WO2025217225A1 - Compositions and methods for effective delivery of lipid nanoparticles operably linked to therapeutic molecules to multiple fetal muscle tissues and other organs for the treatment of genetic disorders - Google Patents

Abstract

Compositions and methods are provided for the treatment of genetic disorders are disclosed.

Description

Compositions and Methods for Effective Delivery of Lipid Nanoparticles Operably Linked to Therapeutic Molecules to Multiple Fetal Muscle Tissues and Other Organs for the Treatment of Genetic Disorders

William Peranteau

Kiran Musunuru

Rajan Jain Mohamad-Gabriel Alameh

Cross-reference to Related Application

This application claims priority to US Provisional Application No. 63/575,915 filed April 8, 2024, the entire contents being incorporated herein by reference as though set forth in full.

Statement of Federally Sponsored Research

This invention was made with government support under HL 152427, NS132301 and DK 123049 awarded by the National Institutes of Health. The government has certain rights in the invention.

Incorporation-by-Reference of Material Submitted in Electronic Form

The Contents of the electronic sequence listing (CHOP-153-PCT.xml; Size: 5,278 bytes; and Date of Creation: April 08, 2025) is herein incorporated by reference in its entirety.

Field of the Invention

This invention relates to the fields of gene therapy and lipid nanoparticle (LNP) mediated delivery to multiple targeted tissues and organs. More specifically, the invention provides lipid nanoparticles conjugated to genetic constructs and methods for delivery of the same to heart, diaphragm, skeletal muscle, kidney, and CD34+ hematopoietic progenitor cells for the treatment of disease.

Background of the Invention

Several publications and patent documents are cited throughout the specification in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated by reference herein as though set forth in full. Advances in gene editing technology now provide the possibility of single shot, one-and- done therapies for many genetic diseases. In vivo gene editing requires an effective delivery vehicle to target the organs and cells of interest. Traditionally, viral vectors have been used to delivery CRISPR-Cas 9 technology, including Cas9 for homology directed repair (HDR) and nonhomologous end joining (NHEJ) and modified Cas9 editing approaches such as adenine and cytosine base editing and prime editing. Although beneficial, viral vector delivery is limited by an immune response to the viral vector and, in the case of adeno-associated viral vectors, the limited packaging capacity of the vector. As an alternative, nonviral delivery vectors, including ionizable lipid nanoparticles (LNPs), to deliver mRNA encoding the editing technology and gRNAs, have emerged as a therapeutically relevant delivery approach for in vivo gene editing.

Specifically, many studies in the postnatal mouse model have demonstrated the ability to target the liver in vivo with LNPs and perform efficient gene editing in hepatocytes. Additional studies in the adult nonhuman primate (NHP) model have also demonstrated the ability to successfully edit hepatocytes following in vivo delivery of LNPs containing CRISPR-Ca9 technology, specifically adenine base editor (ABE) mRNA and gRNA. As a result of these preclinical studies, several clinical trials are currently underway in which LNPs are used to deliver CRISPR-Cas9 technology for in vivo editing in adult recipients - including LNP mediated delivery of SpCas9 mRNA and gRNA targeting the TTR gene in the liver as a treatment for transthyretin amyloidosis (Gillmore et al., 2021; Clinical trial: NCT04601051), LNP mediated delivery of SpCas9 mRNA and gRNA targeting the KLKB1 gene in the liver as a treatment for hereditary angioedema (Longhurst et al., 2024; Clinical trial: NCT05120830), and LNP mediated delivery of ABE mRNA and gRNA targeting the PCSK9 gene in the liver as a treatment for familial hypercholesterolemia (Clinical trial: NCT05398029). Although encouraging, these studies have highlighted the inability of LNPs delivered in vivo in adult recipients to efficiently target and instituted targeted edits via CRISPR-Cas9 technology, including base editing, in organs other than the liver. This is particularly true in the NHP model - which is much more rigorous than the murine model and, critically, which best recapitulates expected findings in patients at the time of clinical translation. For example, in the NHP studies in which LNPs containing ABE mRNA and a gRNA targeting the PCSK9 gene were delivered to adult NHP recipients, no editing was noted in the kidney, lymph nodes, testes, epididymis, skeletal muscle, duodenumjejunum, lung, or brain (Musunuru et al., 2021 ; Rothgangl et al., 2021).

Clearly, a need exists in the art for improved compositions and methods for broadening the scope of tissues and organs that can be successfully targeted with gene editing molecules for the treatment of a wide variety of genetic diseases.

Summary of the Invention

In accordance with the present invention, a sterile injectable composition for systemic injection into a vein selected from an umbilical vein, an intrahepatic umbilical vein, or a cephalic vein of a fetal subject in need thereof for treatment of genetic disorder is disclosed. An exemplary composition comprises lipid nanoparticles (LNP) containing ionizable lipid and an RNA of interest which forms a ribonucleoprotein complex upon expression in a target tissue or organ in a fetal subject in need thereof for correction of a genetic mutation associated with disease, said composition effecting correction in a fetal target tissue or fetal organ in utero selected from one or more of heart, diaphragm, skeletal muscle, kidney, and CD34+ hematopoietic progenitor cells in the absence of a viral vector. In certain embodiments, the ionizable lipid is selected from LP01, SM102, Alc315. In yet another embodiment, the LNP comprises ionizable lipid LP01, an amine lipid, DSPC, cholesterol, and a PEG-DMG lipid at an N:P ratio of 6 combined at a final ratio of 51.68:9.0:36.32:3.0, said LNP having a size and poly dispersity of 65nm, and 0.05 (Z-average), respectively. In another embodiment, the LNP comprises ionizable lipid SM102, DSPC, cholesterol and PEG-DMG at the percentages of 50: 10:38.5:1.5 and an N:P ratio of 5.67, said LNP having a size and poly dispersity of 80nm (Z- average), and 0.12 respectively. The ribonucleoprotein complex formed can be an adenine base editor or a cytosine base editor. The ribonucleoprotein can also comprise a guide RNA and form part of a Crispr-Cas complex. In certain aspects, the composition is effective for treatment of a disease selected from hereditary tyrosinemia type 1, Duchenne muscular dystrophy, familial hypercholesterolemia and said genetic mutation is in a gene selected from HPD, dystrophin or PCSK9 respectively.

