Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
  • C12N15/90—Stable introduction of foreign DNA into chromosome
  • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
  • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/04—Immunostimulants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
  • C12N2750/00011—Details
  • C12N2750/14011—Parvoviridae
  • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
  • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
  • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
  • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]

Definitions

• Alternatives herein relate generally to gene editing in primary human hematopoietic stem cells. More particularly, alternatives herein relate to nucleic acids and vectors that are configured to provide efficient homology directed repair of genes, and methods of repairing genetic deficiencies, such as X-linked recessive disorders.
• WAS Wiskott-Aldrich syndrome
• WAS Wiskott-Aldrich syndrome
• XLT X-linked thrombocytopenia
• XLT primarily affects the circulatory system by reducing the platelet counts in the blood, and also reducing the platelet size compared to normal or healthy individuals, which compromises the clotting process.
• XLT is caused by a mutation in the WAS gene, resulting in decreased, absent, or altered WASp.
• WASp is an activator of the actin nucleator Arp2/3 complex in vitro and is expressed exclusively in hematopoietic cells. WASp is believed to serve as a key integrator between surface receptors and the cytoskeleton of leukocytes.
• Some alternatives relate to therapeutic approaches designed to correct or repair the endogenous WAS locus in autologous hematopoietic stem cells. Some alternatives include compositions and methods, which comprise transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas that are configured to edit the WAS locus in primary human hematopoietic cells. Some alternatives also relate to co-delivery of a nuclease and an AAV donor for modifying endogenous WAS locus in primary human hematopoietic cells.
• TALENs transcription activator-like effector nucleases
• CRISPR clustered regularly interspaced short palindromic repeats
• nucleic acid for homology directed repair (HDR) of the Wiskott-Aldrich Syndrome (WAS) gene comprises a first sequence encoding a WAS gene, a second sequence encoding one or more guide RNA cleavage sites, and a third sequence encoding one or more nuclease binding sites.
• WAS gene comprises the nucleic acid sequence set forth in SEQ ID NO: 4.
• the second sequence comprises the nucleic acid sequence set forth in SEQ ID NO: 17.
• the one or more nuclease binding sites comprises a forward and reverse transcription activator-like effector nuclease (TALEN) binding site.
• the one or more nuclease binding sites is a clustered regularly interspaced short palindromic repeats (CRISPR) associated protein 9 (Cas9) binding site.
• the nucleic acid further comprises one or more enhancer elements.
• the nucleic acid further comprises homology arm sequences.
• the nucleic acid further comprises a nucleic acid sequence encoding a promoter.
• the vector comprises a first sequence encoding a WAS gene, a second sequence encoding one or more guide RNA cleavage sites, and a third sequence encoding one or more nuclease binding sites.
• the WAS gene comprises the nucleic acid sequence set forth in SEQ ID NO: 4.
• the second sequence comprises the nucleic acid sequence set forth in SEQ ID NO: 17.
• the one or more nuclease binding sites comprises a forward and reverse transcription activator-like effector nuclease (TALEN) binding site.
• the one or more nucleic binding sites is a clustered regularly interspaced short palindromic repeats (CRISPR) associated protein 9 (Cas9) binding site.
• the vector further comprises one or more enhancer elements.
• the vector is an adeno-associated viral vector (AAV).
• the vector is a self- complementary AAV (scAAV).
• the cell is a human cell.
• the cell is a primary cell.
• the cell is an autologous cell.
• the cell is a T cell.
• the cell is a hematopoietic stem cell (HSC).
• the cell is a CD34 + HSC.
• the system comprises a vector and a nucleic acid encoding a nuclease.
• the nuclease is a TALEN nuclease.
• the nuclease is a Cas nuclease.
• the vector and nucleic acid are configured for co-delivery to the cell.
• co-delivery to the cell modifies endogenous WAS locus.
• the cell is a primary human hematopoietic cell.
• the cell comprises a nucleic acid.
• the nucleic acid comprises a first sequence encoding a WAS gene, a second sequence encoding a promoter, a third sequence encoding one or more guide RNA cleavage sites, and a fourth sequence encoding one or more nuclease binding sites.
• the nucleic acid is in a vector.
• the vector is an AAV.
• the vector is a scAAV.
• the cell is a human cell.
• the cell is a primary cell.
• the cell is an autologous cell.
• the cell is a T cell.
• the cell is a HSC.
• the cell is a CD34 + HSC.
• Some alternatives relate to methods of promoting HDR of a WAS gene in a subject in need thereof (e.g., a subject identified or selected as one that would receive a benefit from HDR of a WAS gene, such as a subject having XLT).
• the method comprises administering to a subject in need thereof a cell as described herein or a vector as described herein and administering to the subject a nuclease.
• the nuclease is a TALEN nuclease.
• the nuclease is a Cas nuclease.
• the nuclease is co-administered to the subject with the cell or with the vector.
• the cell is from the subject. In some alternatives, the cell is genetically modified by introducing a nucleic acid as described herein or a vector as described herein into the cell. In some alternatives, the administering is performed by adoptive cell transfer. In some alternatives, the cell is a human cell. In some alternatives, the cell is a primary cell. In some alternatives, the cell is an autologous cell. In some alternatives, the cell is a T cell. In some alternatives, the cell is a HSC. In some alternatives, the cell is a CD34 + HSC. In some alternatives, the subject is male. In some alternatives, the subject is identified or selected as one that is suffering from Wiskott-Aldrich syndrome (WAS). In some alternatives, the subject is identified or selected as one that is suffering from X-linked thrombocytopenia (XLT).
• WAS Wiskott-Aldrich syndrome
• XLT X-linked thrombocytopenia
• Some alternatives provided herein relate to a method of treating, inhibiting, or ameliorating WAS and/or XLT or disease symptoms associated with WAS and/or XLT in a subject in need thereof.
• the method comprises administering to a subject a cell as described herein or a vector as described herein, administering to the subject a nuclease, and optionally identifying or selecting the subject as one that would benefit from receiving a therapy for WAS and/or XLT or disease symptoms associated with WAS and/or XLT and/or, optionally measuring an improvement in the progression of WAS and/or XLT or an improvement in a disease symptom associated with WAS and/or XLT in said subject.
• the nuclease is a TALEN nuclease. In some alternatives, the nuclease is a CRISPR Cas nuclease. In some alternatives, the nuclease is co-administered to the subject with the cell or with the vector. In some alternatives, the cell is from the subject. In some alternatives, the cell is genetically modified by introducing a nucleic acid as described herein or a vector as described herein into the cell. In some alternatives, the administering is performed by adoptive cell transfer. In some alternatives, the cell is a human cell. In some alternatives, the cell is a primary cell. In some alternatives, the cell is an autologous cell. In some alternatives, the cell is a T cell.
• the cell is a HSC. In some alternatives, the cell is a CD34 " HSC In some alternatives, the subject is male. In some alternatives, the method improves thrombocytopenia. In some alternatives, the method increases platelet counts. In some alternatives, the subject is identified or selected as one that is suffering from Wiskott-Aldrich syndrome (WAS). In some alternatives, the subject is identified or selected as one that is suffering from X-linked thrombocytopenia (XLT).
• WAS Wiskott-Aldrich syndrome
• XLT X-linked thrombocytopenia
• a nucleic acid for homology directed repair (HDR) of Wiskott-Aldrich Syndrome (WAS) gene comprising: a first sequence encoding a WAS gene; a second sequence encoding one or more guide RNA cleavage sites; and a third sequence encoding one or more nuclease binding sites.
• the WAS gene comprises the nucleic acid sequence set forth in SEQ ID NO: 4.
• the second sequence comprises the nucleic acid sequence set forth in SEQ ID NO: 17.
• the one or more nuclease binding sites comprises a forward and reverse transcription activator-like effector nuclease (TALEN) binding site.
• the one or more nucleic binding sites is a clustered regularly interspaced short palindromic repeats (CRISPR) associated protein 9 (Cas9) binding site.
• the nucleic acid further comprises one or more enhancer elements.
• the nucleic acid further comprises homology arm sequences.
• the nucleic acid further comprises a nucleic acid sequence encoding a promoter.
• a vector for promoting HDR of WAS protein (WASp) expression in a cell comprising: a first sequence encoding a WAS gene; a second sequence encoding one or more guide RNA cleavage sites; and a third sequence encoding one or more nuclease binding sites.
• the WAS gene comprises the nucleic acid sequence set forth in SEQ ID NO: 4.
• the second sequence comprises the nucleic acid sequence set forth in SEQ ID NO: 17.
• the one or more nuclease binding sites comprises a forward and reverse transcription activator-like effector nuclease (TALEN) binding site.
• the one or more nucleic binding sites is a clustered regularly interspaced short palindromic repeats (CRISPR) associated protein 9 (Cas9) binding site.
• the vector further comprises one or more enhancer elements.
• the vector is an adeno-associated viral vector (AAV).
• the vector is a self- complementary AAV (scAAV).
• the cell is a human cell.
• the cell is a primary cell.
• the cell is an autologous cell.
• the cell is a T cell.
• the cell is a hematopoietic stem cell (HSC).
• the cell is a CD34+ HSC.
• a system for promoting HDR of WAS protein (WASp) expression in a cell comprising a vector of any one of the alternatives herein and a nucleic acid encoding a nuclease.
• the nuclease is a TALEN nuclease.
• the nuclease is a Cas nuclease.
• the vector and nucleic acid are configured for co-delivery to the cell.
• co- delivery to the cell modifies endogenous WAS locus.
• the cell is a primary human hematopoietic cell.
• a cell for expressing a WASp comprising:a nucleic acid, which comprises: a first sequence encoding a WAS gene; a second sequence encoding a promoter; a third sequence encoding one or more guide RNA cleavage sites; anda fourth sequence encoding one or more nuclease binding sites.
• the nucleic acid is in a vector.
• the vector is an AAV.
• the AAV is a scAAV.
• cell is a human cell.
• the cell is a primary cell.
• the cell is an autologous cell.
• the cell is a T cell.
• the cell is a HSC.
• the cell is a CD34+ HSC.
• a method of promoting HDR of a WAS gene in a subject in need thereof comprising: administering to a subject the cell or a vector of any one of the alternatives herein; and administering to the subject a nuclease.
• the cell comprises: a nucleic acid, which comprises: a first sequence encoding a WAS gene; a second sequence encoding a promoter; a third sequence encoding one or more guide RNA cleavage sites; and a fourth sequence encoding one or more nuclease binding sites.
• the vector comprises: a first sequence encoding a WAS gene; a second sequence encoding one or more guide RNA cleavage sites; and a third sequence encoding one or more nuclease binding sites.
• the WAS gene comprises the nucleic acid sequence set forth in SEQ ID NO: 4.
• the second sequence comprises the nucleic acid sequence set forth in SEQ ID NO: 17.
• the one or more nuclease binding sites comprises a forward and reverse transcription activator-like effector nuclease (TALEN) binding site.
• the one or more nucleic binding sites is a clustered regularly interspaced short palindromic repeats (CRISPR) associated protein 9 (Cas9) binding site.
• CRISPR clustered regularly interspaced short palindromic repeats
• the vector further comprises one or more enhancer elements.
• the vector is an adeno-associated viral vector (AAV).
• the vector is a self-complementary AAV (scAAV).
• the cell is a human cell.
• the cell is a primary cell.
• the cell is an autologous cell.
• the cell is a T cell.
• the cell is a hematopoietic stem cell (HSC).
• the cell is a CD34+ HSC.
• the nucleic acid is in a vector.
• the vector is an AAV.
