins having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions.
In some embodiments, an expression construct including, for example, a promoter and a gene of interest, is flanked on each side by an inverted terminal repeat sequence. In some embodiments, the expression construct comprises one or more regions comprising a sequence that facilitates expression of the coding sequence of the gene of interest, e.g., expression control sequences operably linked to the coding sequence. Non-limiting examples of expression control sequences include promoters, insulators, silencers, response elements, introns, enhancers, initiation sites, termination signals, and poly(A) tails. Any combination of such control sequences is contemplated herein (e.g., a promoter and an enhancer). In some embodiments, the expression construct includes other regulatory elements such as, e.g., WPRE.
In some embodiments, a nucleic acid of the present disclosure is a plasmid (e.g., a circular nucleic acid comprising one or more of an origin of replication, a selectable marker, and a reporter gene). In some embodiments, a nucleic acid described herein, such as a plasmid, may also contain marker or reporter genes, e.g., LacZ or a fluorescent protein, and an origin of replication. In some embodiments, a plasmid is transfected into a producer cell that produces AAV particles comprising the expression construct.
In some embodiments, a nucleic acid of the present disclosure is a nucleic acid vector such as a recombinant adeno-associated virus (rAAV) genome.
In addition to the major elements identified above for an AAV vector, the vector can also include conventional control elements operably linked to the transgene in a manner that permits its transcription, translation and/or expression in a cell transfected with the vector or infected with the virus produced by the disclosure. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product. A number of expression control sequences, including promoters that are native, constitutive, inducible and/or tissue-specific, are known in the art and may be included in a vector described herein.
In some embodiments, one or more elements of one or more technologies as provided herein comprises one or more promoters.
For example, in some embodiments, the present disclosure provides a nucleic acid, which nucleic acid comprises an expression construct further comprising a promoter operably linked to a coding sequence of one or more genes of interest. In some embodiments, a promoter is a natural or naturally-derived promoter. In some embodiments a promoter can be a truncated natural promoter. In some embodiments, a promoter can include an enhancer and/or basal promoter elements from a natural promoter. In some embodiments, a promoter is a constitutive promoter as described herein. In some embodiments, a promoter is a cell- or tissue-specific promoter as described herein.
Examples of constitutive promoters include but are not limited to the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer), the SV40 promoter, and the dihydrofolate reductase promoter. Inducible promoters allow regulation of gene expression and can be regulated by exogenously supplied compounds, environmental factors such as temperature, or the presence of a specific physiological state, e.g., acute phase, a particular differentiation state of the cell, or in replicating cells only. Inducible promoters and inducible systems are available from a variety of commercial sources, including, without limitation, Invitrogen, Clontech and Ariad. Many other systems have been described and can be readily selected by one of skill in the art. Examples of inducible promoters regulated by exogenously supplied promoters include the zinc-inducible sheep metallothionine (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system, the ecdysone insect promoter, the tetracycline-repressible system, the tetracycline-inducible system, the RU486-inducible system and the rapamycin-inducible system. Still other types of inducible promoters which may be useful in this context are those which are regulated by a specific physiological state, e.g., temperature, acute phase, a particular differentiation state of the cell, or in replicating cells only. In another embodiment, a native promoter, or fragment thereof, for a transgene will be used. In a further embodiment, other native expression control elements, such as enhancer elements, polyadenylation sites or Kozak consensus sequences may also be used to mimic the native expression.
In some embodiments, regulatory sequences impart tissue-specific gene expression capabilities. In some cases, the tissue-specific regulatory sequences bind tissue-specific transcription factors that induce transcription in a tissue specific manner. Such tissue-specific regulatory sequences (e.g., promoters, enhancers, etc.) are well known in the art. In some embodiments, the promoter is a chicken j-actin promoter, a Cbh promoter, a pol II promoter, or a pol III promoter.
In some embodiments, a promoter is a tissue or cell-specific promoter. For example, in some embodiments, neuronal-specific promoters may include, but are not limited to, human synapsin I (SYN) promoter (e.g., as described in Li et al., Proc Natl Acad Sci USA 1993; 90:1460-1464), mouse calcium/calmodulin-dependent protein kinase II (CaMKII) promoter (e.g., as described in Mayford et al., Proc Natl Acad Sci USA 1996; 93: 13250-13255), rat tubulin alpha I (Tal) promoter (e.g., as described in Gloster et al., J Neurosci 1994; 14: 7319-7330), rat neuron-specific enolase (NSE) promoter (e.g., as described in Forss-Petter et al, Neuron 1990; 5: 187-197), and human platelet-derived growth factor-beta chain (PDGF) promoter (e.g., as described in Sasahara et al, Cell 1991; 64: 217-227).
In some embodiments, a viral vector is designed for expressing a transgene described herein in neurons or neuronal progenitors, and viral vector (e.g., an AAV vector) includes one or more neuron-specific regulatory elements, which substantially limit expression of the transgene to neuronal cells or progenitors thereof. Generally, neuron-specific regulatory elements can be derived from any gene known to be exclusively expressed in neurons or neuronal progenitors of the central and/or peripheral nervous system. In some embodiments, a viral vector described herein includes a neuron-specific regulatory element derived from the genomic loci of one or more neuronal proteins.
In some embodiments, the present disclosure provides transfected host cells. A number of transfection techniques are generally known in the art (see, e.g., Graham et al. (1973) Virology, 52:456; Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier; and Chu et al. (1981) Gene 13:197). Such techniques can be used to introduce one or more exogenous nucleic acids, such as a nucleotide integration vector and other nucleic acid molecules, into suitable host cells.
In some embodiments, the present disclosure provides host cells that comprise one or more of rAAV particles, expression construct(s), or nucleic acid vector(s) as described herein. In some embodiments, host cells may be any cell type (e.g., mammalian, insect, etc.). In some embodiments, host cells are mammalian host cells. In some embodiments, host cells are non-human primate cells. In some preferred embodiments of the present disclosure, host cells are human host cells. In some embodiments, host cells are primary cells. In some embodiments, host cells are derived from a cell line (e.g., an immortalized cell line). In some embodiments, host cells may be isolated (e.g., using cell or tissue culture) or obtained via commercially available means that will be known to those of skill in the art. In some embodiments, host cells may be within a body of an animal (e.g., a non-human animal, e.g., a genetically modified mouse). In some embodiments, a host cell may be used as a recipient of an AAV helper construct, an AAV minigene plasmid, an accessory function vector, and/or other transfer DNA associated with the production of recombinant AAVs.
In some embodiments, a transgene or other genomic component of interest is flanked by ITRs and rep/cap genes are introduced into insect host cells by infection with baculovirus-based vectors. Such production systems are known in the art (see generally, e.g., Zhang et al., 2009, Human Gene Therapy 20:922-929). Methods of making and using these and other AAV production systems are also described in U.S. Pat. Nos. 5,139,941; 5,741,683; 6,057,152; 6,204,059; 6,268,213; 6,491,907; 6,660,514; 6,951,753; 7,094,604; 7,172,893; 7,201,898; 7,229,823; and 7,439,065. As will be appreciated by one of skill in the art, the foregoing methods for producing recombinant vectors are not meant to be limiting, and other suitable methods will be apparent to such individuals.
Methods for obtaining viral vectors are generally known in the art.
Methods of producing one or more rAAV vectors, particles and/or nucleic acid vectors are also known in the art and such vectors, particles, and/or nucleic acids or components thereof are commercially available (see, e.g., Zolotukhin et al. Production and purification of serotype 1, 2, and 5 recombinant adeno-associated viral vectors. Methods 28 (2002) 158 167; and U.S. Patent Publication Numbers US20070015238 and US20120322861; and plasmids and kits available from ATCC and Cell Biolabs, Inc.). General principles of rAAV production are reviewed in, for example, Carter, 1992, Current Opinions in Biotechnology, 1533-539; and Muzyczka, 1992, Curr. Topics in Microbial, and Immunol., 158:97-129). Various approaches are described in Ratschin et al, Mol. Cell. Biol. 4:2072 (1984); Hermonat et al, Proc. Natl. Acad. Sci. USA, 81:6466 (1984); Tratschin et al, Mol. Cell. Biol. 5:3251 (1985); McLaughlin et al, J. Virol., 62: 1963 (1988); and Lebkowski et al, 1988 Mol. Cell. Biol., 7:349 (1988). Samulski et al (1989, J. Virol., 63:3822-3828); U.S. Pat. No. 5,173,414; WO 95/13365 and corresponding U.S. Pat. No. 5,658,776; WO 95/13392; WO 96/17947; PCT/US98/18600; WO 97/09441 (PCT/US96/14423); WO 97/08298 (PCT/US96/13872); WO 97/21825 (PCT/US96/20777); WO 97/06243 (PCT/FR96/01064); WO 99/11764; Perrin et al. (1995) Vaccine 13: 1244-1250; Paul et al. (1993) Human Gene Therapy 4:609-615; Clark et al. (1996) Gene Therapy 3: 1124-1132; U.S. Pat. Nos. 5,786,211; 5,871,982; and 6,258,595.
For example, to produce AAV vectors, methods typically involve culturing a host cell which contains a nucleic acid sequence encoding an AAV capsid protein or fragment thereof, a functional rep gene; a recombinant AAV vector composed of AAV inverted terminal repeats (ITRs) and a transgene; and/or sufficient helper functions to permit packaging of the recombinant AAV vector into the AAV capsid proteins. In some embodiments, a nucleic acid vector (e.g., as a plasmid) may be combined with one or more helper plasmids, e.g., that contain a rep gene (e.g., encoding Rep78, Rep68, Rep52 and Rep40) and a cap gene (encoding VP1, VP2, and VP3), and transfected into a producer cell line such that rAAV particles can be packaged and purified.
In some embodiments, components to be cultured in a host cell to package an AAV vector in an AAV capsid may be provided to the host cell in trans. In some embodiments, any one or more of the required components (e.g., recombinant AAV vector, rep sequences, cap sequences, and/or helper functions) may be provided by a stable host cell that has been engineered to contain one or more of the required components using methods known to those of skill in the art. In some embodiments, such a stable host cell contains the required component(s) under the control of an inducible promoter. In other embodiments, the required component(s) may be under the control of a constitutive promoter. In other embodiments, a selected stable host cell may contain selected component(s) under the control of a constitutive promoter and other selected component(s) under the control of one or more inducible promoters. For example, a stable host cell may be generated that is derived from HEK293 cells (which contain E1 helper functions under the control of a constitutive promoter), but that contain the rep and/or cap proteins under the control of inducible promoters. In some embodiments, stable host cells may be generated by one of skill in the art using routine methods.
In some embodiments, the one or more helper plasmids include a first helper plasmid comprising a rep gene and a cap gene and a second helper plasmid comprising other genes that assist in AAV production, such as a Ela gene, a Elb gene, a E4 gene, a E2a gene, and a VA gene. In some embodiments, the rep gene is a rep gene derived from AAV2 and the cap gene is derived from AAV5. Helper plasmids, and methods of making such plasmids, are known in the art and commercially available (see, e.g., pDM, pDG, pDP1rs, pDP2rs, pDP3rs, pDP4rs, pDP5rs, pDP6rs, pDG(R484E/R585E), and pDP8.ape plasmids from PlasmidFactory, Bielefeld, Germany; other products and services available from Vector Biolabs, Philadelphia, PA; Cellbiolabs, San Diego, CA; Agilent Technologies, Santa Clara, Ca; and Addgene, Cambridge, MA; pxx6; Grimm et al. (1998), Novel Tools for Production and Purification of Recombinant Adenoassociated Virus Vectors, Human Gene Therapy, Vol. 9, 2745-2760; Kern, A. et al. (2003), Identification of a Heparin-Binding Motif on Adeno-Associated Virus Type 2 Capsids, Journal of Virology, Vol. 77, 11072-11081.; Grimm et al. (2003), Helper Virus-Free, Optically Controllable, and Two-Plasmid-Based Production of Adeno-associated Virus Vectors of Serotypes 1 to 6, Molecular Therapy, Vol. 7, 839-850; Kronenberg et al. (2005), A Conformational Change in the Adeno-Associated Virus Type 2 Capsid Leads to the Exposure of Hidden VP1 N Termini, Journal of Virology, Vol. 79, 5296-5303; and Moullier, P. and Snyder, R.O. (2008), International efforts for recombinant adenoassociated viral vector reference standards, Molecular Therapy, Vol. 16, 1185-1188).
By way of non-limiting example, in some embodiments, a method of rAAV particle production comprises use of one or more helper plasmids, which helper plasmids comprise rep and cap ORFs for the desired AAV serotype and the adenoviral VA, E2A (DBP), and E4 genes under the transcriptional control of their native promoters. In some such embodiments, cells (e.g., HEK293 cells, which are widely commercially available, e.g., such as from ATCC®) are transfected via, e.g., CaP04-mediated transfection.
In some embodiments, an agent such as polyethylenimine (PEI) is used, in conjunction with the helper plasmid(s) and a plasmid containing a nucleic acid vector described herein. In some embodiments, recombinant AAV vector, rep sequences, cap sequences, and/or helper functions required for producing an AAV in accordance with the present disclosure may be delivered to a packaging host cell using any appropriate genetic element (e.g., vector). In some embodiments, a selected genetic element may be delivered by any suitable method known in the art, e.g., to those with skill in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques (see, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.). Similarly, methods of generating AAV virions are well known and any suitable method may be used in accordance with the present disclosure (see, e.g., K. Fisher et al, J. Virol., 70:520-532 (1993) and U.S. Pat. No. 5,478,745).
In some embodiments, recombinant AAVs may be produced using a triple transfection method (e.g., as described in U.S. Pat. No. 6,001,650). In some embodiments, recombinant AAVs are produced by transfecting a host cell with a recombinant AAV vector (comprising a transgene) to be packaged into AAV particles, an AAV helper function vector, and an accessory function vector. In some embodiments, the AAV helper function vector encodes AAV helper function sequences (i.e., rep and cap), which function in trans for productive AAV replication and encapsidation. In some embodiments, the AAV helper function vector supports efficient AAV vector production without generating any detectable wild-type AAV virions (i.e., AAV virions containing functional rep and cap genes).