Also disclosed is method for delivery of an RNA of interest as discussed above to a target tissue or organ in a fetal subject in need thereof for correction of a genetic mutation associated with disease, comprising accessing the umbilical cord of a fetus or fetal intrahepatic vein and administering one or more doses of an effective amount of a pharmaceutical formulation comprising a carrier operably linked to a nucleic acid encoding a gene editing ribonucleoprotein complex under conditions where said formulation enters one or more fetal tissues or organs selected from heart, diaphragm, skeletal muscle, kidney, and CD34+ hematopoietic progenitor cells, and said ribonucleoprotein complex editing corrects said mutation, thereby treating said disease in the absence of a viral vector. In certain approaches the administration occurs after 12 weeks and before 40 weeks of gestation. In other approaches administration ocurs after 16 weeks of gestation, after 20 weeks of gestation or after 30 weeks of gestation. The formulation requires the presence of a carrier. Carriers include, without limitation, a lipid nanoparticle (LNP) and extracellular vesicles. Carriers may or may not be carrier is modified to contain a targeting molecule which binds to a tissue or organ of interest. Diseases to be treated include, without limitation, hereditary tyrosinemia type 1, Duchenne muscular dystrophy, familial hypercholesterolemia. In these diseases, the genetic mutation is in a gene selected from HPD, dystrophin or PCSK9 respectively.

In certain protocols, a single dose is administered. In other protocols, multiple doses are administered after 12 weeks of gestation, e.g., 2 or 3 or more doses are administered at least 2 weeks apart.

The compositions can be administered at doses between 0.1 to 5 mg/kg. Other dosing ranges include without limitation, 0.1-0.2 mg/kg, 0.25-0.30 mg/kg, 0.50-0.75 mg/kg, 0.75-1 mg/kg, 1-1.5 mg/kg, 2-2.5 mg/kg, 3-3.5 mg/kg, 4-4.5 mg/kg and 5 mg/kg.

In yet another aspect, a sterile injectable composition for treatment of hereditary tyrosinemia type 1 formulated for systemic injection into the intrahepatic umbilical vein or the cephalic vein of a subject in need thereof is disclosed. An exemplary composition comprises i) lipid nanoparticles (LNP) containing 48% ionizable lipid, 9% DSPC, 3% DOPE, 38.5% cholesterol and 1.5% PEG-DMA, an N:/P ratio of 4, an adenine base editor ABE8.8 mRNA and a gRNA targeting an HPD gene, or ii) LNP containing ionizable lipid LP01, an amine lipid, DSPC, cholesterol, and a PEG-DMG lipid at an N:P ratio of 6 combined at a final ratio of 51.68:9.0:36.32:3.0, said LNP having a size and polydispersity of 65nm, and 0.05 respectively, an adenine base editor ABE8.8 mRNA and a gRNA targeting an HPD gene; wherein an editor mRNA and gRNA ratio of i) or ii) being 1 : 1 by mass, said composition effecting base editing in a target tissue or organ selected from one or more of heart, diaphragm, skeletal muscle, kidney, and CD34+ hematopoietic progenitor cells. The composition can be administered at a dose selected from .0.1 -0.2 mg/kg, 0.25-0.3 mg/kg, 0.5-0.75 mg/kg, 0.75-1 mg/kg, 1-1.5 mg/kg, 2-2.5 mg/kg, 3-3.5 mg/kg, 4-4.5 mg/kg and 5 mg/kg25-.30 mg/kg, ,50-.75mg/kg, ,75-lmg/kg, 1- 1.5mg/kg, 2-25mg/kg, 3-3.5mg/kg, 4-4.5mg/kg and 5mg/kg and can be formulated for a single or multiple administrations.

The methods described above are useful for the treatment of a variety of disorders. In one embodiment, the mutation is present in an exon in a dystrophin gene selected from exons 44, 45, 50, 51 or 53, the target tissue is selected from one or more of muscle, heart, and diaphragm, the disease is Duchenne muscular dystrophy and the mutation is edited to correct the mutation or edited to effect exon skipping of the mutation-containing exon.

In another embodiment, the mutation comprises an expansion of CTG repeats in a 3’ untranslated region (UTR) of a dystrophia myotonica protein kinase (DMPK) gene located at 19ql 3.32, the target tissue is muscle selected from heart, skeletal muscle and diaphragm, the disease is Myotonic dystrophy type 1 and said mutation is edited via nuclease mediated excision of CTG repeats or insertion of a termination signal prior to the CTG repeats via prime editing.

In another approach, the mutation is present in a gene selected from PTPN11, S0S1, RAFI, and RIT1, the disease of Noonan syndrome, the target tissue is selected from muscle, and heart and the editing strategy is the correction of the disease causing mutation.

In another embodiment, the mutation is duplication of a chromosome selected from chromosome 21, 13, or 18, the target tissue selected from muscle, and heart, said disease is trisomy 21, trisomy 13 or trisomy 18 respectively, and the editing approach involves nuclease mediated degradation of one of the three copies of the chromosome via targeting of alleles specific to that copy of chromosome 21, 13 or 18, or via epigenetic silencing of one of the three copies of chromosome 21, 13, or 18, via induction of Barr body formation.

In another aspect, the mutation is C.2560OT in the GAA gene, the target tissue is liver and heart, the disease is Pompe disease and said mutation is corrected via adenine base editing or prime editing.

In another embodiment, the mutation is a GAA triplet repeat in the frataxin (FXN) gene, the target tissue is heart and the editing occurs via nuclease editing which excises the triplet repeats or prime editing for insertion of a termination signal prior to the GAA repeats.

In another aspect of the method, the mutation is a single point mutation associated with i) a beta7^{Glu Xal} substitution in the beta globin gene, and said disease is sickle cell disease; ii) a mutation in a beta globin gene causative of beta thalassemia; or iii) a mutation in an alpha globin gene associated with alpha thalassemia, wherein said target tissue is CD34+ cells and editing is performed via prime editing or base editing to correct the underlying mutation, or base editing to change the mutation to a less severe disease causing mutation, or base editing or nuclease editing to upregulate at least one of fetal gamma globin or zeta globin expression.

In yet another approach, the mutation is present in a PKD1 or PKD2 gene, the disease is autosomal dominant polycystic kidney disease, the target tissue is kidney and the editing occurs via at a method selected from base editing, prime editing or nuclease editing to disrupt the PDK1 or PDK2 3’UTR miR-17 binding element in the PKD1 or PKD2 gene.