• the AAV is a scAAV.
• cell is a human cell. In some alternatives, the cell is a primary cell. In some alternatives, the cell is an autologous cell. In some alternatives, the cell is a T cell. In some alternatives, the cell is a HSC. In some alternatives, the cell is a CD34+ HSC In some alternatives, the nuclease is a TALEN nuclease. In some alternatives, the nuclease is a Cas nuclease. In some alternatives, the nuclease is co-administered to the subject with the cell or with the vector. In some alternatives, the cell is from the subject and, wherein the cell is genetically modified by introducing the nucleic acid or the vector of any one of the alternatives described herein into the cell.
• the administering is performed by adoptive cell transfer.
• the cell is a human cell.
• the cell is a primary cell.
• the cell is an autologous cell.
• the cell is a T cell.
• the cell is a HSC.
• the cell is a CD34+ HSC.
• the subject is male.
• the subject is suffering from Wiskott- Aldrich syndrome (WAS).
• the subject is suffering from X-linked thrombocytopenia (XLT).
• a method of treating, inhibiting, or ameliorating WAS and/or XLT or disease symptoms associated with WAS and/or XLT in a subject in need thereof comprising: administering to a subject the cell or a vector of any one of the alternatives described herein; administering to the subject a nuclease; andoptionally identifying the subject as one that would benefit from receiving a therapy for WAS and/or XLT or disease symptoms associated with WAS and/or XLT and/or, optionally measuring an improvement in the progression of WAS and/or XLT or an improvement in a disease symptom associated with WAS and/or XLT in said subject.
• the cell comprises: a nucleic acid, which comprises: a first sequence encoding a WAS gene; a second sequence encoding a promoter; a third sequence encoding one or more guide RNA cleavage sites; and a fourth sequence encoding one or more nuclease binding sites.
• the vector comprises: a first sequence encoding a WAS gene; a second sequence encoding one or more guide RNA cleavage sites; and a third sequence encoding one or more nuclease binding sites.
• the WAS gene comprises the nucleic acid sequence set forth in SEQ ID NO: 4.
• the second sequence comprises the nucleic acid sequence set forth in SEQ ID NO: 17.
• the one or more nuclease binding sites comprises a forward and reverse transcription activator-like effector nuclease (TALEN) binding site.
• the one or more nucleic binding sites is a clustered regularly interspaced short palindromic repeats (CRISPR) associated protein 9 (Cas9) binding site.
• the vector further comprises one or more enhancer elements.
• the vector is an adeno-associated viral vector (AAV).
• the vector is a self-complementary AAV (scAAV).
• the nuclease is a TALEN nuclease
• the nuclease is a CRISPR Cas nuclease
• the nuclease is co-administered to the subject with the cell or with the vector
• the cell is from the subject, wherein the cell is genetically modified by introducing the nucleic acid or the vector of any one of the alternatives described herein into the cell.
• the administering is performed by adoptive cell transfer.
• the cell is a human cell.
• the cell is a primary cell.
• the cell is an autologous cell.
• the cell is a T cell.
• the cell is a HSC.
• the cell is a CD34+ HSC.
• the subject is male.
• the method improves thrombocytopenia.
• the method increases platelet counts.
• Figures 1A and IB depict the assessment of cleavage efficiency of TALENs and guide RNAs in primary T cells.
• Figure 1A shows a schematic of the WAS locus with guides and TALENs annotated. Shown are the location of the WAS TALENs (TALEN #1 (Tl) and TALEN #2 (T2)) and guide RNA (Gl, G2, G3, and G4) cleavage sites within the human WAS gene. Scheme not drawn to scale.
• Figure IB depicts the disruption of the WAS locus with TALENs and CRISPR guides.
• Figure 2 depicts human primary T cell editing with TALENs and rationally designed AAV donor templates.
• Figure 2A shows a schematic representation of the WAS locus and AAV donor templates used in targeting with WAS TALENs. Small boxes represent TALEN forward (T-for) and reverse (T-rev) binding sites. Homology arms are also depicted.
• X represents mutation of the T preceding the TALEN binding site designed to abolish cleavage by TALEN in #1244 vector.
• the #1262 vector has the entire region between the exon 1 up to the reverse TALEN binding site deleted.
• Figure 2B depicts a timeline of gene editing procedure beginning with bead stimulation of primary T cells. AAVs were added at 20% of culture volume.
• Figures 2C and 2D depict time course of GFP expression indicative of HDR ( Figure 2C) and cell viability ( Figure 2D).
• Figure 2E shows cells that were also transfected with TALENs and transduced with an AAV vector with an MND promoter driven blue fluorescent protein (BFP) without any homology arms. Fluorescence from this vector at day 15 is indicative of random integration of the vector. Fluorescence from this vector at Day 15 is indicative of random integration of the vector.
• Figure 2F shows representative FACS plots with GFP expression at day 15 post co-delivery of TALEN mRNA and AAV donor templates.
• Figure 2H shows results from a test for nuclease specificity utilizing AAV without homology arms.
• n 3 and represents the number of independent experiments performed using cells from 3 different donors. Data are presented as mean ⁇ SEM.
• Figure 21 shows representative FACS plots with GFP expression at day 15 post co-delivery of TALEN mRNA and AAV donor templates.
• N 3 and represents the number of independent experiments performed using cells from three different donors.
• Figure 3 depicts human primary T cell editing with CRISPR and AAV donors.
• Figure 3A shows a schematic of editing of the WAS locus using CRISPR in primary T cells.
• Shown is a schematic illustration of the scAAV guide RNA vector (#1189 (G)), donor template (#1201 (PAM mutated)), and AAV vector containing both guide and donor sequences (#1215 (DTG)).
• FIGS 3B and 3C provide graphs showing time course of % GFP+ cells (Figure 3B) and cell viability (Figure 3C). 10% and 20% represent the % culture volume #1215 AAV added. All other AAVs were added at 10% of culture volume.
• Figure 3D provides graphs depicting BFP expression when Cas9, guide and MND.BFP vector with no homology arms were delivered. 20% represent the % of culture volume AAVs were added at. AAVs were added at 10% of culture volume unless otherwise specified.
• Figure 3H provides representative FACS plots showing GFP expression at day 15.
• Figure 4 depicts the editing mobilized adult CD34 + cells using co- delivery of TALEN and AAV.
• Figure 4A depicts a timeline of gene editing procedure for human mobilized adult CD34 + cells.
• Figures 4B and 4C depicts time course of GFP expression indicative of HDR ( Figure 4B) and viability (Figure 4C).
• Figure 4D depicts representative FACS plots showing GFP expression at Day 5.
• FIG. 5A-G depicts the editing mobilized adult CD34 + cells using co- delivery of TALEN mRNA or CRISPR guide delivered as RNP and AAV donor and shows disruption of the WAS locus in human CD34 + cells using TALENs or RNP.
• Mobilized human CD34 + cells were cultured in SCGM media supplemented with TPO, SCF, FLT3L (100 ng/mL) and IL3 (60 ng/mL) for 48 hours, followed by electroporation using Neon electroporation system with either lug of each TALEN monomer or Ribonucleoprotein complex (RNP) of Cas9 protein and single guide RNA mixed in 1 : 1.2 ratio.
• Figure 5A shows disruption of the WAS locus in human CD34 + cells using TALENs or RNP.
• Mobilized human CD34 + cells were cultured in SCGM media supplemented with TPO, SCF, FLT3L (100 ng/mL) and IL3 (60 ng/mL) for 48 hours, followed by electroporation using Neon electroporation system with either lug of each TALEN monomer mRNA or Ribonucleoprotein complex (RNP) of Cas9 protein and single guide RNA mixed in 1 : 1.2 ratio.
• the sgRNA was purchased from Trilink BioTechnologies and has chemically modified nucleotides at the three terminal positions at 5' and 3' ends. The cells were cultured for 5 days and genomic DNA was extracted.
• FIG. 5B shows results from HDR editing of the WAS locus in CD34 + HSCs using co-delivery of TALEN mRNA or RNP and AAV donor template.
• Figure 5C shows FACS plots depicting GFP expression from Mock, AAV or AAV plus TALEN treated CD34+ cells (top row) or AAV+RNP treated cells (bottom row) 5 days post editing.
• Figure 5D shows the cell viability of the edited cells.
• FIG. 5E shows colony forming unit (CFU) assay for TALEN edited CD34 + cells
• Figure 5F shows the results from a CFU assay for RNP edited CD34 + cells. Briefly, 500 cells from edited or untreated (mock) were plated in duplicate in Methocult H4034 media (Stemcell Technologies), incubated at 37 ° C for 12-14 days and colonies enumerated based on their morphology and GFP expression.
• CFU colony forming unit
• CFU-E Colony forming unit erythroid
• M Macrophage
• GM Granulocyte
• macrophage G
• GEMM Granulocyte
• erythroid macrophage
• n 3 experiments and 2 donors. Data are presented as mean ⁇ SEM.
• Figure 5G shows results from a digital droplet PCR assay or flow cytometry staining for determining HDR.
• Figure 6 shows editing of the WAS locus in CD34 + HSCs using co- delivery of TALEN mRNA RNP and AAV donor template.
• Adult mobilized human CD34 + cells that were cultured in SCGM media as described in Figure 5, followed by electroporation using Neon electroporation system with either TALEN mRNA or RNP complex.
• AAV vector carrying the donor template was added immediately after electroporation.
• Controls included un-manipulated cells (mock) and cells transduced with AAV only without transfection of a nuclease (AAV).
• Figure 6A shows Graphs showing %GFP at day 5 indicative of HDR.
• Figures 6C and 6D show edited and mock cells that were plated one day post editing onto Methocult media for colony formation unit (CFU) assay. Briefly, 500 cells were plated in duplicate in Methocult H4034 media (Stemcell Technologies, incubated at 37°C for 12-14 days and colonies enumerated based on their morphology and GFP expression.
• Figure 6C shows data from a TALEN experiment and Figure 6D from an RNP experiment.
• CFU-E Colony forming unit erythroid
• M Macrophage
• GM Granulocyte
• macrophage G
• Granulocyte G
• GEMM Granulocyte
• erythroid macrophage
• megakaryocyte BFU-E: Burst forming unit erythroid.
• Figure 7 shows engraftment of edited human CD34 + cells into immune deficient NSG mice. 2 million human CD34 + cells untreated, AAV treated or treated with AAV and RNP were injected into immune deficient NSG mice preconditioned with 25mg/kg busulfan. The mice were sacrificed 10 weeks post-transplant and BM was harvested.
• Figure 7A depicts total engraftment of edited cells as defined by expression of human CD45 marker.
• Figure 7B illustrates %GFP+ cells within the engrafted cells.
• Figure 7C depicts FACS plots from representative mice.
• Figure 8 shows the design of AAV vectors expressing cDNA for human codon optimized WAS gene.
• Figure 9A shows data representative of assays for cell viability and %HR in input cells used for engraftment in NSG mice.
• Adult mobilized human CD34 + cells were cultured in SCGM media as previously described with the exception that IL-6 (lOOng/ml) was added instead of IL-3.
• Figure 9B shows the data representing the engraftment of edited cells in bone marrow of transplanted NSG mice.
• Six to 10-week old NSG mice were treated with 25 or 35 mg/kg of BUSULFEX (Henry Schein Inc.) via intraperitoneal injection, diluted 1 : 1 in phosphate-buffered saline.
• Twenty-four hours later 2 x 10 6 mock or gene edited hematopoietic stem cells (cultured as described in Fig 5A) in phosphate-buffered saline were delivered via retro-orbital injection.