Non-limiting examples of vectors suitable for use with the present disclosure include, but are not limited to, pHLP19 (see, e.g., U.S. Pat. No. 6,001,650) and pRep6cap6 vector (see, e.g., U.S. Pat. No. 6,156,303). In some embodiments, an accessory function vector encodes nucleotide sequences for non-AAV derived viral and/or cellular functions upon which AAV is dependent for replication (i.e., “accessory functions”). Accessory functions include those functions required for AAV replication, including, without limitation, those moieties involved in activation of AAV gene transcription, stage specific AAV mRNA splicing, AAV DNA replication, synthesis of cap expression products, and AAV capsid assembly. In some embodiments, viral-based accessory functions can be derived from any known helper viruses such as adenovirus, herpesvirus (other than herpes simplex virus type-1), and vaccinia virus.
In some embodiments, the present disclosure provides, among other things, methods for generating and isolating AAV viral vectors suitable for delivery to a subject are described in, e.g., U.S. Pat. Nos. 7,790,449; 7,282,199; WO 2003/042397; WO 2005/033321, WO 2006/110689; and U.S. Pat. No. 7,588,772. In some embodiments, a producer cell line is transiently transfected with a construct that encodes the transgene flanked by ITRs and a construct(s) that encodes rep and cap. In some embodiments, a packaging cell line that stably supplies rep and cap is transiently transfected with a construct encoding the transgene flanked by ITRs. In some embodiments, AAV virions are produced in response to infection with helper adenovirus or herpesvirus, and AAVs are separated from contaminating virus. In some embodiments, infection with helper virus is not required to recover the AAV. In some such embodiments, the helper functions (i.e., adenovirus E1, E2a, VA, and E4 or herpesvirus UL5, UL8, UL52, and UL29, and herpesvirus polymerase) are also supplied, in trans. In some embodiments, helper functions can be supplied by transient transfection of the cells with constructs that encode the helper functions, or the cells can be engineered to stably contain genes encoding the helper functions, the expression of which can be controlled at the transcriptional or posttranscriptional level.
For example, in some embodiments, Sf9-based producer stable cell lines are infected with a single recombinant baculovirus containing a nucleic acid vector of the present disclosure. In some embodiments, a mammalian cell line (e.g., HEK293 or BHK) is infected with an HSV containing a nucleic acid vector of the present disclosure and optionally one or more helper HSVs comprising rep and cap ORFs as described herein and adenoviral VA, E2A (DBP), and E4 genes under the transcriptional control of their native promoters. In some embodiments, infected cells (e.g., HEK293, BHK, or Sf9 cells) are then incubated for a time period (e.g., at least 20, 30, 40, 50, or 60 hours or more) to allow for sufficient rAAV particle production. In some such embodiments, the rAAV particles can then be purified using any method known in the art or described herein, e.g., by iodixanol step gradient, CsCl gradient, chromatography, or polyethylene glycol (PEG) precipitation.
The present disclosure provides, among other things, compositions and methods of use thereof for ameliorating one or more signs, symptoms, and/or disorders associated with SCN9A and/or SCN10A gene(s), as described herein. In some embodiments, principal targets for gene editing are mammalian cells. In some embodiments, principal targets for gene editing are human cells. For example, in some embodiments, an ex-vivo method of gene editing may be used in conjunction with somatic cells, which, after being modified using technologies provided herein, can give rise to committed and/or differentiated cells (e.g., neuronal progenitor cells or neurons of the peripheral nervous system). In some embodiments, an in-vivo method of gene editing as described herein comprises human cells (e.g., human neurons of the peripheral nervous system) and/or affected organs.
In some embodiments, gene editing is performed on autologous cells. In some such embodiments, the present disclosure appreciates that by performing gene editing in autologous cells that are derived from and therefore already completely matched with the patient in need, it is possible to generate cells that can be safely re-introduced into the patient. In some such embodiments, such re-introduction of autologous cells may more effectively give rise to a population of cells that will be able to ameliorating or prevent one or more clinical conditions (e.g., one or more symptoms) associated with the patient's disease (e.g., a pain disorder and/or disorder associated with pain).
Stem cells are capable of both proliferation and giving rise to more progenitor cells, these in turn having the ability to generate a large number of mother cells that can in turn give rise to differentiated or differentiable daughter cells. In some embodiments, a stem cell may be, but does not have to be, multipotent (e.g., able to give rise to cells of different lineages, such as, e.g., skin cells, liver cells, bone cells, etc.). For example, a stem cell such as a neural stem cell may only give rise to cells of a neural lineage (e.g., neurons, glia, etc.). In some embodiments, a stem cell may be, e.g., an iPSC, a mesenchymal stem cell, a hematopoietic stem cell, a neural stem cell, etc. The daughter cells themselves can be induced to proliferate and produce progeny that subsequently differentiate into one or more mature cell types, while also retaining one or more cells with parental developmental potential. In some embodiments, a stem cell is a cell with the capacity or potential, under particular circumstances, to differentiate to a more specialized or differentiated phenotype (e.g., a progenitor cell), and which retains the capacity, under certain circumstances, to proliferate without substantially differentiating. As is known to one of skill in the art, cellular differentiation is a complex process typically occurring through many cell divisions. Thus, in some embodiments, a differentiated cell may derive from a multipotent cell that, itself, is derived from a multipotent cell, and so on. While each of these multipotent cells may be considered stem cells, the range of cell types that each can give rise to may vary considerably. In some embodiments, some differentiated cells also have the capacity to give rise to cells of greater developmental potential. In some embodiments, some differentiated cells are more restricted and may only give rise to cells of a particular lineage (e.g., neuronal lineage). Such capacity may be natural or may be induced artificially upon treatment with various factors.
In some embodiments, self-renewal can be another important aspect of the stem cell. In theory, self-renewal can occur by either of two major mechanisms. Stem cells can divide asymmetrically, with one daughter retaining the stem state and the other daughter expressing some distinct other specific function and phenotype. Alternatively, some of the stem cells in a population can divide symmetrically into two stems, thus maintaining some stem cells in the population as a whole, while other cells in the population give rise to differentiated progeny only. Generally, “progenitor cells” have a cellular phenotype that is more primitive (i.e., is at an earlier step along a developmental pathway or progression than is a fully differentiated cell). Often, progenitor cells also have significant or very high-proliferative potential. Progenitor cells can give rise to multiple distinct differentiated cell types or to a single differentiated cell type, depending on the developmental pathway and on the environment in which the cells develop and differentiate.
In the context of cell ontogeny, the adjective “differentiated,” or “differentiating” is a relative term. A “differentiated cell” is a cell that has progressed further down the developmental pathway than the cell to which it is being compared. Thus, stem cells can differentiate into lineage-restricted precursor cells (such as a myocyte progenitor cell), which in turn can differentiate into other types of precursor cells further down the pathway (such as a myocyte precursor), and then to an end-stage differentiated cell, such as a myocyte, which plays a characteristic role in a certain tissue type, and may or may not retain the capacity to proliferate further.
In some embodiments, genetically engineered human cells described herein can be induced pluripotent stem cells (iPSCs). Without wishing to be bound by any particular theory, the present disclosure contemplated that an advantage of using iPSCs is that the cells can be derived from the same subject to which the progenitor cells are to be administered. That is, for example, a somatic cell can be obtained from a subject, reprogrammed to an induced pluripotent stem cell, and then re-differentiated into a progenitor cell to be administered to the subject (e.g., autologous cells). Because the progenitors are essentially derived from an autologous source, the risk of engraftment rejection or allergic response can be reduced compared to the use of cells from another subject or group of subjects. In addition, the use of iPSCs negates the need for cells obtained from an embryonic or other source. Accordingly, in some embodiments, cells used in accordance with the present disclosure are autologous cells. In some embodiments, cells used in accordance with the present disclosure are stem cells. In some embodiments, cells used in accordance with the present disclosure are not embryonic stem cells.
In some embodiments, differentiation of a cell is generally irreversible under most physiological contexts; however, several methods have been recently developed to reprogram somatic cells to iPSCs. Such methods are known to those of skill in the art and briefly described, in part, herein. For example, in some embodiments, a cell is reprogrammed. In some such embodiments, reprogramming involves a change that alters or reverses a differentiation state of a given differentiated cell (e.g., a somatic cell), for example, into an undifferentiated or more primitive type of cell. In some embodiments, such reprogrammed or dedifferentiated cells may gain potential to differentiate into several different types of cells under appropriate conditions. It will be understood by those of skill in the art that an act of placing many primary cells in culture can lead to some loss of fully differentiated characteristics. Accordingly, such culturing alone does not render these cells non-differentiated cells (e.g., undifferentiated cells) or pluripotent cells. Rather, a transition of a differentiated cell to a more primitive state (e.g., pluripotency) requires certain reprogramming stimulus/stimuli beyond that which can lead to partial loss of differentiated character in culture. In some embodiments, reprogrammed cells also have the characteristic of the capacity of extended passaging without loss of growth potential, relative to primary cell parents, which generally have capacity for only a limited number of divisions in culture.
In some embodiments, a cell to be reprogrammed can be either partially or terminally differentiated prior to reprogramming. In some embodiments, reprogramming can encompass complete reversion of the differentiation state of a differentiated cell (e.g., a somatic cell) to a pluripotent state or a multipotent state. In some embodiments, reprogramming can encompass complete or partial reversion of the differentiation state of a differentiated cell (e.g., a somatic cell) to an undifferentiated cell (e.g., an embryonic-like cell). In some embodiments, reprogramming can result in expression of particular genes by the cells, the expression of which further contributes to reprogramming. In some embodiments, reprogramming of a differentiated cell (e.g., a somatic cell) can cause the differentiated cell to assume an undifferentiated state (e.g., is an undifferentiated cell), which may be referred to as an induced pluripotent stem cell (iPSC).
In some embodiments, reprogramming can involve alteration, e.g., reversal, of at least some of the heritable patterns of nucleic acid modification (e.g., methylation), chromatin condensation, epigenetic changes, genomic imprinting, etc., that occur during cellular differentiation. Reprogramming is distinct from simply maintaining the existing undifferentiated state of a cell that is already pluripotent or maintaining the existing less than fully differentiated state of a cell that is already a multipotent cell (e.g., a myogenic stem cell). Reprogramming is also distinct from promoting the self-renewal or proliferation of cells that are already pluripotent or multipotent, although, in some embodiments, the compositions and methods described herein can also be of use for such purposes.
One of ordinary skill in the art will be aware of many methods known to generate pluripotent stem cells from somatic cells. Any such method that reprograms a somatic cell to the pluripotent phenotype would be appropriate for use in accordance with the present disclosure.
In addition, reprogramming methodologies for generating pluripotent cells using defined combinations of transcription factors have been described and will be known to those of skill in the art. For example, mouse somatic cells can be converted to ES cell-like cells with expanded developmental potential by the direct transduction of Oct4, Sox2, Klf4, and c-Myc; see, e.g., Takahashi and Yamanaka, Cell 126(4): 663-76 (2006). In some embodiments, iPSCs resemble ES cells, as they restore the pluripotency-associated transcriptional circuitry and much of the epigenetic landscape. In some embodiments, mouse iPSCs satisfy all standard assays for pluripotency known to those in the art. For example, mouse iPSCs are capable of in-vitro differentiation into cell types of the three germ layers, teratoma formation, contribution to chimeras, germline transmission (see, e.g., Maherali and Hochedlinger, 2008, Cell Stem Cell. 3(6):595-605), and tetraploid complementation.
In some embodiments, human iPSCs can be obtained using similar transduction methods, and the transcription factor trio, OCT4, SOX2, and NANOG, has been established as the core set of transcription factors that govern pluripotency; see, e.g., Budniatzky and Gepstein, Stem Cells Transl Med. 3(4):448-57 (2014); Barrett et al, Stem Cells Trans Med 3: 1-6 sctm.2014-0121 (2014); Focosi et al., Blood Cancer Journal 4: e211 (2014); and references cited therein. In some embodiments, production of iPSCs can be achieved by the introduction of nucleic acid sequences encoding stem cell-associated genes into an adult, somatic cell, historically using viral vectors.
In some embodiments, iPSCs can be generated or derived from terminally differentiated somatic cells, as well as from adult stem cells, or somatic stem cells. That is, for example, a non-pluripotent progenitor cell can be rendered pluripotent or multipotent by reprogramming. In some such embodiments, it may not be necessary to include as many reprogramming factors as required to reprogram a terminally differentiated cell. Further, in some embodiments, reprogramming can be induced by the non-viral introduction of reprogramming factors, e.g., by introducing the proteins themselves, or by introducing nucleic acids that encode the reprogramming factors, or by introducing messenger RNAs that upon translation produce the reprogramming factors (see e.g., Warren et al., Cell Stem Cell, 7(5):618-30 (2010).
In some embodiments, reprogramming can be achieved by introducing a combination of nucleic acids encoding stem cell-associated genes, including, for example, Oct-4 (also known as Oct-3/4 or Pouf51), Sox1, Sox2, Sox3, Sox 15, Sox 18, NANOG, KM, Klf2, Klf4, Klf5, NR5A2, c-Myc, 1-Myc, n-Myc, Rem2, Tert, and LIN28. Reprogramming in accordance with the present disclosure can further comprise introducing one or more of Oct-3/4, a member of the Sox family, a member of the Klf family, and a member of the Myc family to a somatic cell. In some embodiments, such reprogramming may further comprise introducing one or more of each of Oct-4, Sox2, Nanog, c-MYC and Klf4 for reprogramming. It will be appreciated by one of skill in the art that, the exact method used for reprogramming is not necessarily critical to technologies provided by the present disclosure. However, where cells differentiated from the reprogrammed cells are to be used in, e.g., human therapy, in some embodiments, reprogramming is not effected by a method that alters the genome. Thus, in some such embodiments, reprogramming can be achieved, e.g., without the use of viral or plasmid vectors.