Brief Description of the Drawings

Figures 1A- 1G. Biodistribution of systemically delivered LNPs in fetal, day of life 1, and adult mouse recipients. LNPs made from SM102, Alc315, or C24 ionizable lipids containing Cre recombinase mRNA were injected at 1 mg/kg via the vitelline vein, facial vein, or tail vein into gestational day 14, day of life 1, or 6-8 week old SunlGFP Cre reporter mouse recipients respectively. One month after injection, organs were harvested from mice and the nuclei of individual organs were analyzed by flow cytometry for GFP expression and co-stains as indicated. Fig. 1A % of Dapi+ heart cells that are GFP+. Fig. IB % of PCM1+ (a myocyte marker) heart cells that are GFP+. Fig. 1C % of Dapi+ diaphragm cells that are GFP+. Fig. ID % of PCM1+ skeletal muscle cells from the biceps or quadriceps that are GFP+. Fig. IE % of kidney cells that are GFP+. Fig. IF % of Dapi+ lung cells that are GFP+. Fig. 1G % of ERG+ (an endothelial cell marker) lung cells that are GFP+. PO, day of life 1 (developmentally equivalent to a late gestation human fetus). Fig. 1H % of Dapi+ heart cells that are GFP+. Fig. II % of PCM1+ (a myocyte marker) heart cells that are GFP+ Fig. 1J % of Dapi+ diaphragm cells that are GFP+ Fig. IK % of PCM1+ diaphram cells. Fig. IL GFP expression in mouse muscle satellite cells (PAX7+)(red) in a GFP reporter mouse. Fig. IM % of Dapi+ quadriceps cells that are GFP+ Fig. IN % of PCM1+ skeletal muscle cells quadriceps that are GFP+. Fig. IO quadricep GFP+ satellite cells.

Figures 2A - 2F. Biodistribution of systemically delivered LNPs in fetal nonhuman primate recipients. Two fetal Macaca fascicularis fetuses were injected via the umbilical vein at its entrance into the liver (the intrahepatic umbilical vein) with either an Alc315 LNP containing SunlGFP mRNA (injected at gestational day 71, dose: 5.5 mg/kg LNPs) or a SM102 LNP containing SunlGFP (injected at gestational day 98, dose: 2.5 mg/kg LNPs). Twenty-four hours after injection organs were harvested and the nuclei of individual organs were analyzed by flow cytometry for GFP expression and co-stains as indicated. Fig. 2A % of Dapi+ heart cells that are GFP+. Fig. 2B % of PCM1+ (a myocyte marker) heart cells that are GFP+. Fig. 2C % of Dapi+ diaphragm cells that are GFP+. Fig. 2D % of PCM1+ diaphragm cells that are GFP+. Fig. 2E % of Dapi+ skeletal muscle cells from the biceps or quadriceps that are GFP+. Fig. 2F % of PCM1+ skeletal muscle cells from the biceps or quadriceps that are GFP+. Fig. 2G % of Dapi+ kidney cells that are GFP+. Fig. 2H % of CD34+ hematopoietic progenitor cells from the central fetal liver, peripheral fetal liver or fetal bone marrow that are GFP+. BM, bone marrow.

Figures 3A -3B. Immunohistochemistry of the heart confirms LNP delivery to cardiomyocytes in mid gestation fetal NHP recipients. Fig. 3A Hearts from the fetal NHP recipient of SM102 LNPs containing Sunl GFP mRNA and from a control NHP fetus that received SM102 LNPs and ABE8.8 mRNA were processed for immunohistochemistry and stained with Dapi (blue), an anti-GFP antibody (green) and an antibody to cardiac troponin (cTnT)(red). Representative images demonstrate GFP nuclear staining in cardiomyocytes stained with cTnT. Fig. 3B GFP expression in mouse muscle satellite cells (PAX7+)(red) in a GFP reporter mouse following injection of SMI 02 LNP containing Cre recombinase mRNA. * and # highlight cells costained for GFP and PAX7.

Figures 4A - 4H. In vivo base editing in non-liver organs in fetal and juvenile NHP recipients of LNPs containing ABE8.8 mRNA and HPD20 gRNA. Four fetal and two juvenile Macaca fascicularis NHPs were systemically injected via the intrahepatic umbilical vein or the cephalic vein respectively with SM102 LNPs containing ABE8.8 mRNA and a gRNA targeting the HPD gene. The age at injection and the dose of LNP mRNA are indicated in Table 1. One to two weeks following injection, organs were harvested, and genomic DNA was isolated for individual organs. Next generation sequencing was performed to assess on-target A-to-G editing efficiency in the (Fig. 4A) heart, (Fig. 4B) diaphragm, (Fig. 4C) quadriceps, (Fig. 4D) spleen, (Fig. 4E) kidney, (Fig. 4F) lung, (Fig. 4G) gonad, and (Fig. 4H) hematopoietic cells. CD34, CD34+ hematopoietic progenitor cells; MNC, mononuclear cells; BM, bone marrow; FL, fetal liver.

Figure 5. In vivo liver base editing in fetal and juvenile NHP recipients of LNPs containing ABE8.8 mRNA and HPD20 gRNA. Four fetal and two juvenile Macaca fascicularis NHPs were systemically injected via the intrahepatic umbilical vein or the cephalic vein respectively with SM102 LNPs containing ABE8.8 mRNA and a gRNA targeting the HPD gene. The age at injection and the dose of LNP mRNA are indicated in Table 1. One to two weeks following injection, multiple sections of the livers were harvested, and genomic DNA was isolated. Next generation sequencing was performed to assess on-target A-to-G editing efficiency.

Figures 6A- 6E. Editing of the mouse DMD gene following delivery of ABE8e and gRNA in SM102 LNP. Gestational day (E) 14 fetuses, E16 fetuses and 6-8 week old adult Balb/c mice were injected with an LNP consisting of the SMI 02 ionizable lipid, the ABE8e mRNA and a gRNA targeting the splice acceptor adenine of exon 45 of the mouse dystrophin gene at an RNA dose of 2 mg/kg (Figs. 6A-D) and 3 mg/kg (Figs. 6B-D). One month post injection, indicated organs were harvested, and genomic DNA was isolated. Next generation sequencing was performed to assess on-target A-to-G editing efficiency. Editing in liver (Fig. 6A), heart (Figs. 6A and 6B), diaphragm (Figs. 6A and 6C), quadricep (Figs. 6A -6D), and tibialis anterior (Fig 6A) are shown. Nuclei from the diaphragm and quadriceps of E14 injected fetuses were sorted for PCM1+ nuclei (a marker for myocytes) at the one-month harvest time point and next generation sequencing was performed to assess levels of whole tissue versus myocyte editing at dystrophin exon 45 (Fig. 6E). . Controls (Ctrl) consists of age matched uninjected mice.