• Animals were euthanized 10 to 16 weeks post- transplant, bone marrow and spleens were harvested and analyzed for human cell engraftment.
• Dot plot depicts total engraftment of edited cells as defined by expression of human CD45 marker in BM of sacrificed mice. Dots represent individual mice. Data are presented as mean ⁇ SEM.
• Figure 9C shows data that represent the engraftment of edited cells in bone marrow of NSG mice 16 weeks post transplantation. Representative flow plots of cells harvested from the bone marrow of NSG mice 16 weeks following transplant. On left, bone marrow harvested from mouse transplanted with untreated cells. On right, bone marrow harvested from mouse transplanted with cells treated with AAV plus RNP. Top row, from left to right: hCD45:mCD45 chimerism, human CD45-gated CD33 + and CD19 + staining Bottom row, from left to right: GFP expression among hCD45 + , CD33 + and CD19 + cells.
• Figure 9D shows data that represent the engraftment of edited cells in spleens of transplanted mice.
• Graph depicts total engraftment of edited cells at 10-16 weeks as defined by expression of human CD45 marker in spleens of sacrificed mice. Dots represent individual mice. Data are presented as mean ⁇ SEM.
• Figure 9E shows data that represent the engraftment of edited cells in spleens of NSG Mice. Representative flow plots of cells harvested from the spleens of NSG mice 16 weeks following transplant. On left, spleen harvested from mouse transplanted with untreated cells. On right, spleen harvested from mouse transplanted with cells treated with AAV plus RNP. Top row, from left to right: hCD45:mCD45 chimerism, Human CD45- gated CD33 + and CD19 + staining. Bottom row, from left to right: GFP expression among hCD45 + , CD33 + and CD19 + cells.
• Figure 9F shows data that represent the engraftment of GFP + cells in NSG mice. HDR-editing rate (%GFP + ) among hCD45 + cells recovered from the bone marrow and spleens of NSG mice sacrificed at 10-16 weeks. Data are presented as mean ⁇ SEM.
• Figure 10A shows data that represent the engraftment of edited cells in bone marrow of NSGW41 mice 16 weeks post transplantation.
• Figure 10B shows data that represent the engraftment of edited cells in spleens of NSGW41 mice 16 weeks post transplantation.
• On left spleen harvested from mouse transplanted with untreated cells.
• On right spleen harvested from mouse transplanted with cells treated with AAV plus RNP.
• Top row from left to right: hCD45:mCD45 chimerism, Human CD45-gated CD33 + and CD19 + staining.
• FIG 11A shows data that represent the design of AAV vectors expressing GFP or cDNA for codon optimized WAS gene from endogenous WAS promoter.
• AAV vectors with 0.6kb homology arms flanking either a promoter-less GFP (top) or WAS cDNA (bottom) followed by a shorter WPRE, designated WPRE3 followed by SV40 polyadenylation signal are shown.
• FIG 11B shows data that represent the digital droplet PCR to determine editing rates in WAS targeted promoter-less GFP construct.
• Figure 11C shows data that represent the digital droplet PCR to determine editing rates with WAS targeted promoter-less coWAS expressing AAV vectors.
• “about” is meant a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.
• the terms “comprise(s)” and “comprising” are to be interpreted as having an open-ended meaning. That is, the terms are to be interpreted synonymously with the phrases “having at least” or “including at least.”
• the term “comprising” means that the process comprises at least the recited steps, but may include additional steps.
• the term “comprising” means that the compound, composition or device comprises at least the recited features or components, but may also include additional features or components.
• a "subject” or a “patient” as described herein have their plain and ordinary meaning when read in light of the specification, and may include but is not limited to, for example, an animal that is the object of treatment, observation or experiment.
• “Animal” comprises cold- and warm-blooded vertebrates and invertebrates such as fish, shellfish, reptiles and, in particular, mammals.
• “Mammal” comprises, without limitation, mice, rats, rabbits, guinea pigs, dogs, cats, sheep, goats, cows, horses, primates, such as monkeys, chimpanzees, and apes, and, in particular, humans. In some alternatives, the subject is human.
• Some alternatives disclosed herein relate to selecting a subject or patient in need.
• a patient is selected who is in need of treatment, amelioration, inhibition, progression, or improvement in disease symptoms or who is in need of curative therapy.
• a patient is selected who has symptoms of Wiskott-Aldrich Syndrome (WAS) or X-linked thrombocytopenia (XLT), or who has been diagnosed with WAS or XLT.
• WAS Wiskott-Aldrich Syndrome
• XLT X-linked thrombocytopenia
• treatment has their plain and ordinary meaning when read in light of the specification, and may include but is not limited to, for example, an intervention made in response to a disease, disorder or physiological condition manifested by a subject, particularly a subject suffering from an X- linked disorder, such as WAS or XLT.
• the aim of treatment may include, but is not limited to, one or more of the alleviation or prevention of symptoms, slowing or stopping the progression or worsening of a disease, disorder, or condition, curative treatment of the disease, disorder, or condition, and the remission of the disease, disorder, or condition.
• “treatment” refers to both treatment of the underlying disease or treatment of the disease symptoms.
• treatments reduce, alleviate, ameliorate, or eradicate the symptom(s) of the disease and/or provide curative therapy of the disease.
• adoptive cellular therapy or “adoptive cell transfer,” as described herein, have their plain and ordinary meaning when read in light of the specification, and may include but is not limited to, for example, transfer of cells, most commonly immune- derived cells, back into the same patient or into a new recipient host with the goal of transferring the immunologic functionality and characteristics into the new host.
• adoptive cellular therapy or adoptive cell transfer comprises administering cells for promoting homology directed repair of a WAS gene in a subject.
• nucleic acid and “polynucleotide” are interchangeable and refer to any nucleic acid, whether composed of phosphodiester linkages or modified linkages such as phosphotriester, phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate, carbamate, thioether, bridged phosphoramidate, bridged methylene phosphonate, bridged phosphoramidate, bridged phosphoramidate, bridged methylene phosphonate, phosphorothioate, methylphosphonate, phosphorodithioate, bridged phosphorothioate or sulfone linkages, and combinations of such linkages.
• the terms “nucleic acid” and “polynucleotide” also specifically include nucleic acids composed of bases other than the five biologically occurring bases (adenine, guanine, thymine, cytosine and uracil).
• fusion or “fused” as described herein, have their plain and ordinary meaning when read in light of the specification, and may include but is not limited to, for example, a first nucleic acid linked to a second nucleic acid by a phosphodiester bond, so that a coding sequence at the 3' end of the first nucleic acid is in frame with a coding sequence at the 5' end of the second nucleic acid, and by extension can further refer to a first polypeptide linked by a peptide bond to a second polypeptide at the C- terminus of the first polypeptide.
• a “fused” (or “fusion of a”) nucleic acid or peptide as used herein refers to a configuration of molecules, and does not necessarily involve performing the act of joining two molecules together.
• the fusion of a first nucleic acid to a second nucleic acid can encode a single polypeptide in which a first polypeptide sequence (encoded by the first nucleic acid) is fused to a second polypeptide sequence (encoded by the second nucleic acid).
• the molecule comprising the fused nucleic acids is referred to as a fusion nucleic acid.
• vector as described herein, have their plain and ordinary meaning when read in light of the specification, and may include but is not limited to, for example, a polynucleotide construct, typically a plasmid or a virus, used to transmit genetic material to a host cell.
• Vectors can be, for example, viruses, plasmids, cosmids, or phage.
• a vector as used herein can be composed of either DNA or RNA. In some alternatives, a vector is composed of DNA.
• An "expression vector” is a vector that is capable of directing the expression of a protein encoded by one or more genes carried by the vector when it is present in the appropriate environment. Vectors are preferably capable of autonomous replication.
• an expression vector comprises a transcription promoter, a gene, and a transcription terminator. Gene expression is usually placed under the control of a promoter, and a gene is said to be "operably linked to" the promoter.
• AAV system or “AAV expression system” as described herein, have their plain and ordinary meaning when read in light of the specification, and may include but is not limited to, for example, nucleic acids for expressing at least one transcript-encoding nucleic acid, and which are disposed on one or more AAV vectors.
• activity-dependent expression refers to nucleic acid expression that will be induced upon a change in a particular type of activity of a cell containing the nucleic acid, for example depolarization of the cell.
• the cell is a neuron, and depolarization of the neuron in response to a stimulus induces "activity-dependent" nucleic acid expression.
• an AAV vector includes a sequence as set forth in SEQ ID NOs: 5, 6, 7, 8, 9, 9, 10, 11, 12, 13,14, 15, 21, 22, 23, or 26.
• operably linked is used to describe the connection between regulatory elements and a gene or its coding region.
• gene expression is placed under the control of one or more regulatory elements, for example, without limitation, constitutive or inducible promoters, tissue-specific regulatory elements, and enhancers.
• a gene or coding region is said to be “operably linked to” or “operatively linked to” or “operably associated with” the regulatory elements, meaning that the gene or coding region is controlled or influenced by the regulatory element.
• a promoter is operably linked to a coding sequence if the promoter effects transcription or expression of the coding sequence.
• upstream as described herein, have their plain and ordinary meaning when read in light of the specification, and may include but is not limited to, for example, positions 5' of a location on a polynucleotide, and positions toward the N- terminus of a location on a polypeptide.
• downstream refers to positions 3' of a location on nucleotide, and positions toward the C-terminus of a location on a polypeptide.
• construct as used herein, as described herein, have their plain and ordinary meaning when read in light of the specification, and may include but is not limited to, for example, a recombinant nucleic acid that has been generated for the purpose of the expression of a specific nucleotide sequence(s), or that is to be used in the construction of other recombinant nucleotide sequences.
• promoter is a nucleotide sequence that permits binding of RNA polymerase and directs the transcription of a gene.
• a promoter is located in the 5' non-coding region of a gene, proximal to the transcriptional start site of the gene. Sequence elements within promoters that function in the initiation of transcription are often characterized by consensus nucleotide sequences. Examples of promoters include, but are not limited to, promoters from bacteria, yeast, plants, viruses, and mammals (including humans).
• a promoter can be inducible, repressible, and/or constitutive. Inducible promoters initiate increased levels of transcription from DNA under their control in response to some change in culture conditions, such as a change in temperature.
• the promoter described herein can be a U6 promoter or an MND promoter.
• the term “enhancer” refers to a type of regulatory element that can modulate the efficiency of transcription, regardless of the distance or orientation of the enhancer relative to the start site of transcription.
• variants as described herein, have their plain and ordinary meaning when read in light of the specification, and may include but is not limited to, for example, a polynucleotide (or polypeptide) having a sequence substantially similar to a reference polynucleotide (or polypeptide).
• a variant can have deletions, substitutions, or additions of one or more nucleotides at the 5' end, 3' end, and/or one or more internal sites in comparison to the reference polynucleotide.
• variants and/or differences in sequences between a variant and the reference polynucleotide can be detected using conventional techniques known in the art, for example polymerase chain reaction (PCR) and hybridization techniques.
• variant polynucleotides also include synthetically derived polynucleotides, such as those generated, for example, by using site-directed mutagenesis.
• a variant of a polynucleotide including, but not limited to, a DNA, can have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference polynucleotide as determined by sequence alignment programs known by skilled artisans, or an amount within a range defined by any two of the aforementioned values.
• a variant can have deletions, substitutions, or additions of one or more amino acids in comparison to the reference polypeptide.
• a variant of a polypeptide can have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%), 98%o, 99%) or more sequence identity to the reference polypeptide as determined by sequence alignment programs known by skilled artisans, or an amount within a range defined by any two of the aforementioned values.