In some embodiments, efficiency of reprogramming (i.e., the number of reprogrammed cells) derived from a population of starting cells can be enhanced by the addition of various agents, e.g., small molecules, as shown by Shi et al, Cell-Stem Cell 2:525-528 (2008); Huangfu et al, Nature Biotechnology 26(7):795-797 (2008) and Marson et al, Cell-Stem Cell 3 132-135 (2008). For example, in some embodiments, an agent or combination of agents that enhance the efficiency or rate of induced pluripotent stem cell production can be used in the production of patient-specific or disease-specific iPSCs. Examples of agents that enhance reprogramming efficiency include, but are not limited to, soluble Wnt, Wnt conditioned media, BIX-01294 (a G9a histone methyltransferase), PD0325901 (a MEK inhibitor), DNA methyltransferase inhibitors, histone deacetylase (HDAC) inhibitors, valproic acid, 5′-azacytidine, dexamethasone, suberoylanilide, hydroxamic acid (SAHA), vitamin C, and trichostatin (TSA), Suberoylanilide Hydroxamic Acid (SAHA (e.g., MK0683, vorinostat) and other hydroxamic acids), BML-210, Depudecin (e.g., (-)-Depudecin), HC Toxin, Nullscript (4-(1,3-Dioxo-lH, 3H-benzo[de]isoquinolin-2-yl)-N-hydroxybutanamide), Phenylbutyrate (e.g., sodium phenylbutyrate) and Valproic Acid ((VP A) and other short chain fatty acids), Scriptaid, Suramin Sodium, Trichostatin A (TSA), APHA Compound 8, Apicidin, Sodium Butyrate, pi valoyloxy methyl butyrate (Pivanex, AN-9), Trapoxin B, Chlamydocin, Depsipeptide (also known as FR901228 or FK228), benzamides (e.g., CI-994 (e.g., N-acetyl dinaline) and MS-27-275), MGCD0103, NVP-LAQ-824, CBHA (m-carboxycinnaminic acid bishydroxamic acid), JNJ16241199, Tubacin, A-161906, proxamide, oxamflatin, 3-Cl-UCHA (e.g., 6-(3-chlorophenylureido) caproic hydroxamic acid), AOE (2-amino-8-oxo-9, 10-epoxydecanoic acid), CHAP31 and CHAP 50, dominant negative forms of the HDACs (e.g., catalytically inactive forms), siRNA inhibitors of the HDACs, and antibodies that specifically bind to the HDACs. As will be appreciated by one of skill in the art, such agents are generally available, including, but not limited to, commercial sources such as BIOMOL International, Fukasawa, Merck Biosciences, Novartis, Gloucester Pharmaceuticals, Titan Pharmaceuticals, MethylGene, and Sigma Aldrich.
In some embodiments, to confirm induction of pluripotent stem cells for use in accordance with the present disclosure, isolated clones can be tested for the expression of a stem cell marker. In some embodiments, such expression in a cell derived from a somatic cell identifies the cells as induced pluripotent stem cells. In some embodiments, stem cell markers include, but are not limited to, SSEA3, SSEA4, CD9, Nanog, Fbxl5, Ecatl, Esgl, Eras, Gdf3, Fgf4, Cripto, Daxl, Zpf296, Slc2a3, Rexl, Utfl, and Natl. For example, in some embodiments, a cell that expresses Oct4 or Nanog is identified as pluripotent. As will be appreciated by one of skill in the art, methods for detecting the expression of such markers can include, for example, RT-PCR and immunological methods that detect the presence of the encoded polypeptides, such as Western blots or flow cytometric analyses. In some embodiments, intracellular markers may be best identified via RT-PCR, or protein detection methods such as western blot or immunocytochemistry, while cell surface markers may be most readily identified and/or localized, e.g., by immunocytochemistry.
In some embodiments, pluripotent stem cell character of isolated cells can be confirmed by tests evaluating the ability of the iPSCs to differentiate into cells of each of the three germ layers. For example, in some embodiments, teratoma formation in nude mice can be used to evaluate pluripotent character of isolated clones. In some such embodiments, cells can be introduced into nude mice and histology and/or immunohistochemistry can be performed on a tumor arising from the introduced cells; growth of a tumor comprising cells from all three germ layers, for example, further indicates that the cells are pluripotent stem cells.
The peripheral nervous system includes neurons, nerves, and other nervous system cells (e.g., glia, e.g., Schwann cells) that are outside of the central nervous system (brain and spinal cord). Neurons, which process information, and glial cells, which provide mechanical and metabolic support to the nervous system, are the two main classes of cells of the peripheral nervous system. In some embodiments, neurons include, but are not limited to, sensory neurons (collect impulses from the sensory receptors in areas such as skin, muscles, and organs and carries those impulses through the nerves to the CNS) and motor neurons (collect outgoing messages from the CNS and delivers them to the appropriate body organs, instructing them what action needs to be taken). In some embodiments, glial cells include, but are not limited to Schwann cells in and/or apposed to or contacting nerves or satellite cells in ganglia. In some embodiments, genetically engineered cells of the present disclosure may be neurons and nerves of the peripheral nervous system. In some such embodiments, neurons and nerves are human neurons and nerves.
Generation and/or Isolation of Cells for Genetic Modification
Creating Patient Specific iPSCs
In some embodiments of the present disclosure, patient specific cells are harvested. In some embodiments, such harvested cells may be used to create or derive one or more types of progenitor or stem cells. For example, in some embodiments, patient specific iPS cell, cells, or cell lines are generated. As is known to one of skill in the art, there are many methods for creating patient specific iPS cells (e.g., for non-limiting examples, see Takahashi and Yamanaka 2006; Takahashi, Tanabe et al. 2007). For example, in some embodiments, a generating or creating step can comprise: i) isolating a somatic cell, such as a skin cell or fibroblast, from the patient; and ii) introducing a set of pluripotency-associated genes into the somatic cell in order to induce the cell to become a pluripotent stem cell. As described herein, in some embodiments, the set of pluripotency-associated genes can be one or more of OCT4, SOX1, SOX2, SOX3, SOX15, SOX18, NANOG, KLF1, KLF2, KLF4, KLF5, c-MYC, n-MYC, REM2, TERT and LIN28.
In some embodiments, mesenchymal stem cells can be isolated according to any method known in the art, such as from a patient's bone marrow or peripheral blood. For example, in some embodiments, marrow aspirate can be collected into a syringe with heparin. Cells can be washed and centrifuged on a Percoll. The cells can be cultured in Dulbecco's modified Eagle's medium (DMEM) (low glucose) containing 10% fetal bovine serum (FBS), such as in, e.g., Pittinger M F, Mackay A M, Beck S C et al., Science 1999; 284: 143-147.
Biopsy and Isolation of Tissue and/or Cells from Subjects
As described herein, in some embodiments, one or more cells is harvested from a subject. In some such embodiments, cells may be obtained through a biopsy (sample of tissue) or aspirate (sample of fluid) that is removed from the body of a subject. There are many different kinds of biopsies or aspirates. If the biopsy will be performed with any type of sharp tool and/or will be on the skin or other sensitive area, numbing medicine can be applied first. A biopsy or aspirate may be performed according to any of the known methods in the art. For example, in some embodiments, an aspirate is a bone marrow aspirate and a large needle is used to enter the pelvis bone to collect bone marrow. In some embodiments, a nerve biopsy is performed with tissue taken from skin or leg to isolate neurons of the peripheral nervous system. In some such embodiments, the nerve segment is excised inflicting minimal mechanical injury, for example, squeezing or stretching the nerve is avoided and excessive removal of fat or connective tissue is not attempted.
In some embodiments, biopsy samples are processed to isolate one or more cells. In some such embodiments, an isolated cell is a cell that has been removed from an organism in which it was originally found, or a descendant of such a cell. In some embodiments, the cell can be cultured in-vitro, e.g., under defined conditions or in the presence of other cells. In some embodiments, the cell can be later introduced into a second organism or re-introduced into the organism from which it (or the cell from which it is descended) was isolated. In some embodiments, an isolated population of cells comprises a population of cells that has been removed and separated from a mixed or heterogeneous population of cells. In some embodiments, an isolated population can be a substantially pure population of cells, as compared to a heterogeneous population from which the isolated population of cells was isolated or enriched. In some embodiments, the isolated population can be an isolated population of human progenitor cells, e.g., a substantially pure population of human progenitor cells, as compared to a heterogeneous population of cells comprising human progenitor cells and cells from which the human progenitor cells were derived.
In some embodiments, a population of cells is considered substantially enhanced relative to pre-existing or reference levels. In some embodiments, the increase is at least 2-fold, at least 3-, 4-, 5-, 6-, 7-, 8-, 9, 10-, 20-, 30-, 40-, 50-, 60-, 70-, 80-, 90-100-, 150-, 200-, 250-, 300-, 350-, 400-, 450-, 500-, 600-, 700-, 800-, 900-, 1000-, 5000-, 10000-, 20000-, 50000-, 100000- or more fold depending, e.g., on the desired levels of such cells for ameliorating pain.
In some embodiments, a population or type of cell is considered substantially enriched when it accounts for at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more with respect to total number of cells making up a given cell population.
In some embodiments, a population of cells is considered substantially pure with respect to a particular cell population, when the population is at least about 75%, 85%, 90%, 95% pure, with respect to the cells making up a total cell population. For example, in some embodiments, the terms “substantially pure” or “essentially purified,” with regard to a population of progenitor cells, describe a population of cells that contain fewer than about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, or less than 1%), of cells that are not progenitor cells as described herein.
In some embodiments, neurons of the peripheral nervous system may be isolated according to any known method in the art. For example, in some embodiments, a nerve segment is excised inflicting minimal mechanical injury under aseptic conditions. Squeezing or stretching the nerve is strictly avoided and excessive removal of fat or connective tissue is not attempted. Since nerve fibers are generally very sensitive to mechanical injury, in some embodiments, a proximal nerve cut is performed first, when cutting a nerve segment for removal. By way of non-limiting example, following harvest, a nerve segment is processed using mechanical and chemical steps. In some embodiments following the isolation, the outermost connective tissue layer, the epineurium, is removed and collected for enzymatic digestion. In some embodiments, nerve fibers are teased apart with the help of fine forceps until all fascicles are separated into individual fibers. In some embodiments, epineurium and teased fibers are then subjected to enzymatic digestion overnight with dispase II and type I collagenase. The digested products are filtered and collected by centrifugation and the resulting cell suspensions are plated onto adhesive substrates (e.g. PLL/laminin). Adherent cells may be cultured for analysis such as in, e.g., Andersen et al., Scientific Reports-Nature, 2016, 6:31781. In some embodiments, one or more neurons or progenitors is isolated. In some embodiments, a population of neurons and/or progenitors is isolated.
In some embodiments, a genetically modified cell comprises at least one genetic modification introduced by gene editing (e.g., using the CRISPR/Cas9 or CRISPR/Cpf1 system). Such gene editing techniques are described herein and are known to those of skill in the art. In some embodiments, a genetically modified cell can be a genetically modified stem cell. In some embodiments, a genetically modified cell can be a genetically modified progenitor cell. In some embodiments, a genetically modified cell can be a genetically modified neuron or neuronal progenitor of the peripheral nervous system. In some embodiments, a genetically modified cell comprising an exogenous genome-targeting nucleic acid and/or an exogenous nucleic acid encoding a genome-targeting nucleic acid is contemplated herein.
In some embodiments, such as, for example, methods comprising ex-vivo modification of cells, a control treated population of cells is used. In some such embodiments, control treated cells are treated with identical media, viral induction, nucleic acid sequences, temperature, confluency, flask size, pH, etc., with the exception of the addition of one or more gene editing components. Any method known in the art can be used to measure transcription of SCN9A and/or SCN10A gene or NaV1.7 and/or NavV1.8 protein expression or activity, for example Western Blot analysis of the SCN9A and/or SCN10A protein or real time PCR for quantifying SCN9A or SCN10A mRNA.
In some embodiments, technologies of the present disclosure may use one or more design elements to achieve precise and selective targeting of pain-causative and/or pain propagating neurons. For example, in some embodiments, the present disclosure provides design elements comprising, e.g., (i) localized delivery of a non-replicative viral vector that requires synaptic terminals, sparing the bulk of somatic tissues near the pain site; (ii) neuron-specific promoters that drive expression of the gene editing construct; and/or (iii) gRNA programmed targeting of non-essential ion channel genes exclusively expressed by DRG neurons to spare other types of neurons (e.g., efferent neurons, interneurons, etc.).
In some embodiments, the present disclosure provides technologies for editing a polynucleotide encoding an ion channel or subunit thereof in a cell. In some embodiments, the cell is a stem cell or progenitor cell. In some embodiments, the stem cell is an iPSC, mesenchymal stem cell, or neural stem cell. In some embodiments, the progenitor cell is a neuronal progenitor cell. In some embodiments, the progenitor cell is a glial progenitor cell. In some embodiments, the progenitor cell is a glial and/or neuronal progenitor cell. In some embodiments, the cell is a glial cell. In some embodiments, the glial cell is a Schwann cell or satellite glial cell. In some embodiments, the cell is a neuron. In some embodiments, the neuron is a DRG neuron.
Differentiation of Genome-Edited iPSCs into Cells of the Peripheral Nervous System
In some embodiments, the present disclosure provides, among other things, a method of differentiating cells using iPSCs. For example, in some embodiments, the present disclosure provides an ex-vivo approach for differentiating genome-edited iPSCs into cells of peripheral nervous system lineage and/or identity (e.g., a neuron or neuronal progenitor such as a DRG or progenitor thereof, or a glial cell such as Schwann cell in nerves or satellite glial cell in ganglia). A differentiating step may be performed according to any method known in the art.
For example, in some embodiments, neuronal differentiation of iPSCs is induced using a combination of factors (e.g., brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), nerve growth factor (NGF) and dibutyryl cyclic AMP (dbcAMP)). In some such embodiments, the iPSCs-derived neuronal cells may be further differentiated into Schwann cells using ciliary neurotrophic factor (CNTF), neuregulin 10 and dbcAMP (see, e.g., Wang et al., Biomaterials. 2011; 32(22): 5023-5032).
In some embodiments, differentiation does not result in a post-mitotic neuronal cell. For example, in some such embodiments, differentiation may result in a neuronally-committed progenitor cell.