Figures 7A -7C. Editing of the NHP PCSK9 gene following delivery of ABE8.8 and gRNA in LP01 LNP. Four 2-3 year old juvenile and two fetal Macaca fascicularis NHPs (gestational day 114 and 123) were systemically injected via the cephalic vein or the intrahepatic umbilical vein respectively with LP01 LNPs containing ABE8.8 mRNA and a gRNA targeting the PCSK9 gene. Two to three weeks following injection, multiple sections of the livers (Fig. 7A), heart (Fig. 7B), diaphragm (Fig. 7B), bicep (Fig. 7B), and quadricep (Fig 7B) were harvested, and genomic DNA was isolated. Next generation sequencing was performed to assess on-target A-to-G editing efficiency. Similarly, CD34+ cells (hematopoietic progenitor cells) and CD34- cells from the liver and bone marrow from the two injected fetuses were obtained by flow cytometry sorting and the genomic DNA from these cells was assessed by next generation sequencing for on-target A-to-G editing (Fig. 7C).

Figure 8. GFP expression in sheep organs following injection of LP01 LNP containing GFP mRNA. Three fetal sheep and one newborn sheep were injected with LP01 LNPs containing GFP mRNA at gestational day ~60 (early gestation fetus 1 and 2), gestational day ~90 (mid gestation fetal sheep) or day of life 3 (newborn sheep) via either the umbilical vein (fetal sheep) or the internal jugular vein (newborn sheep). Twenty-four hours after injection, the indicated organs were harvested and the nuclei were isolated and assessed by flow cytometry for GFP expression, The CD34+ cells were harvested from the liver of the fetal sheep and the bone marrow of the newborn sheep. GFP levels were normalized to un-injected controls.

Detailed Description the Invention

The developing fetus presents ontologic opportunities to take advantage of normal developmental properties to potentially target organs and cell types that are not readily accessible after birth via systemic administration of LNPs. For example, tight junctions in endothelial cells may not be as robustly formed thus allowing access to different organs during development. As such, we hypothesize that organs and cell types, including the heart, diaphragm, skeletal muscle, kidney and CD34+ hematopoietic stem cells, that are untargetable in vivo with LNPs for in vivo gene editing are targetable via LNPs and can undergo in vivo gene editing when the editing technology, including adenine base editor mRNA and gRNA, are delivered via LNPs or other suitable carriers to the fetal subjects, including without limitation mammals, non-human primates (NHP) or human recipients via systemic injection by umbilical or hepatic vein injection in utero prior to birth.

We have previously demonstrated the ability to target the liver in the mouse fetus and adult mouse recipient with viral vectors carrying the SpCas9 or cytosine base editor transgene and gRNA targeting the Hpd gene as a therapeutic modality for hereditary tyrosinemia type 1 (HT1) (See US Patent application no. and 17/052,170, and PCT/US2024/011693, which are incorporated herein by reference). These patent applications describe use of nonviral targeting approaches, such as LNPs, to deliver gene editing technology to the liver of fetal recipients. Furthermore, in this earlier filing, we described organ distribution in mouse fetuses following in vivo adenoviral delivery of SpCas9 and a gRNA in a reporter mouse model as well as in vivo viral delivery of a cytosine base editor and gRNA targeting the mouse Hpd or Pcsk9 gene. Notably, the brain, heart, lung, kidney, and liver were assessed in the treated animals and, we did not observe significant editing in any organ other than the liver with the exception of some editing using the reporter model of the fetal heart (See Figure ID, Figure 4E, Figure 6, Figure 7, Figure 9 of the ‘ 170 application). In a subsequent filing (See PCT/US2024/011993), we reported the ability to use LNPs to deliver an ABE mRNA and a new HPD targeting gRNA (HPD20) as a treatment for HT1. In this filing, we demonstrated in both prenatal and postnatal mouse models, the ability of a SMI 02 LNP containing the HPD20 orthologous mouse gRNA and ABE8.8 mRNA to efficiently edit the mouse Hpd gene in fetal and adult recipients and correct the disease phenotype. We also reported the ability of the SM102 LNP containing ABE8.8 mRNA and the human HPD targeting gRNA (HPD20 gRNA) to efficiently edit the HPD gene in the fetal NHP recipient and reduce the expression of the HPD protein. In the ‘993 PCT filing, the data show the biodistribution of editing in the mouse model and highlight minimal editing in all organs other than the liver (See Fig. 11A - neonatal mice injected with LNPs containing ABE8.8 mRNA and Hpd targeting gRNA, Figure 12B - neonatal mice injected with LNPs containing ABE8.8 mRNA and Hpd targeting gRNA, Table 6). Since these initial filings, we have extended our murine and, critically, our NHP studies to evaluate approaches for increasing LNP biodistribution and editing in additional organs and tissues.

The data in the previous filings demonstrate that in the postnatal NHP model, low level base editing (3-6%) of the target splice site in the HPD gene in hepatocytes is now available. Although low, this level of editing demonstrates a beneficial therapeutic effect as edited cells demonstrated a significant survival advantage (Rossidis et al., 2018). Using the new approaches described herein, we are able to efficiently edit organs in the fetal NHP recipient that are unable to be edited in the juvenile or adult NHP recipient. See for example, the patent applications discussed above and (Musunuru et al., 2021; Rothgangl et al., 2021), in postnatal recipients. Specifically, the heart, diaphragm, skeletal muscle, kidney, and CD34+ hematopoietic progenitor cells can now be successfully targeted for gene editing.

These surprising findings support the use of our novel delivery, timing, and dosing approaches (via the umbilical vein or hepatic vein in the fetus) to facilitate a variety of LNP mediated in vivo gene editing therapies, including traditional Cas9 NHEJ, HDR, base editing and prime editing, for genetic diseases not currently amenable to this treatment because of LNP targeting difficulties. These diseases include, but are not limited to, those involving the heart, diaphragm, and/or skeletal muscles (muscular dystrophies including Duchenne muscular dystrophy, myotonic dystrophy type 1, Noonan syndrome, X-linked myotubular myopathy; familial cardiomyopathies; and trisomy 21), diseases affecting the kidney (autosomal recessive polycystic kidney disease, autosomal dominant polycystic kidney disease), cystic fibrosis, genetic blood diseases (sickle cell disease, beta thalassemia, alpha thalassemia, Fanconi anemia, inborn errors of immunity such as severe combined immunodeficiency), and lysosomal storage diseases.

DEFINITIONS

The present subject matter may be understood more readily by reference to the following detailed description which forms a part of this disclosure. It is to be understood that this invention is not limited to the specific products, methods, conditions or parameters described and/or shown herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting of the claimed invention.

Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

As employed above and throughout the disclosure, the following terms and abbreviations, unless otherwise indicated, shall be understood to have the following meanings.

In the present disclosure the singular forms "a," "an," and "the" include the plural reference, and reference to a particular numerical value includes at least that particular value, unless the context clearly indicates otherwise. Thus, for example, a reference to "a compound" is a reference to one or more of such compounds and equivalents thereof known to those skilled in the art, and so forth. The term "plurality", as used herein, means more than one. When a range of values is expressed, another embodiment includes from the one particular and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it is understood that the particular value forms another embodiment. All ranges are inclusive and combinable. As used herein, the terms "component," "composition," "composition of compounds," "compound," "drug," "pharmacologically active agent, " "active agent," "therapeutic," "therapy," "treatment," or "medicament" are used interchangeably herein to refer to a compound or compounds or composition of matter which, when administered to a subject (human or animal) induces a desired pharmacological and/or physiologic effect by local and/or systemic action.

As used herein, the terms "treatment" or "therapy" (as well as different forms thereof) include preventative (e.g., prophylactic), curative or palliative treatment. As used herein, the term "treating" includes alleviating or reducing at least one adverse or negative effect or symptom of a condition, disease or disorder.

The terms "subject," "individual," and "patient" are used interchangeably herein, and refer to an animal, for example a human, to whom treatment, including prophylactic treatment, with the pharmaceutical composition according to the present invention, is provided. The term "subject" as used herein refers to human and non-human animals. The terms "non-human animals" and "non-human mammals" are used interchangeably herein and include all vertebrates, e.g., mammals, such as non-human primates, (particularly higher primates), sheep, dog, rodent, (e.g. mouse or rat), guinea pig, goat, pig, cat, rabbits, cows, horses and non-mammals such as reptiles, amphibians, chickens, and turkeys.

The terms "polynucleotide", "nucleotide", "nucleotide sequence", "nucleic acid" and "oligonucleotide" are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. A polynucleotide may comprise one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.

As used herein the term "wild type" is a term of the art understood by skilled persons and means the typical form of an organism, strain, gene or characteristic as it occurs in nature as distinguished from mutant or variant forms. As used herein the term "variant" should be taken to mean the exhibition of qualities that have a pattern that deviates from the wild type or a comprises non naturally occurring components.

The terms "non-naturally occurring" or "engineered" are used interchangeably and indicate the involvement of the hand of man. The terms, when referring to nucleic acid molecules or polypeptides mean that the nucleic acid molecule or the polypeptide is at least substantially free from at least one other component with which they are naturally associated in nature and as found in nature.

"Complementarity" refers to the ability of a nucleic acid to form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick or other non-traditional types. A percent complementarity indicates the percentage of residues in a nucleic acid molecule which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary). "Perfectly complementary" means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. "Substantially complementary" as used herein refers to a degree of complementarity that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%. 95%, 97%, 98%, 99%, or 100% over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or more nucleotides, or refers to two nucleic acids that hybridize under stringent conditions.

The term "effective amount" or "therapeutically effective amount" refers to the amount of an agent that is sufficient to effect beneficial or desired results. The therapeutically effective amount may vary depending upon one or more of: the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art. The term also applies to a dose that will provide an image for detection by any one of the imaging methods described herein. The specific dose may vary depending on one or more of: the particular agent chosen, the dosing regimen to be followed, whether it is administered in combination with other compounds, timing of administration, the tissue to be imaged, and the physical delivery system in which it is carried.

The discussion below relates to a variety of different gene editing and carrier (LNPs, plasmid and viral vectors, etc ). Several aspects of the invention relate to vector systems comprising one or more vectors, or vectors as such. Vectors can be designed for expression of CRISPR transcripts (e.g. nucleic acid transcripts, proteins, or enzymes) in prokaryotic or eukaryotic cells. For example, CRISPR transcripts can be expressed in bacterial cells such as Escherichia coli, insect cells (using baculovirus expression vectors), yeast cells, or mammalian cells. Suitable host cells are discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press. San Diego, Calif. (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

In some embodiments, a vector is capable of driving expression of one or more sequences in mammalian cells using a mammalian expression vector. When used in mammalian cells, the expression vector's control functions are typically provided by one or more regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, simian virus 40, and others disclosed herein and known in the art. For other suitable expression systems for both prokaryotic and eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed„ Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y,

1989.

In some embodiments, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissuespecific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert, et al., 1987. Genes Dev. 1 : 268-277), lymphoid-specific promoters (Calame and Eaton, 1988. Adv. Immunol. 43: 235-275), in particular promoters of T cell receptors (Winoto and Baltimore, 1989. EMBO J. 8: 729-733) and immunoglobulins (Baneiji, et al., 1983. Cell 33: 729-740; Queen and Baltimore, 1983. Cell 33: 741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci. USA 86: 5473-5477), pancreas-specific promoters (Edlund, et al., 1985. Science 230: 912-916), and mammary gland-specific promoters (e.g., milk whey promoter, U.S. Pat. No. 4,873,316 and European Application Publication No. 264, 166). Developmentally regulated promoters are also encompassed, e.g., the murine hox promoters (Kessel and Gruss,

1990. Science 249: 374-379) and the a-fetoprotein promoter (Campes and Tilghman, 1989. Genes Dev. 3: 537-546).

In general, "CRISPR system" refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated ("Cas") genes, including sequences encoding a Cas gene, a tracr (trans-activating CRISPR) sequence (e g. tracrRNA or an active partial tracrRNA), a tracr-mate sequence (encompassing a "direct repeat" and a tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system), a guide sequence (also referred to as a "spacer" in the context of an endogenous CRISPR system), or other sequences and transcripts from a CRISPR locus. In some embodiments, one or more elements of a CRISPR system is derived from a type I, type II, or type III CRISPR system. In some embodiments, one or more elements of a CRISPR system is derived from a particular organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes. In general, a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence (also referred to as a protospacer in the context of an endogenous CRISPR system). In the context of formation of a CRISPR complex, "target sequence" refers to a sequence to which a guide sequence is designed to have complementarity, where hybridization between a target sequence and a guide sequence promotes the formation of a CRISPR complex. Full complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization and promote formation of a CRISPR complex. A target sequence may comprise any polynucleotide, such as DNA or RNA polynucleotides. In some embodiments, a target sequence is located in the nucleus or cytoplasm of a cell. In some embodiments, the target sequence may be within an organelle of a eukaryotic cell, for example, mitochondrion or chloroplast. A sequence or template that may be used for recombination into the targeted locus comprising the target sequences is referred to as an "editing template" or "editing polynucleotide" or "editing sequence". In aspects of the invention, an exogenous template polynucleotide may be referred to as an editing template. In an aspect of the invention the recombination is homologous recombination.