• transfection as described herein, have their plain and ordinary meaning when read in light of the specification, and may include but is not limited to, for example, introduction of a nucleic acid into a host cell, such as by contacting the cell with a recombinant AAV vector as described herein.
• transient transfection refers to the introduction of exogenous nucleic acid(s) into a host cell by a method that does not generally result in the integration of the exogenous nucleic into the genome of the transiently transfected host cell.
• the nucleic acid is RNA.
• the nucleic acid is DNA.
• the nucleic acid when the nucleic acid is RNA, the nucleic acid does not generally integrate in the genome of the transiently transfected cell. In some alternatives, when the nucleic acid is DNA, the nucleic acid can integrate in the genome of the transiently transfected cell.
• host cell as described herein, have their plain and ordinary meaning when read in light of the specification, and may include but is not limited to, for example, a cell that is introduced with Cas9-mRNA/AAV-guide RNA according to the present alternatives, as well as, cells that are provided with the systems herein.
• Host cells can be prokaryotic cells or eukaryotic cells. Examples of prokaryotic host cells include, but are not limited to E.
• eukaryotic host cells include, but are not limited to, protozoa, fungi, algae, plant, insect, amphibian, avian and mammalian cells.
• a system for editing at least one target gene in a cell is provided, wherein the cell is a eukaryotic cell.
• the cell is a mammalian cell. In some alternatives, the cell is a human cell. In some alternatives, the cell is a primary cell. In some alternatives, the cell is not a transformed cell. In some alternatives, the cell is a primary lymphocyte. In some alternatives, the cell is a primary lymphocyte, a CD34 + stem cell, a hepatocyte, a cardiomyocyte, a neuron, a glial cell, a muscle cell or an intestinal cell.
• T cell precursors as described herein, have their plain and ordinary meaning when read in light of the specification, and may include but is not limited to, for example, lymphoid precursor cells that can migrate to the thymus and become T cell precursors, which do not express a T cell receptor. All T cells originate from hematopoietic stem cells in the bone marrow. Hematopoietic progenitors (lymphoid progenitor cells) from hematopoietic stem cells populate the thymus and expand by cell division to generate a large population of immature thymocytes.
• lymphoid precursor cells that can migrate to the thymus and become T cell precursors, which do not express a T cell receptor. All T cells originate from hematopoietic stem cells in the bone marrow. Hematopoietic progenitors (lymphoid progenitor cells) from hematopoietic stem cells populate the thymus and expand by cell division to generate a
• CD4XD8 double-negative cells.
• CD4 + CD8 + double-positive thymocytes
• CD4 + CD8 + single-positive thymocytes
• Hematopoietic stem cells or "HSC” as described herein, have their plain and ordinary meaning when read in light of the specification, and may include but is not limited to, for example, precursor cells that can give rise to myeloid cells such as, for example, macrophages, monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets, dendritic cells and lymphoid lineages (such as, for example, T-cells, B-cells, NK-cells).
• precursor cells that can give rise to myeloid cells such as, for example, macrophages, monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets, dendritic cells and lymphoid lineages (such as, for example, T-cells, B-cells, NK-cells).
• HSCs have a heterogeneous population in which three classes of stem cells exist, which are distinguished by their ratio of lymphoid to myeloid progeny in the blood (L/M).
• the cells provided are HSC cells.
• the cell is a primary lymphocyte or a CD34 + stem cell.
• autologous refers to the donor and recipient of the stem cells being the same, for example, the patient or subject is the source of the cells.
• Primary human cells as described herein, are directly cultured from their source organ tissue or blood cells. Compared to immortalized cell lines, primary human cells provide enhanced replication of in vivo. In some alternatives, the cells provided are primary human cells.
• co-delivery as described herein, have their plain and ordinary meaning when read in light of the specification, and may include but is not limited to, for example, delivery of two or more separate chemical entities, whether in vitro or in vivo.
• Co-delivery refers to the simultaneous delivery of separate agents; to the simultaneous delivery of a mixture of agents; as well as to the delivery of one agent followed by delivery of a second agent or additional agents.
• agents that are co-delivered are intended to work in conjunction with each other.
• co- delivery comprises delivery of an mRNA of interest and an AAV vector.
• the term "endonuclease” as described herein, have their plain and ordinary meaning when read in light of the specification, and may include but is not limited to, for example, enzymes that cleave the phosphodiester bond within a polynucleotide chain.
• the polynucleotide may be double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), RNA, double-stranded hybrids of DNA and RNA, and synthetic DNA (for example, containing bases other than A, C, G, and T).
• An endonuclease may cut a polynucleotide symmetrically, leaving “blunt” ends, or in positions that are not directly opposing, creating overhangs, which may be referred to as "sticky ends.”
• the methods and compositions described herein may be applied to cleavage sites generated by endonucleases.
• the system can further provide nucleic acids that encode an endonuclease, including zinc finger nucleases (ZFNs), TAL effector nucleases (TALENs), meganucleases (such as MegaTALs), and CRISPR/Cas9 or a fusion protein comprising a domain of an endonuclease, for example, Cas9, TALEN, or MegaTAL, or one or more portion thereof.
• ZFNs zinc finger nucleases
• TALENs TAL effector nucleases
• MegaTALs meganucleases
• CRISPR/Cas9 or a fusion protein comprising a domain of an endonuclease, for example, Cas9, TALEN, or MegaTAL, or one or more portion thereof.
• TALEN transcription activator-like effector nuclease
• TAL-effector DNA binding domains may be engineered to bind to a desired target and fused to a nuclease domain, such as the Fokl nuclease domain, to derive a TAL effector domain-nuclease fusion protein.
• the methods and systems described herein may be applied to cleavage sites generated by TAL effector nucleases.
• the systems can further comprise a TALEN nuclease or a vector or nucleic acid encoding a TALEN nuclease.
• the method can further comprise providing a nuclease, such as a TALEN nuclease.
• CRISPRs are segments of prokaryotic DNA containing short repetitions of base sequences. Each repetition is followed by short segments of "spacer DNA” from previous exposures to a bacterial virus or plasmid.
• Cas9 CRISPR associated protein 9
• Cas9 is an RNA-guided DNA endonuclease enzyme associated with the CRISPR adaptive immunity system in Streptococcus pyogenes, among other bacteria. S. pyogenes utilizes Cas9 to memorize and later interrogate and cleave foreign DNA, such as invading bacteriophage DNA or plasmid DNA. Cas9 performs this interrogation by unwinding foreign DNA and checking for complementarity to the 20 base pair spacer region of the guide RNA. If the DNA substrate is complementary to the guide RNA, Cas9 cleaves the invading DNA.
• the CRISPR/Cas system as described herein is used for gene editing (adding, disrupting, or changing the sequence of specific genes) and gene regulation.
• the Cas9 protein, a derivative, or fragment thereof and appropriate guide RNAs into a cell, the organism's genome can be cut at any desired location. It can be possible to use CRISPR to build RNA-guided genes capable of altering the genomes of entire populations.
• CRISPR/Cas9 system comprise a target gene, a guide RNA, and a Cas9 endonuclease, derivative, or fragment thereof
• An important aspect of applying CRISPR/Cas9 for gene editing is the need for a system to deliver the guide RNAs efficiently to a wide variety of cell types. This could for example involve delivery of an in vitro generated guide RNA as a nucleic acid (the guide RNA generated by in vitro transcription or chemical synthesis).
• the nucleic acid encoding the guide RNA is rendered nuclease resistant by incorporation of modified bases, such as 2'O-methyl bases.
• the CRISPR/Cas9 system described herein whereby the polynucleotide encoding the Cas9 nuclease or a derivative or functional fragment thereof (for example, a 20 nucleic acid sequence of an mRNA vector with Cas9) is provided with a poly(T) or poly(A) tail of a desired length and prepared in accordance with the teachings described herein, for example, is provided with a guide RNA that comprises one or more modified bases, such as any one or more of the modified bases described herein.
• Exemplary guide RNAs useful with the alternatives described herein which may contain one or more of the modified bases set forth herein.
• the modified guide RNA includes the sequences provided in SEQ ID NO: 10.
• AAV adeno- associated virus
• an important system for expressing guide RNAs in this context is based on the use of adeno- associated virus (AAV) vectors because AAV vectors are able to transduce a wide range of primary cells. AAV vectors do not cause infection and are not known to integrate into the genome. Therefore, the use of AAV vectors has the benefits of being both safe and efficacious.
• AAV adeno- associated virus
• complementary to means that the complementary sequence is homologous to all or one or more portions of a reference polynucleotide sequence.
• the nucleotide sequence "CAT” corresponds to a reference sequence “CAT” and is complementary to a reference sequence "GTA.”
• HDR homology-directed repair
• DSB double-stranded break
• HDR requires nucleotide sequence homology and uses a donor polynucleotide to repair the sequence where the DSB (e.g., within a target DNA sequence) occurred.
• the donor polynucleotide generally has the requisite sequence homology with the sequence flanking the DSB so that the donor polynucleotide can serve as a suitable template for repair.
• HDR results in the transfer of genetic information from, for example, the donor polynucleotide to the DNA target sequence.
• HDR may result in alteration of the DNA target sequence (e.g., insertion, deletion, mutation) if the donor polynucleotide sequence differs from the DNA target sequence and part or all of the donor polynucleotide is incorporated into the DNA target sequence.
• an entire donor polynucleotide, a portion of the donor polynucleotide, or a copy of the donor polynucleotide is integrated at the site of the DNA target sequence.
• a "guide” as described herein have their plain and ordinary meaning when read in light of the specification, and may include but is not limited to, for example, any polynucleotide that site-specifically guides a nuclease to a target nucleic acid sequence.
• a guide comprises RNA, DNA, or combinations of RNA and DNA.
• a "genomic region” is a segment of a chromosome in the genome of a host cell that is present on either side of the target nucleic acid sequence site or, alternatively, also comprises a portion of the target site.
• the homology arms of the donor polynucleotide have sufficient homology to undergo homologous recombination with the corresponding genomic regions.
• the homology arms of the donor polynucleotide share significant sequence homology to the genomic region immediately flanking the target site; it is recognized that the homology arms can be designed to have sufficient homology to genomic regions farther from the target site.
• non-homologous end joining as described herein, have their plain and ordinary meaning when read in light of the specification, and may include but is not limited to, for example, repair of a DSB in DNA by direct ligation of one end of the break to the other end of the break without a requirement for a donor polynucleotide.
• NHEJ is a DNA repair pathway available to cells to repair DNA without the use of a repair template. NHEJ in the absence of a donor polynucleotide often results in nucleotides being randomly inserted or deleted at the site of the DSB.
• cleavage site refers to a sequence that mediates the separation of a first polypeptide that would otherwise be in cis to a second polypeptide. Accordingly, for simplicity, "cleavage,” “cleavage site,” and the like as used herein refer to the separation of any two polypeptides that are encoded by a single polynucleotide in cis. Thus, “cleavage” and “cleavage site,” can, but do not necessarily refer to proteolytic sites and events, and can also refer to other mechanisms for mediating the separation of polypeptides, for example ribosomal skipping.
• label refers to a detectable molecule.
• a number of suitable labels comprise polypeptides.
• label nucleic acid refers to a nucleic acid encoding a label.
• the AAV vector systems comprise a label polynucleotide.
• a promoter such as an MND promoter
• Example labels that are suitable in accordance with alternatives herein include, but are not limited to, green fluorescent protein (GFP), including, for example, Aequoria victoria GFP, Renilla muelleri GFP, Renilla reniformis GFP, Renilla ptilosarcus, blue fluorescent protein (BFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP), or orange fluorescent proteins (OFP).