Differentiation of Genome-Edited Mesenchymal Stem Cells into Cells of the Peripheral Nervous System
In some embodiments, the present disclosure provides, among other things, a method of differentiating cells using mesenchymal stem cells (MSCs). For example, in some embodiments, the present disclosure provides an ex-vivo approach for differentiating genome-edited mesenchymal stem cells into cells of peripheral nervous system lineage and/or identity (e.g., a neuron or neuronal progenitor, such as a DRG cell or progenitor thereof, or a glial cell such as Schwann cell in nerves or satellite glial cell in ganglia). A differentiating step may be performed according to any method known in the art. For example, in some embodiments, MSCs are treated with various factors and/or hormones, including, e.g., basic fibroblast growth factor, human recombinant platelet derived growth factor, forskolin, and glial growth factor-2 (see, e.g., Ladak et al., Experimental Neurology 228 (2011) 242-252).
As discussed above, the genome editing systems of this disclosure can be implemented in any suitable manner, meaning that the components of such systems, including without limitation the RNA-guided nuclease, gRNA, and optional donor template nucleic acid, can be delivered, formulated, or administered in any suitable form or combination of forms that results in the transduction, expression or introduction of a genome editing system and/or causes a desired repair outcome in a cell, tissue or subject. Table 1 sets forth several, non-limiting examples of genome editing system implementations. Those of skill in the art will appreciate, however, that these listings are not comprehensive, and that other implementations are possible. With reference to Table 1 in particular, the table lists several exemplary implementations of a genome editing system comprising a single gRNA and an optional donor template. However, genome editing systems according to this disclosure can incorporate multiple gRNAs, multiple RNA-guided nucleases, and other components such as proteins, and a variety of implementations will be evident to the skilled artisan based on the principles illustrated in the table. In the table, [N/A] indicates that the genome editing system does not include the indicated component.
Nucleic acids encoding the various elements of a genome editing system according to the present disclosure can be administered to subjects or delivered into cells by art-known methods or as described herein. For example, RNA-guided nuclease-encoding and/or gRNA-encoding DNA, as well as donor template nucleic acids can be delivered by, e.g., vectors (e.g., viral or non-viral vectors described herein), non-vector based methods (e.g., using naked DNA or DNA complexes), or a combination thereof.
Nucleic acids encoding genome editing systems or components thereof can be delivered directly to cells as naked DNA or RNA, for instance by means of transfection or electroporation, or can be conjugated to molecules (e.g., N-acetylgalactosamine) promoting uptake by the target cells (e.g., erythrocytes, HSCs).
Viral vectors can comprise one or more sequences encoding genome editing system components, such as an RNA-guided nuclease, a gRNA and/or a donor template. A viral vector can also comprise a sequence encoding a signal peptide (e.g., for nuclear localization, nucleolar localization, or mitochondrial localization), associated with (e.g., inserted into or fused to) a sequence coding for a protein. As one example, a viral vectors can include a Cas9 coding sequence that includes one or more nuclear localization sequences (e.g., a nuclear localization sequence from SV40).
In addition to viral vectors, non-viral vectors can be used to deliver nucleic acids encoding genome editing systems according to the present disclosure. One important category of non-viral nucleic acid vectors are nanoparticles, which can be organic or inorganic. Nanoparticles are well known in the art, and are summarized in WO 2016/073990. Any suitable nanoparticle design can be used to deliver genome editing system components or nucleic acids encoding such components. For instance, organic (e.g. lipid and/or polymer) nonparticles can be suitable for use as delivery vehicles in certain embodiments of this disclosure. Exemplary lipids for use in nanoparticle formulations, and/or gene transfer are shown in Table 2, and Table 3 lists exemplary polymers for use in gene transfer and/or nanoparticle formulations.
Non-viral vectors optionally include targeting modifications to improve uptake and/or selectively target certain cell types. These targeting modifications can include e.g., cell specific antigens, monoclonal antibodies, single chain antibodies, aptamers, polymers, sugars (e.g., N-acetylgalactosamine (GalNAc)), and cell penetrating peptides. Such vectors also optionally use fusogenic and endosome-destabilizing peptides/polymers, undergo acid-triggered conformational changes (e.g., to accelerate endosomal escape of the cargo), and/or incorporate a stimuli-cleavable polymer, e.g., for release in a cellular compartment. For example, disulfide-based cationic polymers that are cleaved in the reducing cellular environment can be used.
In certain embodiments, one or more nucleic acid molecules (e.g., DNA molecules) other than the components of a genome editing system, e.g., the RNA-guided nuclease component and/or the gRNA component described herein, are delivered. In certain embodiments, the nucleic acid molecule is delivered at the same time as one or more of the components of the Genome editing system. In certain embodiments, the nucleic acid molecule is delivered before or after (e.g., less than about 30 minutes, 1 hour, 2 hours, 3 hours, 6 hours, 9 hours, 12 hours, 1 day, 2 days, 3 days, 1 week, 2 weeks, or 4 weeks) one or more of the components of the Genome editing system are delivered. In certain embodiments, the nucleic acid molecule is delivered by a different means than one or more of the components of the genome editing system, e.g., the RNA-guided nuclease component and/or the gRNA component, are delivered. The nucleic acid molecule can be delivered by any of the delivery methods described herein. For example, the nucleic acid molecule can be delivered by a viral vector, e.g., an integration-deficient lentivirus, and the RNA-guided nuclease molecule component and/or the gRNA component can be delivered by electroporation, e.g., such that the toxicity caused by nucleic acids (e.g., DNAs) can be reduced. In certain embodiments, the nucleic acid molecule encodes a therapeutic protein, e.g., a protein described herein. In certain embodiments, the nucleic acid molecule encodes an RNA molecule, e.g., an RNA molecule described herein.
Delivery of RNPs and/or RNA Encoding Genome Editing System Components
RNPs (complexes of gRNAs and RNA-guided nucleases) and/or RNAs encoding RNA-guided nucleases and/or gRNAs, can be delivered into cells or administered to subjects by art-known methods, some of which are described in WO 2016/073990. In-vitro, RNA-guided nuclease-encoding and/or gRNA-encoding RNA can be delivered, e.g., by microinjection, electroporation, transient cell compression or squeezing. Lipid-mediated transfection, peptide-mediated delivery, GalNAc- or other conjugate-mediated delivery, and combinations thereof, can also be used for delivery in-vitro and in-vivo.
In-vitro, delivery via electroporation comprises mixing the cells with the RNA encoding RNA-guided nucleases and/or gRNAs, with or without donor template nucleic acid molecules, in a cartridge, chamber or cuvette and applying one or more electrical impulses of defined duration and amplitude. Systems and protocols for electroporation are known in the art, and any suitable electroporation tool and/or protocol can be used in connection with the various embodiments of this disclosure.
Genome editing systems, or cells altered or manipulated using such systems, can be administered to subjects by any suitable mode or route, whether local or systemic. Systemic modes of administration include oral and parenteral routes. Parenteral routes include, by way of example, intravenous, intramarrow, intrarterial, intramuscular, intradermal, subcutaneous, intranasal, and intraperitoneal routes. Components administered systemically can be modified or formulated to target, e.g., HSCs, hematopoietic stem/progenitor cells, or erythroid progenitors or precursor cells.
Local modes of administration include, by way of example, intramarrow injection into the trabecular bone or intrafemoral injection into the marrow space, and infusion into the portal vein. In certain embodiments, significantly smaller amounts of the components (compared with systemic approaches) can exert an effect when administered locally (for example, directly into the bone marrow) compared to when administered systemically (for example, intravenously). Local modes of administration can reduce or eliminate the incidence of potentially toxic side effects that may occur when therapeutically effective amounts of a component are administered systemically.
Administration can be provided as a periodic bolus (for example, intravenously) or as continuous infusion from an internal reservoir or from an external reservoir (for example, from an intravenous bag or implantable pump). Components can be administered locally, for example, by continuous release from a sustained release drug delivery device.
In some embodiments, compositions described herein comprise AAV vectors at known vector genome concentrations and/or for a final vector genome delivery concentration. In some embodiments, the total vector genome titer administered is between about 1×109 and about 1×1014 VG, e.g., 1×109 VG, about 1×1010 VG, about 1×1011VG, about 1×1012VG, about 1×1013 VG, or about 1×1014 VG.
In some embodiments, compositions described herein are delivered as a liquid medium and/or solution. In some embodiments, delivery of specific volumes are known to be within safe and/or efficacious ranges. In some embodiments, a volume for delivery is between about 1 μl and about 100 μl. In some embodiments, a volume for delivery is about 1 μl, 2 μl, 3 μl, 4 μl, 5 μl, 6 μl, 7 μl, 8 μl, 9 μl, 10 μl, 11 μl, 12 μl, 13 μl, 14 μl, 15 μl, 16 μl, 17 μl, 18 μl, 19 μl, 20 μl, 21 μl, 22 μl, 23 μl, 24 μl, 25 μl, 26 μl, 27 μl, 28 μl, 29 μl, 30 μl, 31 μl, 32 μl, 33 μl, 34 μl, 35 μl, 36 μl, 37 μl, 38 μl, 39 μl, 40 μl, 41 μl, 42 μl, 43 μl, 44 μl, 45 μl, 46 μl, 47 μl, 48 μl, 49 μl, 50 μl, 51 μl, 52 μl, 53 μl, 54 μl, 55 μl, 56 μl, 57 μl, 58 μl, 59 μl, 60 μl, 61 μl, 62 μl, 63 μl, 64 μl, 65 μl, 66 μl, 67 μl, 68 μl, 69 μl, 70 μl, 71 μl, 72 μl, 73 μl, 74 μl, 75 μl, 76 μl, 77 μl, 78 μl, 79 μl, 80 μl, 81 μl, 82 μl, 83 μl, 84 μl, 85 μl, 86 μl, 87 μl, 88 μl, 89 μl, 90 μl, 91 μl, 92 μl, 93 μl, 94 μl, 95 μl, 96 μl, 97 μl, 98 μl, 99p1, or 100 μl.
In some embodiments, compositions as described herein are administered locally directly to Dorsal root ganglion (DRG) neurons. In some embodiments, delivery is between the L1 and S3 segments of the spinal cord. In some embodiments, delivery is at the L1 segment. In some embodiments, delivery is at the L2 segment. In some embodiments, delivery is at the L3 segment. In some embodiments, delivery is at the L4 segment. In some embodiments, delivery is at the L5 segment. In some embodiments, delivery is at the S1 segment. In some embodiments, delivery is at the S2 segment. In some embodiments, delivery is at the S3 segment. In some embodiments, delivery is at the transforaminal space surrounding the DRG (L1-S3).
In some embodiments, local administration is achieved through precision injection. In some embodiments, precision injection may involve only one needle, wherein the needle has a gauge of between about 18G and about 35G, e.g., about 16G, 17G, 18G, 19G, 20G, 21G, 22G, 23G, 24G, 25G, 26G, 27G, 28G, 29G, 30G, 31G, 32G, 33G, 34G, or 35G. In some embodiments, precision injection may involve the use of two needles. In some embodiments, the gauge of these needles can range from 18-35G. In some embodiments, a first needle is utilized as a guide needle and is used to approach the site for local administration. In some embodiments, a guide needle has a gauge in the range of 16-26G, e.g., about 16G, 17G, 18G, 19G, 20G, 21G, 22G, 23G, 24G, 25G, or 26G. In some embodiments, a second needle is utilized as a penetrating needle, where such penetrating needle is of a finer gauge than the first needle and is will be threaded through the guide needle to penetrate the tissue of interest. In some embodiments, a penetrating needle has a gauge in the range of 26-35G, e.g., about 26G, 27G, 28G, 29G, 30G, 31G, 32G, 33G, 34G, or 35G. Needle tips for use in precision injection are known in the art, any of which can be used in methods of the disclosure. Nonlimiting needle tips include, e.g., a Quincke, Whitacre, Pitkin, Greene, Tuohy, Pencil-point, Sprotte needle tip and/or a blunt-ended needled tip. In some embodiments, wherein a guide needle is utilized for precision injection, the guide needle tip may be but is not limited to a Quincke, Pitkin, and/or Tuohy needle tip. In some embodiments, wherein a penetrating needle is utilized for precision injection, the penetrating needle tip may be a pencil-point and/or Whitacre needle tip.
In some embodiments, compositions comprising AAV particles and constructs as described herein are locally administered (e.g., directly to DRG neurons) utilizing a controlled flow rate. In some embodiments, a controlled flow rate may be between 0.2 μl/min to 10 μl/min, e.g., a controlled flow rate may be 0.2 μl/min, 0.3 μl/min, 0.4 μl/min, 0.5 μl/min, 0.6 μl/min, 0.7 μl/min, 0.8 μl/min, 0.9p1/min, 1.0 μl/min, 1.25 μl/min, 1.5 μl/min, 1.75 μl/min, 2 μl/min, 2.25 μl/min, 2.5 μl/min, 2.75 μl/min, 3 μl/min, 3.5 μl/min, 4 μl/min, 4.5 μl/min, 5 μl/min, 5.5 μl/min, 6 μl/min, 6.5 μl/min, 7 μl/min, 7.5 μl/min, 8 μl/min, 8.5 μl/min, 9 μl/min, 9.5 μl/min and/or 10 μl/min. In some embodiments, a controlled flow rate may be controlled by an electronic and/or mechanical pump.
In some embodiments, compositions comprising AAV particles and constructs as described herein are administered via injection. In some embodiments, injection occurs utilizing one continuous flow rate over a period of time. In some embodiments, injection occurs in a staggered and/or stepped delivery pattern (e.g., at a relative high-flowrate, at a relative low flowrate (e.g., zero), and again at a relative high-flowrate, this cycle may be repeated any number of times as appropriate).
In addition, components can be formulated to permit release over a prolonged period of time. A release system can include a matrix of a biodegradable material or a material which releases the incorporated components by diffusion. The components can be homogeneously or heterogeneously distributed within the release system. A variety of release systems can be useful, however, the choice of the appropriate system will depend upon rate of release required by a particular application. Both non-degradable and degradable release systems can be used. Suitable release systems include polymers and polymeric matrices, non-polymeric matrices, or inorganic and organic excipients and diluents such as, but not limited to, calcium carbonate and sugar (for example, trehalose). Release systems may be natural or synthetic. However, synthetic release systems are preferred because generally they are more reliable, more reproducible and produce more defined release profiles. The release system material can be selected so that components having different molecular weights are released by diffusion through or degradation of the material.