In some embodiments, one or more vectors driving expression of one or more elements of a CRISPR system are introduced into a host cell such that expression of the elements of the CRISPR system direct formation of a CRISPR complex at one or more target sites. For example, a Cas enzyme, a guide sequence linked to a tracr mate sequence, and a tracr sequence could each be operably linked to separate regulatory elements on separate vectors. Alternatively, two or more of the elements expressed from the same or different regulatory elements, may be combined in a single vector, with one or more additional vectors providing any components of the CRISPR system not included in the first vector. CRISPR system elements that are combined in a single vector may be arranged in any suitable orientation, such as one element located 5' with respect to ("upstream" of) or 3' with respect to ("downstream" of) a second element. The coding sequence of one element may be located on the same or opposite strand of the coding sequence of a second element, and oriented in the same or opposite direction. In some embodiments, a single promoter drives expression of a transcript encoding a CRISPR enzyme and one or more of the guide sequence, tracr mate sequence (optionally operably linked to the guide sequence), and a tracr sequence embedded within one or more intron sequences (e.g. each in a different intron, two or more in at least one intron, or all in a single intron). In some embodiments, the CRISPR enzyme, guide sequence, tracr mate sequence, and tracr sequence are operably linked to and expressed from the same promoter.

In some embodiments, a vector comprises one or more insertion sites, such as a restriction endonuclease recognition sequence (also referred to as a "cloning site"). In some embodiments, one or more insertion sites (e.g. about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more insertion sites) are located upstream and/or downstream of one or more sequence elements of one or more vectors. In some embodiments, a vector comprises an insertion site upstream of a tracr mate sequence, and optionally downstream of a regulatory element operably linked to the tracr mate sequence, such that following insertion of a guide sequence into the insertion site and upon expression the guide sequence directs sequence-specific binding of a CRISPR complex to a target sequence in a eukaryotic cell. In some embodiments, a vector comprises two or more insertion sites, each insertion site being located between two tracr mate sequences so as to allow insertion of a guide sequence at each site. In such an arrangement, the two or more guide sequences may comprise two or more copies of a single guide sequence, two or more different guide sequences, or combinations of these. When multiple different guide sequences are used, a single expression construct may be used to target CRISPR activity to multiple different, corresponding target sequences within a cell. For example, a single vector may comprise about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more guide sequences. In some embodiments, about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more such guide-sequence-containing vectors may be provided, and optionally delivered to a cell.

In some embodiments, a vector comprises a regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme, such as a Cas protein. Non-limiting examples of Cas proteins include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2. Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl , Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, homologs thereof, or modified versions thereof. These enzymes are known; for example, the amino acid sequence of S. pyogenes Cas9 protein may be found in the SwissProt database under accession number Q99ZW2. In some embodiments, the unmodified CRISPR enzyme has DNA cleavage activity, such as Cas9. In some embodiments the CRISPR enzyme is Cas9, and may be Cas9 from S. pyogenes or S. pneumoniae. In some embodiments, the CRISPR enzyme directs cleavage of one or both strands at the location of a target sequence, such as within the target sequence and/or within the complement of the target sequence. In some embodiments, the CRISPR enzyme directs cleavage of one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of a target sequence.

In some embodiments, a vector encodes a CRISPR enzyme that is mutated to with respect to a corresponding wild-type enzyme such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing a target sequence. For example, an aspartate-to-alanine substitution (D10A) in the RuvC I catalytic domain of Cas9 from S. pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (cleaves a single strand). Other examples of mutations that render Cas9 a nickase include, without limitation, H840A, N854A, and N863A. In aspects of the invention, nickases may be used for genome editing via homologous recombination.

In some embodiments, an enzyme coding sequence encoding a CRISPR enzyme is codon optimized for expression in particular cells, such as eukaryotic cells. The eukaryotic cells may be those of or derived from a particular organism, such as a mammal, including but not limited to human, mouse, rat, rabbit, dog, or non-human primate. In general, codon optimization refers to a process of modifying a nucleic acid sequence for enhanced expression in the host cells of interest by replacing at least one codon (e.g. about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Various species exhibit particular bias for certain codons of a particular amino acid. Codon bias (differences in codon usage between organisms) often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, among other things, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, at the "Codon Usage Database", and these tables can be adapted in a number of ways. See Nakamura, Y, et al. "Codon usage tabulated from the international DNA sequence databases: status for the year 2000” Nucl. Acids Res. 28:292 (2000). Computer algorithms for codon optimizing a particular sequence for expression in a particular host cell are also available, such as Gene Forge (Aptagen; Jacobus, Pa.), are also available. In some embodiments, one or more codons (e.g. 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, or all codons) in a sequence encoding a CRISPR enzyme correspond to the most frequently used codon for a particular amino acid.

In general, a guide sequence is any polynucleotide sequence having sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of a CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence, when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences, non-limiting example of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g. the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).

In general, a tracr mate sequence includes any sequence that has sufficient complementarity with a tracr sequence to promote one or more of: (I) excision of a guide sequence flanked by tracr mate sequences in a cell containing the corresponding tracr sequence; and (2) formation of a CRISPR complex at a target sequence, wherein the CRISPR complex comprises the tracr mate sequence hybridized to the tracr sequence. In general, degree of complementarity is with reference to the optimal alignment of the tracr mate sequence and tracr sequence, along the length of the shorter of the two sequences. Optimal alignment may be determined by any suitable alignment algorithm, and may further account for secondary structures, such as self-complementarity within either the tracr sequence or tracr mate sequence.

In some embodiments, the CRISPR enzyme is part of a fusion protein comprising one or more heterologous protein domains (e.g. about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more domains in addition to the CRISPR enzyme). A CRISPR enzyme fusion protein may comprise any additional protein sequence, and optionally a linker sequence between any two domains. Examples of protein domains that may be fused to a CRISPR enzyme include, without limitation, epitope tags, reporter gene sequences, and protein domains having one or more of the following activities: methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity and nucleic acid binding activity. Non-limiting examples of epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Examples of reporter genes include, but are not limited to, glutathione-5-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT) beta-galactosidase, beta-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and autofluorescent proteins including blue fluorescent protein (BFP). A CRISPR enzyme may be fused to a gene sequence encoding a protein or a fragment of a protein that bind DNA molecules or bind other cellular molecules, including but not limited to maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD) fusions, GAL4 DNA binding domain fusions, and herpes simplex virus (HSV) BP 16 protein fusions. Additional domains that may form part of a fusion protein comprising a CRISPR enzyme are described in US20110059502, incorporated herein by reference. In some embodiments, a tagged CRISPR enzyme is used to identify the location of a target sequence.