• GFP green fluorescent protein
• BFP blue fluorescent protein
• YFP yellow fluorescent protein
• RFP red fluorescent protein
• CFP cyan fluorescent protein
• OFFP orange fluorescent proteins
• Additional reporter genes include, but are not limited to neomycin, phosphoro-transferase, chloramphenicol acetyl transferase, thymidine kinase, luciferase, ⁇ -glucuronidase, aminoglycoside, phosphotransferase, hygromycin B, xanthine-guanine phosphoribosyl, luciferases (e.g., renilla, firefly, etc.), DHFR/methotrexate, ⁇ -galactosidase, alkaline phosphatase, turbo and tagRFP, and nuclear targeted versions of any of the aforementioned reporter genes.
• neomycin phosphoro-transferase
• chloramphenicol acetyl transferase thymidine kinase
• luciferase ⁇ -glucuronidase
• aminoglycoside aminoglycoside
• the polypeptide of interest comprises the label itself, for example when production of label in active cells is desired.
• an AAV construct provided herein comprises a U6 promoter driven guide RNA cassette or an MND promoter driven GFP cassette, or both, and wherein the MND promoter driven GFP cassette provides for tracking of AAV transduction efficiency.
• gene expression as described herein, have their plain and ordinary meaning when read in light of the specification, and may include but is not limited to, for example, biosynthesis of a gene product.
• gene expression involves transcription of the structural gene into mRNA and the translation of mRNA into one or more polypeptides.
• a polypeptide is a polymer of amino acid residues joined by peptide bonds, whether produced naturally or synthetically. Polypeptides of less than 10 amino acid residues are commonly referred to as “peptides.” A polypeptide can be considered as a protein.
• a "protein” is a macromolecule comprising one or more polypeptide chains.
• a protein may also comprise non-peptide components, such as carbohydrate groups. Carbohydrates and other non-peptide substituents may be added to a protein by the cell in which the protein is produced, and will vary with the type of cell. Proteins are defined herein in terms of their amino acid backbone structures; substituents such as carbohydrate groups are generally not specified, but may be present nonetheless.
• a system for editing at least one target gene in a cell comprising a first nucleic acid sequence encoding a CRISPR guide RNA, wherein the CRISPR guide RNA is complimentary to at least one target gene in a cell and, wherein said first nucleic acid sequence is present in a vector; said system also comprising a second nucleic acid sequence encoding a Cas9 protein, a third nucleic acid sequence encoding a first adenoviral protein and a fourth nucleic acid sequence encoding a second adenoviral protein.
• the CRISPR guide sequence is defined by SEQ ID NO: 3, 5, 6, 7 and 8.
• the CRISPR guide sequence is defined by SEQ ID NO: 31,32,33,34.
• the disorder is Wiskott- Aldrich Syndrome (WAS).
• WAS Wiskott- Aldrich Syndrome
• WAS is a severe X-linked genetic disorder with an incidence of around 1 in 250,000 live male births.
• WAS is a result of mutation in the WAS gene.
• WAS is characterized by opportunistic viral and bacterial infections due to abnormal lymphocyte function, thrombocytopenia with small platelets, eczema, and increased risk of autoimmune disorders and malignancies.
• the disorder is X-linked thrombocytopenia (XLT). XLT also results from mutations in the WAS gene leading primarily to thrombocytopenia and bleeding.
• XLT X-linked thrombocytopenia
• HCT Hematopoietic cell transplantation
• HLA human leukocyte antigen
• a French/UK group published the outcome of seven WAS patients who underwent GT using the identical LV. Abina et al. JAMA, 2015, 313, 1550-1563; incorporated by reference herein in its entirety. However, similar to data from Aiuti et al., thrombocytopenia improved but platelet counts were not corrected. In summary, to date WAS LV trials have shown sustained viral marking and WASp expression in multiple hematopoietic lineages with partial clinical improvement. However, LV therapy has failed to rescue thrombocytopenia. Further, the long-term response to immunization and infections challenge and risk for autoimmunity remain to be determined.
• hematopoietic stem cells HSCs
• the systems and methods described herein rescue immunologic and functional defects in WAS and XLT and provide a curative therapy.
• WAS gene replacement using viral-based gene therapy has been used to partially correct this disease
• current viral vector approaches have led to increased risk for malignancy (when using gamma-retroviral vectors) or have failed to correct all hematopoietic deficits, including, most notably, the failure to correct thrombocytopenia.
• the systems and methods provided herein for gene editing of the endogenous WAS gene lead to improved long-term disease correction.
• the systems and methods described herein provide several unique opportunities for treatment of WAS and XLT.
• some alternatives described herein relate to editing a gene product, for example, at the WAS locus.
• the edited gene product remains under control of the endogenous promoter and enhancer elements.
• the editing rates are performed at high efficiency in a cell, such as a hematopoietic stem cell.
• systems and methods for achieving high efficiency editing are also provided.
• systems and methods for engrafting edited cells include use of autologous stem cells.
• WAS gene editing uses autologous stem cells.
• the methods and systems provided herein use the endogenous gene promoter/enhancer to control gene expression.
• the gene editing systems and methods described herein use an endogenous gene promoter/enhancer to control gene expression, which leads to endogenous levels of WASp in all relevant cell lineages.
• Gene editing also eliminates the risk for viral vector mutagenesis that is present in all gene replacement strategies and was observed as a serious adverse event in nearly all WAS patients treated with gamma-retroviral vectors. Gene editing also leads to more appropriate levels of WAS protein expression in comparison to current LV vector therapy.
• the published trials in WAS LV therapy utilized a vector containing the WAS minimal promoter, which partially rescued function in some cell types but did not rescue platelet production most likely because the minimal promoter was not sufficiently robust in platelet progenitor cells.
• Some alternatives provided herein relate to unique nuclease reagents based upon either a TALEN or CRISPR Cas9 platform in parallel with AAV based delivery of novel HDR repair templates.
• the combination of a nuclease and an AAV vector achieves high rates of HDR at the WAS locus in human T cells and CD34 + HSCs, leading to sustained gene expression in vitro and in vivo following engraftment in immunodeficient mice.
• the methods and systems provided herein permit introduction of corrective cDNAs into the WAS locus in autologous cells from control subjects of subjects with WAS or XLT, thereby permitting lineage and developmental specific regulation of the functional gene product in vitro and in vivo.
• the methods and systems provided herein permit long-term sustained cell lineage appropriate therapeutic WASp expression without adverse events within the host genome.
• the systems and methods described herein minimize the risk of mutagenesis by targeting integration of a therapeutic cassette into the WAS locus.
• the systems described herein include an AAV donor template for integrating an expression cassette into the first exon of WAS by homology directed repair.
• nuclease platforms targeting the WAS locus including TALENs and CRISPR/Cas9.
• Some alternatives concern methods for using adult mobilized CD34 + cells and co-delivery of either TALEN mRNA or Cas9/gRNA ribonucleoprotein complexes (R Ps) and an AAV donor for targeted integration of a promoter-driven fluorescent marker.
• R Ps Cas9/gRNA ribonucleoprotein complexes
• the methods provided herein achieve efficient homology directed repair rates across multiple donors at an efficiency of 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%), 60%, 70%, 80%, or greater, or an efficiency within a range defined by any two of the aforementioned values for TALEN and an efficiency of 10%, 15%, 20%, 25%, 30%, 35%, 40%), 45%, 50%, 60%, 70%), 80%, or greater, or an efficiency within a range defined by any two of the aforementioned values for RNP.
• the highest levels of cell viability is observed using RNP/ AAV co-delivery.
• edited HSC retain their potential to give rise to multiple lineages in colony forming unit assays.
• the systems provided herein provide long-term engraftment and differentiation potential in immune-deficient mice.
• AAV vectors carrying WAS cDNA restore expression in WAS deficient cells.
• the systems and methods described herein provide therapeutic correction of the disease or a disease symptom in patients. Homology Directed Repair
• HDR Homology directed repair
• a homologous nucleic acid e.g., a sister chromatid or an exogenous nucleic acid
• HDR typically involves a series of steps such as recognition of the break, stabilization of the break, resection, stabilization of single stranded DNA, formation of a DNA crossover intermediate, resolution of the crossover intermediate, and ligation.
• HDR can be used to alter a target sequence and correct (e.g., repair or edit) a mutation in the genome. While not wishing to be bound by theory, it is believed that alteration of the target sequence occurs by HDR with a donor template or template nucleic acid. For example, the donor template or the template nucleic acid provides for alteration of the target position.
• the gene is a WAS gene.
• the method comprises HDR of the WAS gene in human hematopoietic cells.
• the disorder is an X-linked disorder.
• the disorder is WAS.
• the disorder is XLT.
• the method and systems include nuclease-based HDR of the WAS gene.
• the nuclease based HDR comprises a TALEN based nuclease.
• the nuclease based HDR comprises a CRISPR/Cas based nuclease.
• TALEN Transcription activator-like effector-DNA modifying enzyme
• TALENs are made by fusing a TAL-effector domain to a DNA cleavage domain.
• the WAS locus used in targeting with WAS TALENs is co-delivered with an AAV donor.
• the WAS TALEN forward sequence is defined by SEQ ID NO: 27 or SEQ ID NO: 29.
• the WAS TALEN reverse sequence is defined by SEQ ID NO: 28 or SEQ ID NO: 30.
• the AAV donor comprises a GFP cassette under control of an MND promoter.
• the AAV donor has a 1 kb homology arm flanking an MND promoter driven GFP cassette (SEQ ID NO: 35).
• the AAV donor comprises one or more nucleotide mutations to abolish cleavage by TALENs.
• the nucleotide mutation is a mutation of the T preceding the TALEN binding site (SEQ ID NO: 36).
• the AAV donor comprises deletion of the entire region between exon 1 up to the reverse TALEN binding site (SEQ ID NO: 37).
• TALEN nuclease for use in HDR of a gene of interest.
• the TALEN binds to a TALEN binding site in a gene of interest.
• the gene of interest is a WAS gene (SEQ ID NO: 4).
• a WAS TALEN binds to native WAS sequence (SEQ ID NO: 4).
• the WAS locus used in targeting with WAS TALENs comprises the following components from 5' to 3' : upstream homology arm (SEQ ID NO: 11); exon 1, including a guide RNA (SEQ ID NO: 19); T-for (TALEN forward binding site; SEQ ID NO: 15); cleavage site (SEQ ID NO: 17); T-rev (TALEN reverse binding site; SEQ ID NO: 16); exon 2 (SEQ ID NO: 18); and downstream homology arm (SEQ ID NOs: 12, 13, or 14).
• upstream homology arm SEQ ID NO: 11
• exon 1 including a guide RNA (SEQ ID NO: 19)
• T-for TALEN forward binding site; SEQ ID NO: 15
• cleavage site SEQ ID NO: 17
• T-rev TALEN reverse binding site
• SEQ ID NO: 16 exon 2
• downstream homology arm SEQ ID NOs: 12, 13, or 14
• the WAS locus used in targeting with WAS TALENs is co-delivered with an AAV donor.
• the WAS TALEN forward sequence is defined by SEQ ID NO: 1 or SEQ ID NO: 24.
• the WAS TALEN reverse sequence is defined by SEQ ID NO: 2 or SEQ ID NO: 25.
• the AAV donor comprises a GFP cassette under control of an MND promoter.
• the AAV donor has a 1 kb homology arm flanking an MND promoter driven GFP cassette (SEQ ID NO: 5).