Representative synthetic, biodegradable polymers include, for example: polyamides such as poly(amino acids) and poly(peptides); polyesters such as poly(lactic acid), poly(glycolic acid), poly(lactic-co-glycolic acid), and poly(caprolactone); poly(anhydrides); polyorthoesters; polycarbonates; and chemical derivatives thereof (substitutions, additions of chemical groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art), copolymers and mixtures thereof. Representative synthetic, non-degradable polymers include, for example: polyethers such as poly(ethylene oxide), poly(ethylene glycol), and poly(tetramethylene oxide); vinyl polymers-polyacrylates and polymethacrylates such as methyl, ethyl, other alkyl, hydroxyethyl methacrylate, acrylic and methacrylic acids, and others such as poly(vinyl alcohol), poly(vinyl pyrolidone), and poly(vinyl acetate); poly(urethanes); cellulose and its derivatives such as alkyl, hydroxyalkyl, ethers, esters, nitrocellulose, and various cellulose acetates; polysiloxanes; and any chemical derivatives thereof (substitutions, additions of chemical groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art), copolymers and mixtures thereof.
Poly(lactide-co-glycolide) microsphere can also be used. Typically the microspheres are composed of a polymer of lactic acid and glycolic acid, which are structured to form hollow spheres. The spheres can be approximately 15-30 microns in diameter and can be loaded with components described herein.
Skilled artisans will appreciate, in view of the instant disclosure, that different components of genome editing systems disclosed herein can be delivered together or separately and simultaneously or nonsimultaneously. Separate and/or asynchronous delivery of genome editing system components can be particularly desirable to provide temporal or spatial control over the function of genome editing systems and to limit certain effects caused by their activity.
Different or differential modes as used herein refer to modes of delivery that confer different pharmacodynamic or pharmacokinetic properties on the subject component molecule, e.g., a RNA-guided nuclease molecule or gRNA. For example, the modes of delivery can result in different tissue distribution, different half-life, or different temporal distribution, e.g., in a selected compartment, tissue, or organ.
Some modes of delivery, e.g., delivery by a nucleic acid vector that persists in a cell, or in progeny of a cell, e.g., by autonomous replication or insertion into cellular nucleic acid, result in more persistent expression of and presence of a component. Examples include viral, e.g., AAV or lentivirus, delivery.
By way of example, the components of a genome editing system, e.g., a RNA-guided nuclease and a gRNA, can be delivered by modes that differ in terms of resulting half-life or persistent of the delivered component the body, or in a particular compartment, tissue or organ. In certain embodiments, a gRNA can be delivered by such modes. The RNA-guided nuclease molecule component can be delivered by a mode which results in less persistence or less exposure to the body or a particular compartment or tissue or organ.
More generally, in certain embodiments, a first mode of delivery is used to deliver a first component and a second mode of delivery is used to deliver a second component. The first mode of delivery confers a first pharmacodynamic or pharmacokinetic property. The first pharmacodynamic property can be, e.g., distribution, persistence, or exposure, of the component, or of a nucleic acid that encodes the component, in the body, a compartment, tissue or organ. The second mode of delivery confers a second pharmacodynamic or pharmacokinetic property. The second pharmacodynamic property can be, e.g., distribution, persistence, or exposure, of the component, or of a nucleic acid that encodes the component, in the body, a compartment, tissue or organ.
In certain embodiments, the first pharmacodynamic or pharmacokinetic property, e.g., distribution, persistence or exposure, is more limited than the second pharmacodynamic or pharmacokinetic property.
In certain embodiments, the first mode of delivery is selected to optimize, e.g., minimize, a pharmacodynamic or pharmacokinetic property, e.g., distribution, persistence or exposure.
In certain embodiments, the second mode of delivery is selected to optimize, e.g., maximize, a pharmacodynamic or pharmacokinetic property, e.g., distribution, persistence or exposure.
In certain embodiments, the first mode of delivery comprises the use of a relatively persistent element, e.g., a nucleic acid, e.g., a plasmid or viral vector, e.g., an AAV or lentivirus. As such vectors are relatively persistent product transcribed from them would be relatively persistent.
In certain embodiments, the second mode of delivery comprises a relatively transient element, e.g., an RNA or protein.
In certain embodiments, the first component comprises gRNA, and the delivery mode is relatively persistent, e.g., the gRNA is transcribed from a plasmid or viral vector, e.g., an AAV or lentivirus. Transcription of these genes would be of little physiological consequence because the genes do not encode for a protein product, and the gRNAs are incapable of acting in isolation. The second component, a RNA-guided nuclease molecule, is delivered in a transient manner, for example as mRNA or as protein, ensuring that the full RNA-guided nuclease molecule/gRNA complex is only present and active for a short period of time.
Furthermore, the components can be delivered in different molecular form or with different delivery vectors that complement one another to enhance safety and tissue specificity.
Use of differential delivery modes can enhance performance, safety, and/or efficacy, e.g., the likelihood of an eventual off-target modification can be reduced. Delivery of immunogenic components, e.g., Cas9 molecules, by less persistent modes can reduce immunogenicity, as peptides from the bacterially-derived Cas enzyme are displayed on the surface of the cell by MHC molecules. A two-part delivery system can alleviate these drawbacks.
Differential delivery modes can be used to deliver components to different, but overlapping target regions. The formation active complex is minimized outside the overlap of the target regions. Thus, in certain embodiments, a first component, e.g., a gRNA is delivered by a first delivery mode that results in a first spatial, e.g., tissue, distribution. A second component, e.g., a RNA-guided nuclease molecule is delivered by a second delivery mode that results in a second spatial, e.g., tissue, distribution. In certain embodiments, the first mode comprises a first element selected from a liposome, nanoparticle, e.g., polymeric nanoparticle, and a nucleic acid, e.g., viral vector. The second mode comprises a second element selected from the group. In certain embodiments, the first mode of delivery comprises a first targeting element, e.g., a cell specific receptor or an antibody, and the second mode of delivery does not include that element. In certain embodiments, the second mode of delivery comprises a second targeting element, e.g., a second cell specific receptor or second antibody.
When the RNA-guided nuclease molecule is delivered in a virus delivery vector, a liposome, or polymeric nanoparticle, there is the potential for delivery to and therapeutic activity in multiple tissues, when it may be desirable to only target a single tissue. A two-part delivery system can resolve this challenge and enhance tissue specificity. If the gRNA and the RNA-guided nuclease molecule are packaged in separated delivery vehicles with distinct but overlapping tissue tropism, the fully functional complex is only be formed in the tissue that is targeted by both vectors.
In some embodiments, modified cells, e.g., progenitor cells (e.g., genetically modified progenitor cells) are administered to a subject. In some embodiments, a composition of modified cells can contain a physiologically tolerable carrier together with a cell described herein. In some embodiments, such compositions may further comprise at least one additional bioactive agent as described herein. In some embodiments, the at least one additional bioactive agent may be dissolved or dispersed therein as an active ingredient. In some embodiments, a composition is not substantially immunogenic when administered to a mammal or human patient for therapeutic purposes, unless so desired.
In general, compositions (e.g., comprising modified cells (e.g., progenitor cells)) may be administered as a suspension with a pharmaceutically acceptable carrier. One of skill in the art will recognize that a pharmaceutically acceptable carrier to be used in a cell composition will not any agents (e.g., certain buffers, compounds, cryopreservation agents, preservatives, etc.) that substantially interfere with the viability of the cells to be delivered to the subject.
In some embodiments, a formulation comprising cells can include e.g., osmotic buffers that permit cell membrane integrity to be maintained. In some such embodiments, nutrients to maintain cell viability or enhance engraftment upon administration. In some such embodiments, such formulations and suspensions are known to those of skill in the art and/or can be adapted for use with compositions comprising cells (e.g., progenitor cells), as described herein. In some embodiments, a composition comprising cells may also be emulsified or presented as a liposome composition, provided that emulsification procedure does not adversely affect cell viability. In some such embodiments, the composition comprising cells and any other active ingredient may be mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient, and in amounts suitable for use in the therapeutic methods described herein.
In some embodiments, additional agents included in a composition comprising cells as describe herein may include pharmaceutically acceptable salts of the components therein. Pharmaceutically acceptable salts may include the acid addition salts (formed with the free amino groups of the polypeptide) that are formed with inorganic acids, such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, tartaric, mandelic and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases, such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like.
Physiologically tolerable carriers are well known in the art. In some embodiments, exemplary liquid carriers are sterile aqueous solutions that contain no materials in addition to the active ingredients and water, or contain a buffer such as sodium phosphate at physiological pH value, physiological saline or both, such as phosphate-buffered saline. Still further, aqueous carriers can contain more than one buffer salt, as well as salts such as sodium and potassium chlorides, dextrose, polyethylene glycol and other solutes. In some embodiments, liquid compositions can also contain liquid phases in addition to and to the exclusion of water. By way of non-limiting example, in some such embodiments, additional liquid phases are glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions.
In some embodiments, a composition comprises a least 102 progenitor cells, at least 5×102progenitor cells, at least 103progenitor cells, at least 5×103progenitor cells, at least 104progenitor cells, at least 5×104 progenitor cells, at least 105 progenitor cells, at least 2×105 progenitor cells, at least 3×105 progenitor cells, at least 4×105 progenitor cells, at least 5×105 progenitor cells, at least 6×105 progenitor cells, at least 7×105 progenitor cells, at least 8×105 progenitor cells, at least 9×105 progenitor cells, at least 1×106 progenitor cells, at least 2×106 progenitor cells, at least 3×106 progenitor cells, at least 4×106 progenitor cells, at least 5×106 progenitor cells, at least 6×106 progenitor cells, at least 7×106 progenitor cells, at least 8×106 progenitor cells, at least 9×106 progenitor cells, or multiples thereof. In some embodiments, progenitor cells can be derived from one or more donors. In some embodiments, progenitor cells are obtained from an autologous source. In some embodiments, progenitor cells are obtained from stem cells (e.g., iPSC cells that are differentiated into progenitor cells). In some embodiments, progenitor cells can be expanded in culture prior to administration to a subject.
In some embodiments, treatment according to the present disclosure can ameliorate or prevent one or more symptoms associated with a pain disorder and/or disorder associated with pain by decreasing or altering the amount of an SCN9A and/or SCN10A gene or gene product (e.g., protein) in a subject.
The present disclosure provides, in some embodiments, methods for treating a subject with one or more symptoms of a pain disorder and/or disorder associated with pain, or a patient at risk of a pain disorder and/or disorder associated with pain.
In some embodiments, a method is or comprises ex-vivo cell-based therapy. For example, in some embodiments, a biopsy is performed on a patient. In some embodiments, the biopsy is of a patient's peripheral nerves is performed. In some embodiments, nerve tissue can be isolated from the patient's skin or leg. Then, at least one cell of the peripheral nervous system (e.g., a neuron or a glial cell such as Schwann cell in nerves or satellite glial cell in ganglia, or a progenitor thereof) is isolated from the biopsied material. Concomitant with or following isolation, the chromosomal DNA of the cell of the peripheral nervous system (e.g., a neuron and/or a glial cell such as Schwann cell in nerves or satellite glial cell in ganglia) can be edited using approaches and technologies as provided and described herein. Finally, the edited cell of the peripheral nervous system (e.g., a neuron or a glial cell such as Schwann cell in nerves or satellite glial cell in ganglia) is implanted into the patient.
In some embodiments, a patient specific stem cell may be harvested or generated. For example, in some embodiments, a stem cell may be an induced pluripotent stem cell (iPSC) is generated (e.g., from cells harvested from peripheral nerves, patient skin, etc.). In some embodiments, a stem cell may be a mesenchymal stem cell isolated from a patient's bone marrow or peripheral blood. In some embodiments, stem cells are autologous stem cells. In some such embodiments, chromosomal DNA of these iPSC cells can be edited using approaches and technologies provided and described herein. Concomitant with or following isolated, genome-edited iPSCs can be differentiated into cells of the nervous system (e.g., a neural stem cell, a neuronal progenitor cell, a peripheral neuron or a glial cell such as Schwann cell in nerves or satellite glial cell in ganglia). Finally, the neural stem cell, neuronal progenitor cell, or neuronal/glial cells of the peripheral nervous system can be administered (e.g., via infusion or implantation) to a patient (e.g., a subject in need thereof, a control subject, etc.).
In some embodiments, cells produced for ex-vivo therapy are comprehensively analyzed prior to administration to a subject. For example, in some embodiments, nuclease-based therapeutics may have some level of off-target effects. By performing gene editing ex-vivo, cells are able to be analyzed after modification (e.g., editing), but prior to administration (e.g., implantation) to a subject. In some embodiments, the entire genome of one or more edited cells can be sequenced. In some embodiments, such sequencing provides information regarding off-target effects, if any. In some such embodiments, if any off-target effects are detected, cells with effects in genomic locations associated with minimal risk to the patient may be selected for administration to a subject. In some embodiments, populations of specific cells, including clonal populations, can be isolated prior to implantation.
In some embodiments, an advantage of ex-vivo cell therapy relates to genetic modification in iPSCs compared to other primary cell sources. Generally, iPSCs are prolific, such that obtaining the large number of cells that will be required for a cell-based therapy is not difficult. Furthermore, in some embodiments, iPSCs are an ideal cell type for performing clonal isolations. This allows screening for the correct genomic modification, without risking a decrease in viability.
In some embodiments, non-iPSCs such as differentiated cells (e.g., glial cells), are viable for only a few passages and difficult to clonally expand. Thus, manipulation of iPSCs for the treatment of pain can be much easier, and can shorten the amount of time needed to make the desired genetic modification.
In some embodiments, the present disclosure provides, among other things, methods comprising in-vivo therapy. In some such embodiments, chromosomal DNA of the cells in the patient is edited using the technologies provided and described in accordance with the present disclosure. In some embodiments, the target cell in an in-vivo based therapy is a neuron of the peripheral nervous system. In some embodiments, the target cell in an in-vivo based therapy is a neuronal progenitor cell.
In some embodiments comprising in-vivo therapy, impact in non-target cells can also be prevented by the use of promoters only active in certain cells and or developmental stages. In some embodiments, promoters are inducible, and therefore can be temporally controlled if the nuclease is delivered as a plasmid.