In an aspect of the invention, a reporter gene which includes but is not limited to glutathione-5-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT) beta-galactosidase, beta-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and auto-fluorescent proteins including blue fluorescent protein (BFP), may be introduced into a cell to encode a gene product which serves as a marker by which to measure the alteration or modification of expression of the gene product. In a further embodiment of the invention, the DNA molecule encoding the gene product may be introduced into the cell via a vector. In a preferred embodiment of the invention the gene product is luciferase. In a further embodiment of the invention the expression of the gene product is decreased.

In some aspects, the invention provides methods comprising delivering one or more polynucleotides, such as or one or more vectors as described herein, one or more transcripts thereof, and/or one or proteins transcribed therefrom, to a host cell. In some aspects, the invention further provides cells produced by such methods, and organisms (such as animals, plants, or fungi) comprising or produced from such cells. In some embodiments, a CRISPR enzyme in combination with (and optionally complexed with) a guide sequence is delivered to a cell. Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acids in mammalian cells or target tissues. Such methods can be used to administer nucleic acids encoding components of a CRISPR system to cells in culture, or in a host organism. Non-viral vector delivery systems include DNA plasmids, RNA (e g. a transcript of a vector described herein), naked nucleic acid, and nucleic acid complexed with a delivery vehicle, such as a liposome. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. For a review of gene therapy procedures, see Anderson, Science 256:808-813 (1992); Nabel & Feigner, TIBTECH 11:211-217 (1993); Mitani & Caskey, TIBTECH 11 : 162-166 (1993); Dillon, TIBTECH 11 : 167-175 (1993); Miller, Nature 357:455-460 (1992); Van Brunt, Biotechnology 6(10): 1149-1154 (1988); Vigne, Restorative Neurology and Neuroscience 8:35-36 (1995); Kremer & Perricaudet, British Medical Bulletin 51 (1):31-44 (1995); Haddada et al., in Current Topics in Microbiology and Immunology Doerfler and Bihm (eds) (1995); and Yu et al., Gene Therapy 1 : 13-26 (1994).

Methods of non-viral delivery of nucleic acids include lipofection, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipidmucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA. Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Feigner, WO 91/17424; WO 91/16024. Delivery can be to cells (e.g. in vitro or ex vivo administration) or target tissues (e.g. in vivo administration).

The preparation of lipidmucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to one of skill in the art (see, e g., Crystal, Science 270:404-410 (1995); Blaese et al., Cancer Gene Then 2:291-297 (1995); Behr et al., Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gao et al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res. 52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787). More recently, Kasiewicz, L.N., Biswas, S., Beach, A. et al. described GalNAc-Lipid nanoparticles which enable non-LDLR dependent hepatic delivery of a CRISPR base editing therapy in Nat Commun 14, 2776 (2023). For other suitable lipid nanoparticle formulations, see also US Patent No. 11,801,306 and International Patent Application No. W02022060871. Clinical trials are also being conducted which also employ a LNP described in trial clinical trial identifier no. NCT05398029. Virus-like Particles as Nanocarriers for Intracellular Delivery of Biomolecules and Compounds are described by He, J. et al. in “Virus-like Particles as Nanocarriers for Intracellular Delivery of Biomolecules and Compounds.” Viruses. 2022 Aug 28; 14(9): 1905.

The use of RNA or DNA viral based systems for the delivery of nucleic acids take advantage of highly evolved processes for targeting a virus to specific cells in the body and trafficking the viral payload to the nucleus. Viral vectors can be administered directly to patients (in vivo) or they can be used to treat cells in vitro, and the modified cells may optionally be administered to patients (ex vivo). Conventional viral based systems could include retroviral, lentivirus, adenoviral, adeno-associated and herpes simplex virus vectors for gene transfer. Integration in the host genome is possible with the retrovirus, lentivirus, and adeno-associated virus gene transfer methods, often resulting in long term expression of the inserted transgene. Additionally, high transduction efficiencies have been observed in many different cell types and target tissues.

The tropism of a retrovirus can be altered by incorporating foreign envelope proteins, expanding the potential target population of target cells. Lentiviral vectors are retroviral vectors that are able to transduce or infect non-dividing cells and typically produce high viral titers. Selection of a retroviral gene transfer system would therefore depend on the target tissue. Retroviral vectors are comprised of cis-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimum cis-acting LTRs are sufficient for replication and packaging of the vectors, which are then used to integrate the therapeutic gene into the target cell to provide permanent transgene expression. Widely used retroviral vectors include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immuno deficiency virus (SIV), human immune-deficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al., J. Virol. 66:2731-2739 (1992); Johann et al., J. Virol. 66: 1635-1640 (1992); Sommnerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol. 63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991); PCT/US94/05700). In applications where transient expression is preferred, adenoviral based systems may be used. Adenoviral based vectors are capable of very high transduction efficiency in many cell types and do not require cell division. With such vectors, high titer and levels of expression have been obtained. This vector can be produced in large quantities in a relatively simple system. Adeno-associated virus ("AAV") vectors may also be used to transduce cells with target nucleic acids, e.g., in the in vitro production of nucleic acids and peptides, and for in vivo and ex vivo gene therapy procedures (see, e g., West et al., Virology 160:38-47 (1987); U.S. Pat. No. 4,797,368; WO 93/24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351 (1994). Construction of recombinant AAV vectors are described in a number of publications, including U.S. Pat. No. 5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985); Tratschin, et al., Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81 :6466-6470 (1984); and Samulski et al., J. Virol. 63:03822-3828 (1989).