• the AAV donor comprises one or more nucleotide mutations to abolish cleavage by TALENs.
• the nucleotide mutation is a mutation of the T preceding the TALEN binding site (SEQ ID NO: 6).
• the AAV donor comprises deletion of the entire region between exon 1 up to the reverse TALEN binding site (SEQ ID NO: 7).
• the WAS TALEN forward sequence is defined by SEQ ID NO: 27 or SEQ ID NO: 29.
• the WAS TALEN reverse sequence is defined by SEQ ID NO: 28 or SEQ ID NO: 30.
• the AAV donor comprises a GFP cassette under control of an MND promoter.
• the AAV donor has a 1 kb homology arm flanking an MND promoter driven GFP cassette (SEQ ID NO: 35).
• the AAV donor comprises one or more nucleotide mutations to abolish cleavage by TALENs.
• the nucleotide mutation is a mutation of the T preceding the TALEN binding site (SEQ ID NO: 36).
• the AAV donor comprises deletion of the entire region between exon 1 up to the reverse TALEN binding site (SEQ ID NO: 37).
• Cas nuclease for use in HDR of a gene of interest.
• the Cas nuclease is a Cas9 nuclease.
• Cas9 is an RNA-guided DNA endonuclease enzyme associated with the CRISPR (Clustered Regularly Interspersed Palindromic Repeats) adaptive immunity system in Streptococcus pyogenes, among other bacteria.
• CRISPR Clustered Regularly Interspersed Palindromic Repeats
• S. pyogenes utilizes Cas9 to memorize and later interrogate and cleave foreign DNA, such as invading bacteriophage DNA or plasmid DNA. Cas9 performs this interrogation by unwinding foreign DNA and checking for if it is complementary to the 20 base pair spacer region of the guide RNA. If the DNA substrate is complementary to the guide RNA, Cas9 cleaves the invading DNA.
• the Cas nuclease is delivered in a complex with a single guide RNA as a ribonucleoprotein complex (RNP).
• the CRISPR guide sequence is defined by SEQ ID NO: 3.
• the RNP is co-delivered with an AAV donor.
• the AAV donor is a self-complementary AAV (scAAV).
• the AAV donor comprises a GFP cassette under control of an MND promoter wherein a protospacer adjacent motif (PAM) site is deleted (SEQ ID NO: 5).
• the AAV donor comprises a U6 promoter driven guide RNA cassette (SEQ ID NO: 8).
• the AAV donor comprises both the donor and guide sequences (SEQ ID NO: 9).
• the AAV donor is an scAAV done including guide sequences, and includes a sequence defined by SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23.
• the Cas nuclease is delivered in a complex with a single guide RNA as a ribonucleoprotein complex (RNP).
• the CRISPR guide sequence is defined by SEQ ID NO: 31,32,33,34.
• the RNP is co- delivered with an AAV donor.
• the AAV donor is a self-complementary AAV (scAAV).
• the AAV donor comprises a GFP cassette under control of an MND promoter wherein a protospacer adjacent motif (PAM) site is deleted (SEQ ID NO: 9).
• the AAV donor comprises a U6 promoter driven guide RNA cassette (SEQ ID NO: 38).
• the AAV donor comprises both the donor and guide sequences (SEQ ID NO: 39).
• the cell is a mammalian cell. In some alternatives, the cell is a human cell. In some alternatives, the cell is an autologous cell. In some alternatives, the cell is a primary cell. In some alternatives, the cell is a lymphocyte. In some alternatives, the cell is not a transformed cell. In some alternatives, the cell is a primary lymphocyte. In some alternatives, the cell is a lymphocyte precursor cell. In some alternatives, the cell is a T cell. In some alternatives, the cell is a hematopoietic cell. In some alternatives, the cell is a CD34 + cell. In some alternatives, the cell is a primary human hematopoietic cell.
• the cell is transformed by co-delivery of a nuclease, such as a TALEN nuclease or Cas nuclease, and an AAV donor template to modify endogenous WAS locus in a cell.
• a nuclease such as a TALEN nuclease or Cas nuclease
• a method of editing a WAS gene in a cell comprises introducing into a cell a first vector that comprises a first nucleic acid sequence encoding a guide RNA, such as a TALEN guide RNA or a CRISPR guide RNA, wherein the guide RNA is complimentary to at least one target gene in said cell, and introducing into said cell a second nucleic acid sequence encoding a nuclease, such as a TALEN nuclease or a Cas nuclease, a derivative, or fragment thereof.
• a cell is provided, wherein the cell is manufactured by the said methods.
• the method comprises selecting or identifying a subject in need thereof.
• a selected or identified subject in need thereof is a subject that presents with symptoms of an X-linked disorder, such as WAS or XLT, or a subject that has been diagnosed with an X-linked disorder, such WAS or XLT.
• Such evaluations can be made clinically or by diagnostic test.
• the method comprises adoptive cellular therapy or adoptive cell transfer of treated cells to a subject in need.
• adoptive cellular therapy or adoptive cell transfer comprises administering cells for promoting homology directed repair of a WAS gene in a subject.
• the method comprises obtaining cells from the subject in need thereof.
• the cells from the subject in need are primary human hematopoietic cells.
• the cells are transformed by co-delivery of a nuclease, such as a TALEN nuclease or a Cas nuclease, and an AAV donor, which modifies the endogenous WAS locus in the cell.
• the method comprises expanding the transformed cells.
• the method comprises selecting transformed cells that have successful modification of the WAS locus in the cell.
• the transformed cells are administered to the patient.
• administration of the transformed cells to the patient comprises administration of autologous cells to the patient.
• administration of the transformed cells to the patient treats, inhibits, or ameliorates symptoms of WAS and/or XLT.
• administration of the transformed cells to the patient treats WAS and/or XLT.
• the method improves thrombocytopenia. In some alternatives, the method increases platelet counts.
• an amount of treated cells is administered to the composition.
• the amount of cells administered is 1 X 10 4 , 2X 10 4 , 3X10 4 , 4X10 4 , 5X10 4 , 6X 10 4 , 7X10 4 , 8X10 4 , 9X 10 4 , 1 X10 5 , 2X10 5 , 3X10 5 , 4X 10 5 , 5X10 5 , 6X10 5 , 7X10 5 , 8X 10 5 , 9X10 5 , 1 X10 6 , 2X 10 6 , 3X10 6 , 4X10 6 , 5X10 6 , 6X 10 6 , 7X10 6 , 8X10 6 , 9X10 6 , 1 X 10 7 , 2X10 7 , 3X10 7 , 4X 10 7 , 5X10 7 , 6X10 7 , 7X10 7 , 8X 10 7 , 9X10 7 , 1 X10 8
• the treated cells are administered to a subject as a co- therapy with an additional therapy that is used to treat the symptoms of the disorder or used to treat the disorder.
• the additional therapy includes immunoglobulin therapy, an antibiotic therapy, corticosteroid therapy, or transfusion therapy.
• Cells prepared by the systems or methods provided herein can be administered directly to a patient for targeted homology directed repair of a WAS locus and for therapeutic or prophylactic applications, for example, for treating, inhibiting, or ameliorating an X-linked disorder, such as WAS or XLT.
• cells are prepared by the systems provided herein.
• a composition is provided, wherein the composition comprises the cell.
• the compositions described herein can be used in methods of treating, preventing, ameliorating, or inhibiting an X- linked disorder, such as WAS or XLT or ameliorating a disease condition or symptom associated with an X-linked disorder, such as WAS or XLT.
• compositions comprising the cells are administered in any suitable manner, and in some alternatives with pharmaceutically acceptable carriers. Suitable methods of administering such compositions comprising the cells are available and well known to those of skill in the art, and, although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route.
• compositions are determined in part by the particular composition being administered, as well as, by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions that are available (see, e.g., Remington's Pharmaceutical Sciences).
• Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
• the disclosed compositions can be administered, for example, by intravenous infusion, orally, topically, intraperitoneally, intravesically or intrathecally.
• the formulations of compounds can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials. Injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.
• the composition to be administered can be formulated for delivery via one or more of the above noted routes.
• FIG. 1A depicts the location of the WAS TALENs (TALEN #1 and TALEN #2) and guide RNAs, termed Gl, G2, G3, and G4 within the human WAS gene.
• IL-2 50 ng/mL
• IL-7 5 ng/mL
• IL-15 5 ng/mL
• Beads were removed and cells rested overnight followed by electroporation using Neon Transfection system with either TALEN mRNA (1 ⁇ g of each RNA monomer) or co-delivery of 1 ⁇ g of Cas9 mRNA and scAAV carrying guide RNA.
• Cells were cultured for 5 more days and genomic DNA was extracted. The region surrounding the cut site was amplified and purified using PCR purification kit. 200 ng of purified PCR product was incubated with T7 endonuclease (NEB), analyzed on a gel and percent disruption quantified using Licor Image Studio Lite software.
• NEB T7 endonuclease
• TALEN Tl and Guide Gl were used in the following examples.
• IL-2 50ng/ml
• IL-7 5ng/ml
• IL-15 5 ng/ml
• Beads were removed and cells rested overnight followed by electroporation using Neon Transfection system with either TALEN mRNA ( ⁇ g of each RNA monomer) or co-delivery of ⁇ g of Cas9 mRNA and scAAV carrying guide RNA.
• Cells were cultured for 5 more days and the genomic DNA was extracted. The region surrounding the cut site was amplified and purified using PCR purification kit.
• TALEN Tl and Guide Gl were used in experiments in subsequent figures.
• a WAS gene was prepared using the TALEN Tl and Guide Gl as described in Example 1, and as shown in Figure 2A.
• Figure 2A depicts the WAS locus and AAV donor templates used in targeting with WAS TALENs.
• the WAS locus shows the TALEN binding sites, represented by T-f (TALEN forward) and T-r (TALEN reverse). Homology arms are also shown.
• the three AAV vectors are represented as vectors #1201, #1244, and #1262.
• AAV vector #1201 has 1 kb of homology arms flanking an MND promoter driven green fluorescent protein (GFP) cassette.
• AAV vector #1244 has couple nucleotide mutations (represented by X) to abolish cleavage by TALENs.
• AAV vector #1262 has the entire region between the exon 1 up to the reverse TALEN binding site deleted.
• DT AAV vector has 1 kb of homology arms flanking an MND promoter driven green fluorescent protein (GFP) cassette.
• GFP green fluorescent protein
• DT-M AAV donor has couple nucleotide mutations (represented by X) to abolish cleavage by TALENs.
• the #DT-D vector has the entire region between the exon 1 up to the reverse TALEN binding site deleted.
• FIG. 2B depicts the timescale for the experimental method.
• Primary human T cells were cultured in T cell growth medium supplemented with IL-2 (50 ng/mL), IL-7 (5 ng/mL), and IL-15 (5 ng/mL) and stimulated using CD3/CD28 beads (Dynabeads, Life Technologies) for 48 hours.
• Primary human T cells were cultured in T cell growth medium supplemented with IL-2 (50ng/ml) , IL-7 (5ng/ml), and IL-15 (5 ng/ml) and stimulated using CD3/CD28 beads (Dynabeads, Life Technologies) for 48 hours.
• FIG. 1 depicts the %GFP at days 2, 8, and 15 at the indicated conditions (+/- mRNA and the various AAV vectors).
• Primary human CD3+ T cells were cultured and bead stimulated. Cells were then transfected with TALEN mRNA and AAV donors added two hours later at 20% of culture volume. Cells were analyzed for GFP expression on Days 2, 8 and 15.