In some embodiments, an amount of time that delivered RNA and protein remain in the cell can also be adjusted using treatments or domains added to change the half-life. In some embodiments, use of in-vivo approaches would eliminate a number of treatment steps, but a lower rate of delivery can require higher rates of editing. In some such embodiments, use of In-vivo approaches can eliminate problems and losses from ex-vivo approaches such as e.g. engraftment and post-engraftment integration of neurons and glial cells appropriately into existing neural circuits.
In some embodiments, an advantage of an in-vivo approach can be the ease of therapeutic production and administration. In some embodiments, the same therapeutic approach and therapy will have the potential to be used to treat more than one patient, for example a number of patients who share the same or similar genotype or allele.
In some embodiments, ex-vivo cell therapy typically requires using a patient's own cells, which are isolated, manipulated and returned to the same patient. Accordingly, in some such embodiments, ex-vivo approaches may be more individualized, but less translatable on a large scale than in-vivo approaches.
In some embodiments, the present disclosure provides technologies comprising a cellular method for editing SCN9A and/or SCN10A gene(s) in a cell by gene editing. For example, in some embodiments, a cell can be isolated from subject (e.g., a patient or animal). Then, the chromosomal DNA of the cell can be edited using, among other things, materials and methods described herein.
The present disclosure also provides, among other things, approaches which may, regardless of whether a cellular or ex-vivo or in-vivo method, involve reducing (knock-down) or eliminating (knock-out) the expression of the SCN9A and/or SCN10A gene by introducing one or more modifications, e.g. insertions, deletions, substitutions, mutations, etc. within or near the SCN9A and/or SCN10A gene(s) or other DNA sequences that encode regulatory elements of the SCN9A and/or SCN10A gene(s).
For example, in some embodiments, the knock-down or knock-out strategy can involve disrupting the reading frame in the SCN9A and/or SCN10A gene(s) by introducing random insertions or deletions (indels) that arise due to the imprecise NHEJ repair pathway. This can be achieved by inducing one single stranded break or double stranded break in the SCN9A gene with one or more CRISPR endonucleases and a gRNA (e.g., crRNA+tracrRNA, or sgRNA), or two or more single stranded breaks or double stranded breaks in the SCN9A and/or SCN10A gene(s) with two or more CRISPR endonucleases and two or more sgRNAs. This approach can require development and optimization of sgRNAs for the SCN9A and/or SCN10A gene(s).
In some embodiments, the knock-down or knock-out strategy can also involve deletion of one or more segments within or near the SCN9A and/or SCN10A gene(s) or other DNA sequences that encode regulatory elements of the SCN9A and/or SCN10A gene(s). This deletion strategy requires at least a pair of gRNAs (e.g., crRNA+tracrRNA, or sgRNA) capable of binding to two different sites within or near the SCN9A and/or SCN10A gene(s) and one or more CRISPR endonucleases. The CRISPR endonucleases, configured with the two gRNAs, induce two double stranded breaks at the desired locations. After cleavage, the two ends, regardless of whether blunt or with overhangs, can be joined by NHEJ, leading to the deletion of the intervening segment. In certain aspects, NHEJ repair pathways can lead to insertions, deletions or mutations at the joints.
In addition to the above gene editing strategies, another strategy involves modulating expression, function, or activity of SCN9A and/or SCN10A by editing in the regulatory sequence.
In addition to the editing options listed above, site-specific nucleases, e.g., Cas9 or similar proteins, can be used to target effector domains to the same target sites that can be identified for editing, or additional target sites within range of the effector domain. A range of chromatin modifying enzymes, methylases or demethylases can be used to alter expression of the target gene. One possibility is decreasing the expression of the SCN9A and/or SCN10A protein(s) if a mutation leads to undesirable activity. In some such embodiments, such epigenetic regulation may have some advantages, particularly as they are limited in possible off-target effects.
In some embodiments, a number of types of genomic target sites can be present in addition to the coding and splicing sequences. The regulation of transcription and translation implicates a number of different classes of sites that interact with cellular proteins or nucleotides. Often DNA binding sites of transcription factors or other proteins can be targeted for mutation or deletion to study the role of the site, though they can also be targeted to change gene expression. Sites can be added through non-homologous end joining NHEJ or direct gene editing by homology directed repair (HDR). Increased use of genome sequencing, RNA expression and genome-wide studies of transcription factor binding have increased our ability to identify how the sites lead to developmental or temporal gene regulation. These control systems can be direct or can involve extensive cooperative regulation that can require the integration of activities from multiple enhancers. Transcription factors typically bind 6-12 bp-long degenerate DNA sequences. The low level of specificity provided by individual sites suggests that complex interactions and rules are involved in binding and the functional outcome. Binding sites with less degeneracy can provide simpler means of regulation. Artificial transcription factors can be designed to specify longer sequences that have less similar sequences in the genome and have lower potential for off-target cleavage. In some embodiments, any of these types of binding sites can be mutated, deleted or even created to enable changes in gene regulation or expression (see, e.g., Canver, M. C. et al., Nature (2015)).
Efficacy of a given treatment in accordance with the present disclosure can be determined by the skilled clinician. In some embodiments, a treatment is considered effective if any one or all of the signs or symptoms of or other clinically accepted symptoms or markers of disease are improved or ameliorated. For example, in some embodiments, if a level of SCN9A and/or SCN10A is/are altered in a beneficial manner (e.g., decreased by at least 10%), a treatment may be effective. In some embodiments, efficacy can also be measured by failure of an individual to worsen as assessed by hospitalization or need for medical interventions (e.g., progression of the disease is halted or at least slowed). Methods of measuring these indicators are known to those of skill in the art and/or described herein. Treatment includes any treatment of a disease in an individual or an animal (some non-limiting examples include a human, or a mammal) and includes: (i) inhibiting the disease, e.g., arresting, or slowing the progression of symptoms; or (ii) relieving the disease, e.g., causing regression of symptoms; and (ii) preventing or reducing the likelihood of the development of symptoms.
SCN9A has been associated with one or more symptoms of one or more diseases or disorders having or associated with pain such as, but not limited to, Congenital Pain Insensitivity, Anosmia, As If Personality, Borderline Personality Disorder, Malignant neoplasm of breast, Non-Small Cell Lung Carcinoma, Cold intolerance, Febrile Convulsions, Diabetes, Diabetes Mellitus, Dissociative disorder, Epilepsy, Erythromelalgia, Primary Erythermalgia, Facial Pain, Herpesviridae Infections, Hereditary Sensory Autonomic Neuropathy Type 5, Hyperplasia, Neuralgia, Hereditary Sensory and Autonomic Neuropathies, Degenerative polyarthritis, Pain, Pain in limb, Postoperative Pain, Parkinson Disease, Postherpetic neuralgia, Prostatic Neoplasms, Pruritus, Seizures, Somatoform Disorder, Tobacco Use Disorder, Trigeminal Neuralgia, Synovial Cyst, Chronic pain, Acute onset pain, Paramyotonia Congenita (disorder), Malaise, Sensory Discomfort, Burning Pain, Indifference to pain, Inflammatory pain, Mechanical pain, Scalp pain, Hereditary Motor and Sensory-Neuropathy Type II, Common Migraine, Absence of pain sensation, Malignant neoplasm of prostate, Pain Disorder, Knee Osteoarthritis, Neuropathy, Complex Regional Pain Syndromes, Tonic-clonic seizures, Inherited neuropathies, Prostate carcinoma, Breast Carcinoma, Infantile Severe Myoclonic Epilepsy, Myxoid cyst, Channelopathies, Paroxysmal Extreme Pain Disorder, Painful Neuropathy, Compressive Neuropathies, Congenital Indifference to Pain Autosomal Recessive, Generalized Epilepsy With Febrile Seizures Plus Type 2, Generalized Epilepsy With Febrile Seizures Plus 7, Febrile Seizures Familial 3B, and Small Fiber Neuropathy (Adult-onset is referred to as small fiber neuropathy). (See, e.g., Yang et al., Trends Pharmacol. Sci. 39:258-275 (2018).)
In some embodiments, editing the SCN9A gene using any of the methods described herein may be used to treat, prevent and/or mitigate one or more symptoms of one or more disorders described herein.
The SCN9A gene encodes the alpha subunit of a sodium channel, NaV1.7. NaV1.7 is primarily expressed in sensory neurons and plays a significant role in nociception signaling.
In some embodiments, mutations in the SCN9A gene are known to cause pain perception disorders, including primary erythermalgia, paroxysmal extreme pain disorder, congenital insensitivity to pain, and small fiber neuropathy. For example, humans presenting with homozygous SCN9A loss-of-function mutations may suffer from congenital insensitivity to pain (CIP).
In some embodiments, the SCN9A gene is involved in itching.
In some embodiments, gain-of-function mutations in SCN9A cause congenital pain syndromes, such as primary erythermalgia. In some embodiments, gain-of-function mutations in the SCN9A gene result in spontaneous pain as observed in primary erythermalgia and paroxysmal extreme pain disorder. Thus, in some embodiments, knock-out or knock-down of the SCN9A gene in patients having primary erythromelalgia or paroxysmal extreme pain disorder can be used to treat, prevent and/or mitigate the associated symptoms. In some embodiments, primary erythromelalgia is a rare autosomal dominant disorder characterized by episodes of burning pain in the feet and hands in response to heat and movement. Affected individuals typically develop signs and symptoms in early childhood, although in milder cases symptoms can appear later in life. Management of this condition is mainly symptomatic. Besides avoidance of pain triggers (such as heat, exercise, and alcohol), treatment options include cooling and elevating the extremity, use of anesthetics such as lidocaine and mexilitine, and use of opioid drugs in extreme cases.
In some embodiments, paroxysmal extreme pain disorder is another rare disorder characterized by severe episodic pain in rectal, ocular, and mandibular regions as well as skin redness. Symptoms of this condition often begin in the neonatal period or in the early childhood, and can retain throughout life. Agents for treating chronic neuropathic pain disorders are often used to alleviate the pain episodes caused by the disease. Carbamazepine, a sodium channel blocker, has proven most effective of these treatments.
In some embodiments, technologies of the present disclosure may be combined with one or more other treatments, e.g., gene-editing combined with a pharmacological agent or intervention (e.g., lidocaine, mexilitine, carbamazepine).
SCN10A has been associated with one or more symptoms of one or more diseases or disorders having or associated with pain such as, but not limited to, Congenital Pain Insensitivity, Asthma, Ataxia, Atrial Fibrillation, Malignant neoplasm of breast, Hypertrophic Cardiomyopathy, Cardiovascular Diseases, Cerebellar Diseases, Dyspepsia, Experimental Autoimmune Encephalomyelitis, Primary Erythermalgia, Heart Block, Hereditary Sensory Autonomic Neuropathy Type 5, Hyperalgesia, Acute Myelocytic Leukemia, Multiple Sclerosis, Neuralgia, Neuroblastoma, Pain, Postherpetic neuralgia, Tobacco Use Disorder, Trigeminal Neuralgia, Ventricular Fibrillation, Synovial Cyst, Sciatic Neuropathy, Chronic pain, Fibrillation, Hematologic Neoplasms, Electrocardiogram: P-R interval, Neuropathy, Breast Carcinoma, Central neuroblastoma, Varicella zoster, Brugada Syndrome (disorder), Myxoid cyst, Channelopathies, Compressive Neuropathies, Paroxysmal Extreme Pain Disorder, High grade atrioventricular block, Small Fiber Neuropathy, and Familial Episodic Pain Syndrome 2. (See, e.g., Dib-Hajj et al., Ann. Rev. Neurosci. 42:87-106 (2019).)
In some embodiments, editing the SCN10A gene using any of the methods described herein may be used to treat, prevent and/or mitigate one or more symptoms of one or more disorders described herein.
The gene SCN10A encodes the alpha subunit of a voltage-gated sodium channel, NaV1.8. NaV1.8 is primarily expressed in the sensory neurons of the dorsal root ganglion and plays an important role in neuropathic pain mechanisms.
In some embodiments, mutations in the SCN10A gene have been found in patients with familial episodic pain syndrome type 2. Familial episodic pain syndrome type 2 is a rare autosomal dominant neurologic disorder characterized by adult-onset of paroxysmal pain in the feet region. The episodes are generally triggered by heat, cold, chemicals and certain surfaces. Patients may also develop hypersensitivity to touch and elevated response to pain stimulus. Currently no treatment is available for this disease. Warmth has been shown to relieve the pain episodes.
In some embodiments, SCN10A gene mutations are also responsible for about five percent of the cases of small fiber neuropathy. Small fiber neuropathy is a condition characterized by severe pain attacks and insensitivity to pain. The pain attacks are usually described as numbness, stabbing or burning, or abnormal skin sensations such as tingling or itchiness. Currently, there is no cure for small fiber peripheral neuropathy. Treatment options include intravenous immunoglobulin (IVIG) and plasmapheresis.
In some embodiments, technologies of the present disclosure may be combined with one or more other treatments, e.g., gene-editing combined with a pharmacological agent or intervention (e.g., IVIG plasmapheresis)
Among other things, the present disclosure provides technologies for treatment (e.g., amelioration, prevention) of one or more symptoms of a pain disorder and/or disorder associated with pain. In some embodiments, pain is a symptom of one or more pain disorders or of one or more other diseases with which pain is associated. In some embodiments, pain is the only symptom experienced by a subject. In some embodiments, a subject is at risk of having pain, but has not yet experienced pain. For example, in some embodiments, a subject has one or more mutations known to result in a pain disorder and/or disorder associated with pain as described herein. In some such embodiments, the present disclosure provides technologies that may prevent the subject from experiencing pain as related to manifestation of the disorder which they are at risk of developing.
In some embodiments, one or more symptoms of a pain disorder and/or disorder associated with pain is intractable after one or more traditional treatments used for treating the one or more symptoms and/or underlying disease or disorder.
In some embodiments, a pain disorder or a disorder associated with pain is arthritis, fibromyalgia, headache, migraine, shingles, neuropathy, failed back surgery syndrome, phantom limb pain, post traumatic pain, acute pain, pain from advanced prostate cancer, ankylosing spondylitis, AIDS-related pain, arachnoiditis, arthrofibrosis, ataxic cerebral palsy, autoimmune atrophic gastritis, autoimmune disease, avascular necrosis, back pain, Behcet's Disease/Syndrome, Breakthrough Pain, Burning Mouth Syndrome, Bursitis, CADASIL, Cancer Pain, Carpal Tunnel, Cauda Equina Syndrome, Central Pain Syndrome, Cerebral Palsy, Cerebrospinal Fluid Leak, Cervical Stenosis, Charcot-Marie-Tooth Disease, Chronic Fatigue Syndrome, Chronic Functional Abdominal Pain, Chronic Pancreatitis, Coccyx pain, collapsed lung (pneumothorax), complex regional pain syndrome (CRPS I and II), Corneal Neuropathic Pain, Crohn's disease, degenerative disc disease, Dercum's disease, dystonia, dematomyositis, diabetic peripheral neuropathy, Ehlers-Danlos syndrome, endometriosis, eosinophilia-myalgia syndrome, erythromelalgia, gout, growing pains, hydrocephalus, herniated disc, hip injury, hip replacement, knee injury, knee replacement, intercostal neuralgia, interstitial cystitis, irritable bowel syndrome, juvenile dermatositis, loin pain-haematuria syndrome, lupus, Lyme disease, medullary sponge kidney, meralgia paresthetica, mesothelioma, mitochondrial disorders, musculoskeletal pain, myofascial pain, myositis, neuropathic pain, nociceptive pain, neck pain, occipital neuralgia, osteoarthritis, Paget's disease, Parsonage Turner syndrome, pelvic pain, pinched nerve, polycystic kidney disease, polymyalgia rheumatica, polymyositis, porphyria, post-herniorraphy pain syndrome, post-mastectomy pain syndrome, post-stroke pain, post-thoracotomy pain syndrome, post-amputation stump pain, postherpetic neuralgia, post-polio syndrome, post-surgery pain, post-surgical neuralgia, post-hip surgery pain, post-traumatic stress disorder, primary lateral sclerosis, rheumatoid arthritis, psoriatic arthritis, pudendal neuralgia, radiculopathy, Raynaud's disease, restless leg syndrome, rheumatoid arthritis, sacroiliac joint dysfunction, sarcoidosis, Scheuemann's Kyphosis disease, sciatica, scoliosis, herpes zoster virus, sickle cell pain, Sjogren's syndrome, sleep apnea, spasmodic torticollis, sphincter of Oddi dysfunction, spinal cerebellar ataxia, spinal cord injury, spinal stenosis, syringomyelia, Tarlov cysts, tethered cord syndrome, thoracic outlet syndrome, temporomandibular joint syndrome, transverse myelitis, trigger point pain, ulcerative colitis, vascular pain, vasculitis, vulvodynia, whiplash, pain after hernia repair, pain after total knee replacement, pain after hip replacement, Congenital Pain Insensitivity, Anosmia, As If Personality, Borderline Personality Disorder, Malignant neoplasm of breast, Non-Small Cell Lung Carcinoma, Cold intolerance, Febrile Convulsions, Diabetes, Diabetes Mellitus, Dissociative disorder, Epilepsy, Erythromelalgia, Primary Erythermalgia, Facial Pain, Herpesviridae Infections, Hereditary Sensory Autonomic Neuropathy Type 5, Hyperplasia, Neuralgia, Hereditary Sensory and Autonomic Neuropathies, Degenerative polyarthritis, Pain, Pain in limb, Postoperative Pain, Parkinson Disease, Postherpetic neuralgia, Prostatic Neoplasms, Pruritus, Seizures, Somatoform Disorder, Tobacco Use Disorder, Trigeminal Neuralgia, Synovial Cyst, Chronic pain, Acute onset pain, Paramyotonia Congenita (disorder), Malaise, Sensory Discomfort, Burning Pain, Indifference to pain, Inflammatory pain, Mechanical pain, Scalp pain, Hereditary Motor and Sensory-Neuropathy Type II, Common Migraine, Absence of pain sensation, Malignant neoplasm of prostate, Pain Disorder, Knee Osteoarthritis, Neuropathy, Complex Regional Pain Syndromes, Tonic-clonic seizures, Inherited neuropathies, Prostate carcinoma, Breast Carcinoma, Infantile Severe Myoclonic Epilepsy, Myxoid cyst, Channelopathies, Paroxysmal Extreme Pain Disorder, Painful Neuropathy, Compressive Neuropathies, Congenital Indifference to Pain Autosomal Recessive, Generalized Epilepsy With Febrile Seizures Plus Type 2, Generalized Epilepsy With Febrile Seizures Plus 7, Febrile Seizures Familial 3B, and Small Fiber Neuropathy (Adult-onset is referred to as small fiber neuropathy), Asthma, Ataxia, Atrial Fibrillation, Hypertrophic Cardiomyopathy, Cardiovascular Diseases, Cerebellar Diseases, Dyspepsia, Experimental Autoimmune Encephalomyelitis, Primary Erythermalgia, Heart Block, Hyperalgesia, Acute Myelocytic Leukemia, Multiple Sclerosis, Neuralgia, Neuroblastoma, Pain, Postherpetic neuralgia, Tobacco Use Disorder, Trigeminal Neuralgia, Ventricular Fibrillation, Synovial Cyst, Sciatic Neuropathy, Chronic pain, Fibrillation, Hematologic Neoplasms, Electrocardiogram: P-R interval, Neuropathy, Breast Carcinoma, Central neuroblastoma, Varicella zoster, Brugada Syndrome (disorder), High grade atrioventricular block, Small Fiber Neuropathy, and Familial Episodic Pain Syndrome 2.
It is known that humans presenting with homozygous SCN9A loss-of-function mutations may suffer from congenital insensitivity to pain (CIP). In some embodiments, the SCN9A gene is involved in itching. In some embodiments, gain-of-function mutations in SCN9A and/or SCN10A cause congenital pain syndromes, such as primary erythermalgia.
Pain may occur in different parts of the body and for different reasons. For example, in some embodiments, chronic pain that occurs in most parts of the body and the extremities involve afferent neurons of the dorsal root ganglia (DRG), which reside in clusters of nerve cells near the spinal cord and have long axons extending towards, for example, the skin, muscles, and organs.
In some embodiments, a mechanism of enhanced excitability involves voltage-gated ion channels and background/leak channels that set a resting membrane potential and firing threshold of a neuron (e.g., a DRG). Generally, under conditions in which no disorder or symptom of disorder is present, chemical, mechanical, and/or thermal stimuli are required to activate receptors and ion channels in peripheral nerve endings to initiate action potentials that propagate along axons of DRG neurons. For example, in some embodiments, dendritic termini of the DRG neurons liberate neurotransmitters (e.g., glutamate and substance-P) at synapses in the spinal cord dorsal horn, activating second-order neurons that communicate pain signals to the brain. Generally (i.e., in absence of disorders or genetic changes) human DRG neurons constitutively express specific and specialized ion channels that have been implicated in afferent pain signaling. In some such embodiments, such ion channels may be targeted for modulation of one or more symptoms of a pain disorder.
In some embodiments, NaV1.7 and/or NaV1.8, are constitutively expressed in DRG neurons. In some embodiments, targeting ion channels comprising one or more proteins as described herein in accordance with the present disclosure may lead to changes (e.g., gene ablation, loss-of-function, destabilization of transcript and/or protein folding) of or in the targeted protein and/or ion channel(s). In some such embodiments, such changes in cells of a patient with one or more symptoms of a pain disorder results in reduced pain transmission and/or reduced experience of one or more pain symptoms.
Without being bound by any particular theory the present disclosure appreciates that genomic editing (e.g., disruption) of SCN9A and/or SCN10A is only desirable at a localized level. For example, nociception is essentially a protective mechanism from overextension and deformation of our joints and muscles, and is also necessary for our sense of smell. Accordingly, generalized changes in SCN9A and/or SCN10A may have undesirable consequences in addition to, e.g., amelioration and/or prevention of one or more symptoms of a pain disorder.
In some embodiments, technologies provides by the present disclosure (e.g., genome-editing agents) may be deployed (e.g., administered to or near) to DRG neurons (e.g., dysregulated DRG neurons) to modify the genes responsible for propagation of pain signals in DRG neurons.
In some embodiments, methods of the disclosure comprise treating pain in a subject by administering to a subject in need thereof a composition comprising an AAV5 vector comprising at least one sequence that encodes a guide RNA (gRNA) that targets a gene encoding a voltage-gated sodium channel subunit, wherein the gRNA comprises a spacer comprising or consisting of the nucleotide sequence of any one of SEQ ID NOs: 1-310. In some embodiments, an AAV5 vector comprises at least one gRNA sequence comprising a spacer sequence comprising the nucleotide sequence of SEQ ID NO: 5. In some embodiments, an AAV5 vector comprises at least one gRNA sequence comprising a spacer sequence comprising the nucleotide sequence of SEQ ID NO: 7. In some embodiments, an AAV5 vector comprises at least one gRNA sequence comprising a spacer sequence comprising the nucleotide sequence of SEQ ID NO: 182. In some embodiments, an AAV5 vector comprises at least one gRNA sequence comprising a spacer sequence comprising the nucleotide sequence of SEQ ID NO: 189. In some embodiments, an AAV5 vector comprises two gRNA sequences comprising spacer sequences comprising the nucleotide sequences of SEQ ID NO: 5 and 189. In some embodiments, an AAV5 vector comprises two gRNA sequences comprising spacer sequences comprising the nucleotide sequences of SEQ ID NO: 7 and 182. In some embodiments, an AAV5 vector comprises two gRNA sequences comprising spacer sequences comprising the nucleotide sequences of SEQ ID NO: 5 and 182. In some embodiments, an AAV5 vector comprises two gRNA sequences comprising spacer sequences comprising the nucleotide sequences of SEQ ID NO: 7 and 189.
In some embodiments, methods of the disclosure comprise treating pain in a subject by administering to a subject in need thereof a composition comprising an AAV9 vector comprising at least one sequence that encodes a guide RNA (gRNA) that targets a gene encoding a voltage-gated sodium channel subunit, wherein the gRNA comprises a spacer comprising or consisting of the nucleotide sequence of any one of SEQ ID NOs: 1-310. In some embodiments, an AAV9 vector comprises at least one gRNA sequence comprising a spacer sequence comprising the nucleotide sequence of SEQ ID NO: 5. In some embodiments, an AAV9 vector comprises at least one gRNA sequence comprising a spacer sequence comprising the nucleotide sequence of SEQ ID NO: 7. In some embodiments, an AAV9 vector comprises at least one gRNA sequence comprising a spacer sequence comprising the nucleotide sequence of SEQ ID NO: 182. In some embodiments, an AAV9 vector comprises at least one gRNA sequence comprising a spacer sequence comprising the nucleotide sequence of SEQ ID NO: 189. In some embodiments, an AAV9 vector comprises two gRNA sequences comprising spacer sequences comprising the nucleotide sequences of SEQ ID NO: 5 and 189. In some embodiments, an AAV9 vector comprises two gRNA sequences comprising spacer sequences comprising the nucleotide sequences of SEQ ID NO: 7 and 182. In some embodiments, an AAV9 vector comprises two gRNA sequences comprising spacer sequences comprising the nucleotide sequences of SEQ ID NO: 5 and 182. In some embodiments, an AAV9 vector comprises two gRNA sequences comprising spacer sequences comprising the nucleotide sequences of SEQ ID NO: 7 and 189.
The present disclosure also provides technologies that are superior to any current and/or traditional method of pain management or prevention. For example, in some such embodiments, strategies for treatment or prevention of pain (e.g., pain suppression) described herein are superior to traditional methods of pain management due to their high specificity, efficacy, and safety profile.
In some embodiments, technologies provided by the present disclosure may be combined with one or more current or traditional methods of pain management.
In some embodiments, pain is evaluated by one of skill in the art and/or by a subject experiencing pain. In some such embodiments, pain is evaluated relative to pain experienced before, during, and/or after treatment. In some embodiments, pain is evaluated between subjects, such as between subjects receiving a placebo or control treatment and subject receiving a treatment that comprises one or more genetic modifications to, e.g., SCN9A and/or SCN10A.
In some embodiments, a level or measure of pain reveals reduction, amelioration, prevention, or attenuation in one or more symptoms of pain or a disorder associated with pain, relative to a control. In some such embodiments, a control may be within a single subject (e.g., experience prior to administration of a composition of the present disclosure) or as compared to a control subject (e.g., a subject receiving a placebo or control composition).
By way of non-limiting example, in some embodiments, pain may be measured based on average level of pain over a period of time, relative to average level of pain over a period of time that occurred prior to treatment in accordance with the present disclosure.
In some embodiments, pain is evaluated based on one or more measures of efficacy as described in the present disclosure.
In some embodiments, various tools and measurements of pain will be known to one of skill in the art.
The disclosure is further illustrated by the following example. This example is provided for illustrative purposes only. It is not to be construed as limiting the scope or content of the disclosure in any way.
In the present example, spacer sequences targeting the genes SCN9A and SCN10A respectively, whose products function in tandem to transmit “painful” stimuli, were screened for cleavage efficacy and indel formation frequency.
Ribonucleoproteins (RNPs) were complexed from bacterially expressed and purified protein and chemically synthesized RNA oligonucleotides. crRNAs and tracrRNA for SaCas were made as RNA oligomers using standard oligonucleotide synthesis (made by IDT vendow). RNA molecules were desalted and lyophilized. crRNAs and tracrRNAs were resuspended in H150 buffer (10 mM HEPES, N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid, 150 mM NaCl, pH 7.5) to a concentration of 333 μM and then mixed at a 1:1 ratio. The crRNA and tracrRNA were then heat annealed at 95° C. for 5 minutes and slow cooled to form the full-length gRNA at 200 μM concentration. SaCas9 protein was made by Aldevron, concentrated to 0.4 mM, buffer exchanged to HG300 buffer (50 mM HEPES, 300 mM NaCl, 1 mM TCEP, 20% glycerol (% v/v), pH 7.5), aliquoted, and stored at −80° C. Annealed guides were mixed with 110 μM SaCas9 protein in H300 buffer (50 mM HEPES, 300 mM NaC, 1 mM tris(2-carboxyethyl)phosphine, 20% glycerol (% v/v), pH 7.5) to make a final 55 μM RNP. RNP was then frozen at −80° C. until ready for use.