• Figure 2D shows the cell viability under each condition.
• Figure 2E are the results from a test for nuclease specificity utilizing AAV without homology arms. Primary T cells transfected with TALENs and transduced with an AAV vector with an MND promoter driven blue fluorescent protein (BFP) without any homology arms. Fluorescence from this vector at day 15 is indicative of random integration.
• n 3 and represents the number of independent experiments performed using cells from 3 different donors. Data are presented as mean ⁇ SEM.
• Figure 2F shows representative FACS plots with GFP expression at day 15 post co-delivery of TALEN mRNA and AAV donor templates.
• Figure 2G provides additional representative FACS plots showing GFP expression at Day 15.
• N 3 and represents the number of independent experiments performed using cells from three different donors. Shown in Figure 2H are the results from a test for nuclease specificity utilizing AAV without homology arms. Primary T cells transfected with TALENs and transduced with an AAV vector with an MND promoter driven blue fluorescent protein (BFP) without any homology arms. Fluorescence from this vector at day 15 is indicative of random integration.
• BFP blue fluorescent protein
• n 3 and represents the number of independent experiments performed using cells from 3 different donors. Data are presented as mean ⁇ SEM.
• Figure 3A depicts the scAAV guide RNA vector (#1189), donor template (#1201), and AAV vector containing both guide and donor sequences (#1215). Both #1201 and #1215 AAVs have the PAM site mutated to abolish cleavage by guide.
• FIG. 1 Primary human CD3 T cells were cultured as described in Example 1 and transfected with 1 ⁇ g of Cas9 mRNA followed by transduction with either vector #1189 and #1201 or #1215 AAV vectors.
• Figure 3B shows the %GFP at days 2, 8, and 15, and
• Figure 3C shows the cell viability at days 2, 8, and 15 under the specified conditions. 10% and 20% represent the % of culture volume #1215 AAV was added at. All other AAVs were added at 10%) of culture volume.
• Figure 3D depicts BFP expression when Cas9, guide and MND.BFP vector with no homology arms were delivered.
• Figure 3E shows representative FACS plots showing GFP expression at Day 15.
• N 3 and represents the number of independent experiments performed using cells from 3 donors.
• Figure 3F shows the %GFP at days 2, 8, and 15, and Figure 3G depicts BFP expression when Cas9, guide and MND.BFP vector with no homology arms were delivered.
• Examples 2 and 3 show that using the T7 endonuclease assay, frequencies of 85% and 73% were achieved with TALENs and CRISPR/Cas systems, respectively. These examples also show methods that result in 70% homology directed repair (HDR) in T cells when the nucleases were co-delivered with an AAV donor template.
• HDR 70% homology directed repair
• CD34 + cells were transfected as described in Example 2 with TALEN and AAV.
• Figure 4A shows the timeline for the experimental conditions, where the cells were analyzed at days 2, 5, and 8 with flow cytometry.
• Figure 4B shows the %GFP at days 2 and 5, and the cell viability is depicted in Figure 4C.
• Figure 4D shows representative FACS plots showing GFP expression at day 5.
• T cells were transfected with 1 ⁇ g of forward and reverse WAS TALEN. Five days post-transfection, genomic DNA was extracted, endogenous WAS locus and predicted off target loci were amplified and colony sequenced. Off-targets were predicted using Prognos software. As shown in Table 1 below, potential off-target cleavage sites for TALENs identified using the Prognos software were amplified and sequenced, with no evidence of off-target cleavage observed at any of the predicted loci.
• Mobilized human CD34 + cells were cultured in stem cell growth medium (SCGM) supplemented with thrombopoietin (TPO), stem cell factor (SCF), Fms-related tyrosine kinase 3 ligand (FLT-3L) (100 ng/mL) and IL-3 (60 ng/mL) for 48 hours, followed by electroporation using Neon electroporation system with either 1 ⁇ g of each TALEN monomer or ribonucleoprotein complex (RNP) of Cas9 protein and single guide RNA (sgRNA) mixed in 1 : 1.2 ratio.
• SCGM stem cell growth medium
• TPO thrombopoietin
• SCF stem cell factor
• FLT-3L Fms-related tyrosine kinase 3 ligand
• IL-3 60 ng/mL
• the sgRNA was purchased from Trilink BioTechnologies and has chemically modified nucleotides at the three terminal positions at 5' and 3' ends. The cells were cultured for 5 days and genomic DNA was extracted. The region surrounding the cut site for WAS TALEN and guide was amplified and cloned into pJET cloning vector. Colony sequencing was performed to quantify % cleavage at the cut site by analyzing the indels.
• FIG. 5B adult mobilized human CD34 + cells were cultured in SCGM media as described in Figure 5A, followed by electroporation using Neon electroporation system with either TALEN mRNA or RNP complex ⁇ g).
• AAV vector MOI ranging from 62-300 carrying the donor template was added immediately after electroporation.
• FIG. 5C Shown in Figure 5C, are FACS plots depicting GFP expression from Mock, AAV or AAV plus TALEN treated CD34+ cells (top row) or AAV+RNP treated cells (bottom row) 5 days post editing.
• FIG. 5E Shown in Figure 5E, are results from a CFU assay for TALEN edited CD34+ cells.
• TALEN edited and mock cells were plated one day post editing onto Methocult media for colony formation unit (CFU) assay. Briefly, 500 cells were plated in duplicate in Methocult H4034 media (Stemcell Technologies), incubated at 37 ° C for 12-14 days and colonies enumerated based on their morphology and GFP expression.
• CFU colony formation unit
• CFU-E Colony forming unit erythroid
• M Macrophage
• GM Granulocyte
• macrophage G
• Granulocyte G
• GEMM Granulocyte
• erythroid macrophage
• n 3 independent donors. Data are presented as mean ⁇ SEM.
• FIG. 5F Shown in Figure 5F, are results from a CFU assay for RNP edited CD34+ cells.
• RNP edited and mock cells were plated one day post editing onto Methocult media for colony formation unit (CFU) assay. Briefly, 500 cells were plated in duplicate in Methocult H4034 media (Stemcell Technologies), incubated at 37 ° C for 12-14 days and colonies enumerated based on their morphology and GFP expression.
• CFU colony formation unit
• CFU-E Colony forming unit erythroid
• M Macrophage
• GM Granulocyte
• macrophage G
• Granulocyte G
• GEMM Granulocyte
• erythroid macrophage
• megakaryocyte BFU-E: Burst forming unit erythroid.
• n 3 experiments and 2 donors. Data are presented as mean ⁇ SEM.
• FIG. 5G Shown in Figure 5G are results from the digital droplet PCR assay for determining HDR.
• Genomic DNA was isolated from hematopoietic stem and progenitor cells (HSPCs) using a DNeasy Blood and Tissue kit (Qiagen).
• HSPCs hematopoietic stem and progenitor cells
• Qiagen DNeasy Blood and Tissue kit
• "in-out" droplet digital PCR was performed with the forward primer binding within the AAV insert and the reverse primer binding the WAS locus outside the region of homology.
• a control amplicon of similar size was generated for the ActB gene to serve as a control. All reactions were performed in duplicate.
• the PCR reactions were partitioned into droplets using a QX200 Droplet Generator (Bio-Rad).
• Amplification was performed using ddPCR Supermix for Probes without UTP (Bio-Rad), 900nM of primers, 250nM of Probe, 50 ng of genomic DNA, and 1% DMSO. Droplets were analyzed on the QX200 Droplet Digital PCR System (Bio-Rad) using QuantaSoft software (Bio-Rad). Data are presented as mean ⁇ SEM .
• This alternative demonstrates editing of the WAS locus in CD34 + hematopoietic stem cells using co-delivery of TALEN mRNA or RNP and an AAV donor template.
• FIG. 7A depicts total engraftment of edited cells as defined by expression of human CD45 marker.
• Figure 7B Illustrates %GFP+ cells within the engrafted cells.
• Figure 7C shows FACS plots from representative mice.
• FIG. 9A Shown in Figure 9A is the cell viability and %HR in input cells used for engraftment in NSG mice.
• Adult mobilized human CD34+ cells were cultured in SCGM media as previously described with the exception that IL-6 (lOOng/ml) was added instead of IL-3.
• Figure 9B shows the results from engraftment of edited cells in bone marrow of transplanted NSG mice.
• mice Six to 10 week old NSG mice were treated with 25 or 35 mg/kg of BUSULFEX (Henry Schein Inc.) via intraperitoneal injection, diluted 1 : 1 in phosphate-buffered saline. Twenty- four hours later, 2 x 10 6 mock or gene edited hematopoietic stem cells (cultured as described in Figure 9A) in phosphate-buffered saline were delivered via retro-orbital injection. Animals were euthanized 10 to 16 weeks post-transplant, bone marrow and spleens were harvested and analyzed for human cell engraftment. Dot plot depicts total engraftment of edited cells as defined by expression of human CD45 marker in BM of sacrificed mice.
• Figure 9C shows the result of engraftment of edited Cells in bone marrow of NSG mice 16 weeks post transplantation. Representative flow plots of cells harvested from the bone marrow of NSG mice 16 weeks following cell transplantation. On left panel shows the data of bone marrow harvested from mouse transplanted with untreated cells. On right, bone marrow harvested from mouse transplanted with cells treated with AAV plus RNP. On the top row of the figure, from left to right: hCD45:mCD45 chimerism, human CD45-gated CD33+ and CD 19+ staining. Bottom row, from left to right: GFP expression among hCD45+, CD33+ and CD19+ cells.
• Figure 9D Engraftment of edited cells in spleens of transplanted mice.
• Graph depicts total engraftment of edited cells at 10-16 weeks as defined by expression of human CD45 marker in spleens of sacrificed mice. Dots represent individual mice.
• Figure 9E shows results from engraftment of edited cells in spleens of NSG Mice. Representative flow plots of cells harvested from the spleens of NSG mice 16 weeks following cell transplantation are shown. On left, bone marrow harvested from mouse transplanted with untreated cells. On right, bone marrow harvested from mouse transplanted with cells treated with AAV plus RNP.
• FIG. 10A Shown in Figure 10A are the results from engraftment of edited cells in bone marrows of NSGW41 mice. Representative flow plots of cells harvested from the bone marrows of NSGW41 mice 16 weeks following cell transplantation. On left, bone marrow harvested from mouse transplanted with untreated cells. On right, bone marrow harvested from mouse transplanted with cells treated with AAV plus RNP. Top row, from left to right: hCD45 :mCD45 chimerism, Human CD45-gated CD33 + and CD19 + staining. Bottom row, from left to right: GFP expression among hCD45 + , CD33 + and CD19 + cells.
• FIG. 10B Shown in Figure 10B are the results from engraftment of edited cells in spleens of NSGW41 mice. Representative flow plots of cells harvested from the spleens of NSGW41 mice 16 weeks following cell transplantation. On left, bone marrow harvested from mouse transplanted with untreated cells. On right, bone marrow harvested from mouse transplanted with cells treated with AAV plus R P. Top row, from left to right: hCD45 :mCD45 chimerism, Human CD45-gated CD33 + and CD19 + staining. Bottom row, from left to right: GFP expression among hCD45 + , CD33 + and CD19 + cells .
• FIG. 11A shows the design of AAV vectors expressing cDNA for GFP or codon optimized WAS gene from the endogenous promoter.
• WPRE3 a promoterless GFP
• WPRE3 a shorter WPRE3 followed by SV40 polyadenylation signal.