Human T cells were nucleofected with the RNP, grown for 4 days, and then genomic DNA (gDNA) collected for Next Generation Sequencing (NGS). Primary human CD4+T-cells were isolated from whole blood samples (Massachusetts General Hospital, HemaCare) using Miltenyi CD4 Microbeads (Catalog number 130-045-101). Cells were frozen down once isolated in Cryotstor CS10 cell freezing medium (STEMCELL, Catalog #: 07930) and stored in liquid nitrogen for future use. 250,000 cells were thawed, pelleted, resuspended in buffer P2 (Lonza), and aliquoted into 96 well nucleofection plates at 20 μl per well. 2 μl of individual test RNPs (as described above) were added to each well. The cells were nucleofected using the Lonza 4D-Nucleofector (AAF-1002B 4D-Nucleofector Core unit, 96-well Shuttle™ Device) using the CA-137 program at an RNP dose of 5 μM. After nucleofection, the cells were incubated at room temperature for 5-10 min. Then 30 μl of complete expansion medium (see Media Components Table (Table 6) for additional details) was added, and the cell mix transferred to a 96-well plate containing 100 μl pre-warmed complete expansion medium. The cell plates were incubated at 37° C. 5% CO2 and collected 96 hours post nucleofection. Genomic DNA was isolated using the Agencourt DNAdvance kit (Beckman Coulter) according to manufacturer's instructions and quantified using Qubit fluorometric quantitation (ThermoFisher).
For Illumina amplicon sequencing, two rounds of amplification were performed: round 1 targets the off-target region, and round 2 adds the full-length Illumina adapter sequence. Round 1 was performed in a 12 μL reaction volume, consisting of 6 μL of NEBNext® Ultra™ II Q5@ Master Mix (New England Biolabs), 0.5 μM total primers (0.25 μM forward and 0.25 μM reverse), nuclease free water, and 20 ng of gDNA template. PCR conditions were as follows: 30 s at 98° C. for initial denaturation, followed by 20 cycles of 10s at 98° C. for denaturation, 15 s at 60° C. for annealing, 30s at 72° C. for extension, and 5 min at 72° C. for the final extension. Four μl of round 1 PCR product was added directly into the 12 μl round 2 reaction volume, consisting of 6 μL of NEBNext® Ultra™ II Q5@ Master Mix (New England Biolabs), 2 μM total primers (1 μM forward and 1 μM reverse), and nuclease free water. PCR conditions were as follows: 30 s at 98° C. for initial denaturation, followed by 14 cycles of 10s at 98° C. for denaturation, 15 s at 60° C. for annealing, 30s at 72° C. for extension, and 5 min at 72° C. for the final extension. Equal volumes of each PCR product was pooled and purified using (0.9×) Agencourt AMPure XP beads (Beckman Coulter Agencourt AMPure XP—PCR Purification #A63882) as per the manufacturer's protocol followed by size 300-1200 bp size selection on the BluePippin (Sage Science, Beverly, MA) and loaded on the Illumina MiSeq with 10-20% phiX.
Analysis of indel rates at the predicted cut site (e.g., editing rates) was done as described in Bothmer, et. al, Nat. Communications, 8, 13905 (2017), but reads were aligned to reference sequences using bowtie2, version 2.1.0, instead of needle as in Bothmer et al. Table 4 shows “tiers” of values for measurements of average indel fraction windows and results of the analysis described herein are shown in Table 5.
In the present example, recombinant AAV (rAAV) viral particles comprising constructs as described herein were produced and transduction efficiencies and transcriptional intensities in human explant Dorsal Root Ganglion (DRG) neurons, nociceptor, and non-neuronal cell types were determined.
rAAV Particles
rAAV particles of the AAV5, and AAV9 serotypes, which included a CMV driven eGFP encoding nucleotide sequence, were obtained from Asklepios BioPharmaceutical, Inc. and/or SIRION Biotech International Inc.
Approximately 75 DRG explants were created from human donor DRGs and were randomized across test conditions. Approximately 24 hr after explants were generated, all media was removed from the explants and fresh DRG media containing rAAV at 3×109, 3×1010, or 3×1011 VG/ml was added to each explant, and the plate containing explants was returned to a 37° C. incubator. Approximately 24 hr after infection, 300 μl of fresh DRG media was added to each well. Approximately 72 hr after infection, a half media change was performed with fresh DRG media (300 μl media removed, 300 μl of media added). Additional half media changes were performed three times per week until explants were processed for histology and/or RNA isolation. Alternatively, for bloody explants, initial transduction events were performed 96 hr after explant generation to facilitate an additional media change prior to transduction to wash out the blood. In general, transduction typically occurred 24-48 hr following sectioning.
Explants were cultured for four weeks before formalin-fixed paraffin-embedding (FFPE) fixation using standard methods known in the art. In brief, 1) explants were transferred to 1.7 ml microcentrifuge tubes containing 1 ml 4% PFA in PBS; 2) explants were incubated for 1 hr at room temperature; 3) supernatant was removed and explants were washed with 1 ml PBS for 1 min; 4) supernatant was removed and 1 ml 25% ethanol was added, samples were incubated for 30 minutes; 5) supernatant was removed, and 1 ml 50% ethanol was added, samples were incubated for 30 minutes; 6) supernatant was removed, and 1 ml 70% ethanol was added, samples were incubated for 30 minutes; if sample solution was cloudy, the supernatant was replaced with a new aliquot of 70% ethanol; 7) samples were stored at 4C until additional processing; 8) samples were embedded in paraffin, followed by 5 μm sectioning and slide creation in preparation for histological analysis.
Sections were stained with an anti-TUJ1 (neuron-specific class III P-tubulin) antibody and an anti-peripherin (class-III neuronal intermediate filament protein) antibody. Three slides were created for each explant, with a total of 24 sections per condition, leading to approximately 200 slides for detection and quantification of neuronal and/or nociceptor transduction efficiency and translational intensity determination.
In the present example, parameters for ex-vivo intra-DRG injection of AAV constructs into human and Rhesus macaque whole DRG were assessed.
Whole human donor DRG segments (left and right for L3 through S2) were obtained from vendor Donor NetWork West, at a postmortem interval of <6-8 hr, with a total time from extraction to dissection of <18 hr. DRG segments were cultured and subjected to ex-vivo injection of AAV5/9-CMV-eGFP (3×1012 VG/ml) in 1×PBS buffer with 0.014% tween20 and 0.1% Evans Blue dye. Injection rates varied from ˜0.5 μl/min to ˜2.5 μl/min, with grossly non-physiologically damaging injection volumes ranging from ˜10 μl to ˜50 μl.
Whole NHP (Rhesus macaque) DRG segments (Cauda equine) were subjected to ex-vivo injection of vehicle 1×PBS buffer with 0.014% tween20 and 0.1% Evans Blue dye. Injection rates varied from ˜0.5 μl/min to ˜2.0 μl/min, with grossly non-physiologically damaging injection volumes ranging from ˜10 μl to ˜20 μl.
The series of ex-vivo injected human DRG segments revealed an optimal injection volume of ˜45 μl administered at ˜1 μl/min when delivered to the human DRG left and/or right L4 or L5 segment. Additionally, for NHP DRG injections, the optimal volume to be administered was revealed as ˜10 μl delivered at ˜1 μl/min. These results suggest that intra-DRG in-vivo injection between the L3-L4 segment is feasible (as depicted in
In the present example, the editing efficacy of guide RNA sequences for endonuclease induced SCN9A and SCN10A gene modification were determined utilizing rAAV particles comprising constructs as described herein.
rAAV particles of the AAV5 serotype comprising CMV driven Cas9-encoding nucleotide sequence and either nucleotide sequences encoding gRNAs of SEQ ID NOs: 5 and 189 (“VIR-A”, targeting SCN9A and SCN10A, respectively), or nucleotide sequences encoding gRNAs of SEQ ID NOs: 7 and 182 (“VIR-B”, targeting SCN9A and SCN10A, respectively), were produced using standard methods known in the art. DRG explants were produced from seven human donor DRGs. Approximately 209 samples were created, each sample comprising two explants. DRG explants were randomized across test conditions. Approximately 24 hours following explant production, samples were infected with rAAV vectors. Explants were infected using 300 μl of media with a final concentration of about 3×109, 3×1010, or 3×1011 VG/ml. Explants were maintained as described above for four weeks before DNA and RNA harvesting for analysis using ddPCR and sequencing, targeted RNA sequencing, drop of RTddPCR, and NMD RTddPCR.
Premixed PrimeTime qPCR assays were ordered from Integrated DNA technologies Inc. (IDT). Assays were custom ordered for 3.6:1 primer:probe ratios, and were supplied at 20×. A total of 350ng RNA per sample was utilized for quantification. qPCR reaction mixtures were created according to manufacturer specifications. TRPV1 was utilized as an internal control for qPCR amplification normalization. Droplets were generated using AutoDG prior to combined RT-PCR and amplification PCR conducted in Veriti thermocyclers using standard methods. Following amplification, plates were loaded into a QX200 droplet reader and quantification was performed using the QuantSoft™ software. RNA sequencing was performed using standard methods.
As depicted in
Depicted in
In the present example, molecular and physiological phenotype changes induced by delivery of rAAV constructs are determined in-vivo in rat models. rAAV particles of the AAV5 serotype and comprising CMV driven Cas9 and GFP nucleotide sequences are produced using standard methods known in the art. Additional rAAV constructs include CMV driven Cas9 nucleotide sequence and nucleotide sequences encoding gRNAs targeting SCN9A and SCN10A as described herein.
Adult animals undergo 10p intrathecal injection of rAAV particles (3×1010VG/ml). At day 35 post-injection, each animal's general physiological well-being and coordination are analyzed using the rotarod assessment. At day 41 post-injection, animals undergo surgical incision (brennan model). At day 42 post-injection, animals undergo behavioral analysis (tactile allodynia—Electronic von Frey Test; tactile hyperalgesia—Pinchmeter Test (Randall-Selitto); and thermal hyperalgesia—plantar test (Hargreaves)).
Following the live portion of the experiment, animals are euthanized and DRG segments (L3-L6) are harvested for molecular characterization. Editing percentages are determined utilizing RT-ddPCR; nonsense-mediated-decay (NMD) levels are determined utilizing RT-ddPCR; and alternative NaV channel transcriptional compensation is determined utilizing RT-qPCR.
Treatment with VIR particles comprising nucleotide sequences encoding gRNAs targeting SCN9A and SCN10A as described herein result in alterations to the tactile allodynia response in animals injected with test AAV particles when compared to vehicle or control AAV, Treatment with VIR particles comprising nucleotide sequences encoding gRNAs targeting SCN9A and SCN10A as described herein result in increases in pain threshold at 6-weeks post injection when compared to vehicle or control AAV injection, as determined by One-way ANOVA with Dunnett's/Tukey's multiple comparison test. Treatment with VIR particles comprising nucleotide sequences encoding gRNAs targeting SCN9A and SCN10A as described herein result in murine tactile hyperalgesia assay alterations (pain in response to a robust stimuli) when compared to vehicle or control AAV injection as determined using the pinchmeter test (Randall-Selitto).
In the present example, molecular and physiological phenotype changes induced by delivery of rAAV particles comprising constructs as described herein are determined in-vivo in non-human primate (NHP) models.
Prior to injection, adult Cynomolgus animals undergo functional observational batteries (FOBs) to identify baseline physiological characteristics. FOBs are performed prior to, during, and at the immediate terminus of the live portion of the experiment and include but are not limited to assays to determine: body temperature, reflexes (Chaddock and Baniski), movement, posture, gait, proprioception, paresis, ataxia, dysmetria, slope assessment and/or nerve conduction velocity.
Adult Cynomolgus animals are injected with a single-unilateral intra-DRG injection at the L4 and/or L5 segment as depicted diagrammatically in
Identifying AAV with Ideal Tropism and Biodistribution Characteristics In-Vivo
At the termination of the living stage of the assay, necroscopy is performed to facilitate molecular characterization of tissues of interest. Tissues such as the DRGs (left and right for L4, L5, and S1) spinal cord (L1-L5), and spinal nerve root (L5) are collected and samples are flash frozen and stored at −80° C. for non-histological characterization, and/or fixed and stored for later histological characterization using standard methodologies as are known in the art (e.g., samples are fixed in approximately 4% PFA or approximately 10% formalin at 4° C. for 24 hrs, and are then transferred to 70% EtOH for storage at ambient temperature). Additional tissues of interest (e.g., thoracic segments T1-12, and sacral segments S1-S5, liver biopsies, heart biopsies, brain stem biopsies, brain biopsies, peripheral nerve bundles L4-L5 left/right, and/or dermatome biopsies L4-L5 left/right) are flash-frozen using liquid nitrogen and retained at −80° C. for non-histological characterization.
Fixed tissues are histologically analyzed using various staining assays as known in the art. For example, histological assays may comprise but are not limited to: Cas9 Immunohistochemistry to determine AAV tropism within DRGs in-vivo, Haemotoxylin and Eosin (H&E) staining to detect morphological damage, Neuron-specific class III beta-tubulin (Tuj 1) and Neuronal nuclei antigen (NeuN, also known as RNA binding protein fox-1 homolog 3) staining to determine neuronal cell body toxicity, Platelet endothelial cell adhesion molecule (PECAMI, also known as CD31) staining to determine and detect damage to vascular morphology, density, and/or distribution, Cleaved Caspase 3 (CASP3) staining to detect cell death and/or stress, Ionized calcium binding adapter molecule 1 (iba1) staining to determine and detect activated microglia, T-cell surface glycoprotein CD3 epsilon (CD3) staining to characterize mononuclear cells of the T-lineage, and/or B-lymphocyte antigen CD20 (CD20, also known as MS4A1) staining to characterize mononuclear cells of the B-lineage.
Frozen tissue samples are characterized by DNA and RNA isolation followed by AAV biodistribution analysis utilizing Cas9 mRNA presence as measured by qPCR as a proxy.
All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein.
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
This application claims the benefit of U.S. Provisional Application No. 63/233,445, filed Aug. 16, 2021, the contents of which are hereby incorporated herein by reference in their entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US22/40320 | 8/15/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63233445 | Aug 2021 | US |