• Figure 11B is the results from the digital droplet PCR to determine editing rates in WAS targeted promoterless GFP construct. In-out droplet digital PCR was performed as previously described. Droplets were analyzed on the QX200 Droplet Digital PCR System (Bio-Rad) using QuantaSoft software (Bio-Rad). All experiments were performed on female donors.
• a nucleic acid for homology directed repair (HDR) of Wiskott-Aldrich Syndrome (WAS) gene comprising: a first sequence encoding a WAS gene; a second sequence encoding one or more guide RNA- cleavage sites; and a third sequence encoding one or more nuclease-binding sites.
• the WAS gene comprises the nucleic acid sequence set forth in SEQ ID NO: 4.
• the second sequence comprises the nucleic acid sequence set forth in SEQ ID NO: 17.
• the one or more nuclease binding sites comprises a forward and reverse transcription activator-like effector nuclease (TALEN) binding site.
• the one or more nucleic binding sites is a clustered regularly interspaced short palindromic repeats (CRISPR) associated protein 9 (Cas9) binding site.
• the nucleic acid further comprises one or more enhancer elements.
• the nucleic acid further comprises homology arm sequences.
• the nucleic acid further comprises nucleic acid sequence encoding a promoter.
• a vector for promoting HDR of WAS protein (WASp) expression in a cell comprising: a first sequence encoding a WAS gene; a second sequence encoding one or more guide RNA-cleavage sites; and a third sequence encoding one or more nuclease-binding sites.
• the WAS gene comprises the nucleic acid sequence set forth in SEQ ID NO: 4.
• the second sequence comprises the nucleic acid sequence set forth in SEQ ID NO: 17.
• the one or more nuclease binding sites comprises a forward and reverse transcription activator-like effector nuclease (TALEN) binding site.
• the one or more nucleic binding sites is a clustered regularly interspaced short palindromic repeats (CRISPR) associated protein 9 (Cas9) binding site.
• the vector further comprises one or more enhancer elements.
• the vector is an adeno-associated viral vector (AAV).
• the vector is a self- complementary AAV (scAAV).
• the cell is a human cell.
• the cell is a primary cell.
• the cell is an autologous cell.
• the cell is a T cell.
• the cell is a hematopoietic stem cell (HSC).
• the cell is a CD34 + HSC.
• a system for promoting HDR of WAS protein (WASp) expression in a cell comprising a vector of any one of the alternatives herein and a nucleic acid encoding a nuclease.
• the vector comprises a first sequence encoding a WAS gene; a second sequence encoding one or more guide RNA-cleavage sites; and a third sequence encoding one or more nuclease- binding sites.
• the WAS gene comprises the nucleic acid sequence set forth in SEQ ID NO: 4.
• the second sequence comprises the nucleic acid sequence set forth in SEQ ID NO: 17.
• the one or more nuclease binding sites comprises a forward and reverse transcription activator-like effector nuclease (TALEN) binding site.
• the one or more nucleic binding sites is a clustered regularly interspaced short palindromic repeats (CRISPR) associated protein 9 (Cas9) binding site.
• the vector further comprises one or more enhancer elements.
• the vector is an adeno-associated viral vector (AAV).
• the vector is a self-complementary AAV (scAAV).
• the cell is a human cell.
• the cell is a primary cell.
• the cell is an autologous cell.
• the cell is a T cell.
• the cell is a hematopoietic stem cell (HSC). In some alternatives, the cell is a CD34 + HSC. In some alternatives of the system, the nuclease is a TALEN nuclease. In some alternatives of the system, the nuclease is a Cas nuclease. In some alternatives of the system, the vector and nucleic acid are configured for co-delivery to the cell. In some alternatives of the system, co- delivery to the cell modifies endogenous WAS locus. In some alternatives of the system, the cell is a primary' human hematopoietic cell.
• a cell for expressing a WASp comprising: a nucleic acid, which comprises: a first sequence encoding a WAS gene; a second sequence encoding a promoter; a third sequence encoding one or more guide RNA cleavage sites; and a fourth sequence encoding one or more nuclease binding sites.
• the nucleic acid is in a vector.
• the vector are an AAV.
• the AAV are a scAAV.
• the cell are a human cell.
• the cell are a primary cell.
• the cell are an autologous cell.
• the cell are a T cell.
• the cell are a HSC.
• the cell are a CD34 + HSC.
• a method of promoting HDR of a WAS gene in a subject in need thereof comprising: administering to a subject the cell of any one of alternatives provided herein or a vector of any one of the alternatives provided herein, and administering to the subject a nuclease.
• the nuclease is a TALEN nuclease.
• the nuclease is a Cas nuclease.
• the nuclease is co-administered to the subject with the cell or with the vector.
• the cell is from the subject and, wherein the cell is genetically modified by introducing the nucleic acid of any one of alternatives of any one of the alternatives herein or the vector of any one of the alternatives herein into the cell.
• the administering is performed by adoptive cell transfer.
• the cell is a human cell.
• the cell is a primary cell.
• the cell is an autologous cell.
• the cell is a T cell.
• the cell is a HSC.
• the cell is a CD34 + HSC.
• the subject is male.
• the subject is suffering from Wiskott-Aldrich syndrome (WAS).
• WAS Wiskott-Aldrich syndrome
• the subject is suffering from X-linked thrombocytopenia (XLT).
• a method of treating, inhibiting, or ameliorating WAS and/or XLT or disease symptoms associated with WAS and/or XLT in a subject in need thereof comprising: administering to a subject the cell of any one of the alternatives herein or a vector of any one of the alternatives herein; administering to the subject a nuclease; and optionally identifying the subject as one that would benefit from receiving a therapy for WAS and/or XLT or disease symptoms associated with WAS and/or XLT and/or, optionally measuring an improvement in the progression of WAS and/or XLT or an improvement in a disease symptom associated with WAS and/or XLT in said subject.
• the nuclease is a TALEN nuclease. In some alternatives, the nuclease is a CRISPR/Cas nuclease. In some alternatives, the nuclease is co-administered to the subject with the cell or with the vector. In some alternatives, the cell is from the subject, wherein the cell is genetically modified by introducing the nucleic acid of any one of alternatives 1-8 or the vector of any one of the alternatives herein into the cell. In some alternatives, the administering is performed by adoptive cell transfer. In some alternatives, the cell is a human cell. In some alternatives, the cell is a primary cell. In some alternatives, the cell is an autologous cell. In some alternatives, the cell is a T cell. In some alternatives, the cell is a HSC. In some alternatives, the cell is a CD34 + HSC. In some alternatives, the subject is male. In some alternatives, the method improves thrombocytopenia. In some alternatives, the method increases platelet counts.
• a nucleic acid for homology directed repair (HDR) of Wiskott-Aldrich Syndrome (WAS) gene comprising: a first sequence encoding a WAS gene, a second sequence encoding one or more guide RNA cleavage sites; and a third sequence encoding one or more nuclease binding sites.
• the one or more nuclease binding sites comprises a forward and reverse transcription activator-like effector nuclease (TALEN) binding site.
• the one or more nucleic binding sites is a clustered regularly interspaced short palindromic repeats (CRISPR) associated protein 9 (Cas9) binding site.
• the nucleic acid further comprises one or more enhancer elements, n some alternatives, the nucleic acid further compriseshomology arm sequences, n some alternatives, the nucleic acid further comprisesa nucleic acid sequence encoding a promoter.
• a vector for promoting HDR of WAS protein (WASp) expression in a cell comprising a first sequence encoding a WAS gene, a second sequence encoding one or more guide RNA cleavage sites; and a third sequence encoding one or more nuclease binding sites.
• the one or more nuclease binding sites comprises a forward and reverse transcription activator-like effector nuclease (TALEN) binding site.
• the one or more nucleic binding sites is a clustered regularly interspaced short palindromic repeats (CRISPR) associated protein 9 (Cas9) binding site.
• the vector further comprises one or more enhancer elements.
• the vector is an adeno-associated viral vector (AAV). In some alternatives, the vector is a self-complementary AAV (scAAV). In some alternatives, the cell is a human cell. In some alternatives, the cell is a primary cell. In some alternatives, the cell is an autologous cell. In some alternatives, the cell is a T cell. In some alternatives, the cell is a hematopoietic stem cell (HSC). In some alternatives, the cell is a CD34+ HSC.
• AAV adeno-associated viral vector
• scAAV self-complementary AAV
• the cell is a human cell. In some alternatives, the cell is a primary cell. In some alternatives, the cell is an autologous cell. In some alternatives, the cell is a T cell. In some alternatives, the cell is a hematopoietic stem cell (HSC). In some alternatives, the cell is a CD34+ HSC.
• a system for promoting HDR of WAS protein (WASp) expression in a cell comprising a vector of any one of the alternatives herein and a nucleic acid encoding a nuclease.
• the nuclease is a TALEN nuclease.
• the nuclease is a Cas nuclease.
• the vector and nucleic acid are configured for co-delivery to the cell.
• co-delivery to the cell modifies endogenous WAS locus.
• the cell is a primary human hematopoietic cell.
• a cell for expressing a WASp comprising: a nucleic acid, which comprises a first sequence encoding a WAS gene, a second sequence encoding a promoter, a third sequence encoding one or more guide RNA cleavage sites; and a fourth sequence encoding one or more nuclease binding sites.
• the nucleic acid is in a vector.
• the vector is an AAV.
• the AAV is a scAAV.
• the cell is a human cell.
• the cell is a primary cell.
• the cell is an autologous cell.
• the cell is a T cell.
• the cell is a HSC.
• the cell is a CD34+ HSC.
• a method of promoting HDR of a WAS gene in a subject in need thereof comprising: administering to a subject the cell or a vector of any one of the alternatives herein; and administering to the subject a nuclease.
• the nuclease is a TALEN nuclease.
• the nuclease is a Cas nuclease.
• the nuclease is co-administered to the subject with the cell or with the vector.
• the cell is from the subject and, wherein the cell is genetically modified by introducing the nucleic acid or the vector of any one of the alternatives herein into the cell.
• the administering is performed by adoptive cell transfer.
• the cell is a human cell.
• the cell is a primary cell.
• the cell is an autologous cell.
• the cell is a T cell.
• the cell is a HSC.
• the cell is a CD34+ HSC.
• the subject is male.
• the subject is suffering from Wiskott-Aldrich syndrome (WAS).
• WAS Wiskott-Aldrich syndrome
• XLT X-linked thrombocytopenia
• a method of treating, inhibiting, or ameliorating WAS and/or XLT or disease symptoms associated with WAS and/or XLT in a subject in need thereof comprising: administering to a subject the cell or a vector of any one of the alternatives herein, administering to the subject a nuclease; and optionally identifying the subject as one that would benefit from receiving a therapy for WAS and/or XLT or disease symptoms associated with WAS and/or XLT and/or, optionally measuring an improvement in the progression of WAS and/or XLT or an improvement in a disease symptom associated with WAS and/or XLT in said subject.
• the nuclease is a TALEN nuclease. In some alternatives, the nuclease is a CRISPR/Cas nuclease. In some alternatives, the nuclease is co-administered to the subject with the cell or with the vector. In some alternatives, the cell is from the subject, wherein the cell is genetically modified by introducing the nucleic acid or the vector of any one of the alternatives into the cell. In some alternatives, the administering is performed by adoptive cell transfer. In some alternatives, the cell is a human cell. In some alternatives, the cell is a primary cell. In some alternatives, the cell is an autologous cell. In some alternatives, the cell is a T cell. In some alternatives, the cell is a HSC. In some alternatives, the cell is a CD34+ HSC. In some alternatives, the subject is male. In some alternatives, the method improves thrombocytopenia. In some alternatives, the method increases platelet counts.

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