Packaging cells are typically used to form virus particles that are capable of infecting a host cell. Such cells include 293 cells, which package adenovirus, and v|/2 cells or PA317 cells, which package retrovirus. Viral vectors used in gene therapy are usually generated by producing a cell line that packages a nucleic acid vector into a viral particle. The vectors typically contain the minimal viral sequences required for packaging and subsequent integration into a host, other viral sequences being replaced by an expression cassette for the polynucleotide(s) to be expressed. The missing viral functions are typically supplied in trans by the packaging cell line. For example, AAV vectors used in gene therapy typically only possess ITR sequences from the AAV genome which are required for packaging and integration into the host genome. Viral DNA is packaged in a cell line, which contains a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking ITR sequences. The cell line may also be infected with adenovirus as a helper. The helper virus promotes replication of the AAV vector and expression of AAV genes from the helper plasmid. The helper plasmid is not packaged in significant amounts due to a lack of ITR sequences. Contamination with adenovirus can be reduced by, e.g., heat treatment to which adenovirus is more sensitive than AAV.

In some embodiments, a host cell is transiently or non-transiently transfected with one or more vectors described herein. In some embodiments, a cell is transfected as it naturally occurs in a subject. In some embodiments, a cell that is transfected is taken from a subject. In some embodiments, the cell is derived from cells taken from a subject, such as a cell line.

In one aspect, the invention provides for methods of modifying a target polynucleotide in a eukaryotic cell, which may be in vivo, ex vivo or in vitro. In some embodiments, the method comprises sampling a cell or population of cells from a human or non-human animal, and modifying the cell or cells. Culturing may occur at any stage ex vivo. The cell or cells may be reintroduced into the human or non-human animal.

In one aspect, the invention provides for methods of modifying a target polynucleotide in a eukaryotic cell. In some embodiments, the method comprises allowing a CRISPR complex to bind to the target polynucleotide to effect cleavage of said target polynucleotide thereby modifying the target polynucleotide, wherein the CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence hybridized to a target sequence within said target polynucleotide, wherein said guide sequence is linked to a tracr mate sequence which in turn hybridizes to a tracr sequence.

In one aspect, the invention provides kits containing any one or more of the elements disclosed in the above methods and compositions. In some embodiments, the kit comprises a vector system or components for an alternative delivery system such as those described above and instructions for using the kit. In some embodiments, the vector or delivery system comprises (a) a first regulatory element operably linked to a tracr mate sequence and one or more insertion sites for inserting a guide sequence upstream of the tracr mate sequence, wherein when expressed, the guide sequence directs sequence-specific binding of a CRISPR complex to a target sequence in a eukaryotic cell, wherein the CRISPR complex comprises a CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence; and/or (b) a second regulatory element operably linked to an enzyme-coding sequence encoding said CRISPR enzyme comprising a nuclear localization sequence. Elements may be provided individually or in combinations, and may be provided in any suitable container, such as a vial, a bottle, or a tube. In some embodiments, the kit includes instructions in one or more languages, for example in more than one language.

In some embodiments, a kit comprises one or more reagents for use in a process utilizing one or more of the elements described herein. Reagents may be provided in any suitable container. For example, a kit may provide one or more reaction or storage buffers. Reagents may be provided in a form that is usable in a particular assay, or in a form that requires addition of one or more other components before use (e.g. in concentrate or lyophilized form). A buffer can be any buffer, including but not limited to a sodium carbonate buffer, a sodium bicarbonate buffer, a borate buffer, a Tris buffer, a MOPS buffer, a HEPES buffer, and combinations thereof. In some embodiments, the buffer is alkaline. In some embodiments, the buffer has a pH from about 7 to about 10. In some embodiments, the kit comprises one or more oligonucleotides corresponding to a guide sequence for insertion into a vector so as to operably link the guide sequence and a regulatory element. In some embodiments, the kit comprises a homologous recombination template polynucleotide.

In one aspect, the invention provides methods for using one or more elements of a CRISPR system. The CRISPR complex of the invention provides an effective means for modifying a target polynucleotide. The CRISPR complex of the invention has a wide variety of utility including modifying (e.g., deleting, inserting, translocating, inactivating, activating) a target polynucleotide in a multiplicity of cell types in methods of gene therapy. An exemplary CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence hybridized to a target sequence within the target polynucleotide. The guide sequence is linked to a tracr mate sequence, which in turn hybridizes to a tracr sequence.

Hereditary tyrosinemia type 1 (HT1) is a fatal genetic disease caused by autosomal recessive mutations in the Fah gene, which codes for the fumarylacetoacetate hydroxylase (FAH), leading to the accumulation of toxic fumarylacetoacetate and succinyl acetoacetate, causing liver and kidney damage. Over the past two decades, the disease has been controlled by 2-(2-nitro-4-trifluoromethylbenzoyl)-l,3-cyclohexanedione (NTBC), which inhibits 4- hydroxyphenylpyruvate dioxygenase upstream in the tyrosine degradation pathway, thus preventing the accumulation of the toxic metabolites. However, this treatment requires lifelong management of diet and medication and may eventually require liver transplantation.

The invention provides a method for in vivo genome editing, both prenatal and postnatal, the method comprising: administering to a subject an adenoviral vector, wherein the subject is a fetus, the adenoviral vector comprising CRTSPR-mediated base editor 3 (BE3) and a guide RNA (gRNA), the gRNA targeting an enzyme in the tyrosine catabolic pathway; and introducing a modified codon in the therapeutic gene by base editing the therapeutic gene, wherein the base editing is performed by the adenoviral vector or lipid nanoparticle. Base editing addresses the disadvantage of the need to create double strand breaks (DSBs) to instigate NHEJ or HDR. The base editor can comprise a catalytically impaired Streptococcus pyogenes Cas9 (SpCas9) protein, unable to make DSBs, fused to either a cytosine deaminase domain from a nucleic acid-editing protein (CBE) or a modified tRNA adenosine deaminase (ABE). The SpCas9 and gRNA tether the base editor at the genome target site, and the cytosine deaminase converts a nearby cytosine into uracil and, ultimately, thymine (resulting in either C— >T or G^A changes in the coding sequence of a gene, depending on which strand is targeted). The cytosine deaminase can introduce nonsense mutations in a site-specific fashion. Alternatively, the adenine deaminase converts a nearby adenine into inosine and, ultimately, guanine and can correct a disease-causing G^A mutation. Unlike HDR, base editing does not require proliferating cells to efficiently introduce mutations.

In an embodiment, the method further comprises before step (a) identifying in vitro a target codon for base editing into a nonsense codon; and generating the adenoviral vector by cloning BE3 -encoding gene, a synthetic polyadenylation sequence from pCMV-BE3, CAG reporter from pCas9_GFP, and U6 promoter-driven gRNA cassette with a protospacer sequence into a dual- expression vector. In certain embodiments, the gRNA targets the splice acceptor or the splice donor. In certain embodiments, the gRNA is at least one of those identified in tables 2- 4.

In various embodiments, genome editing is performed in utero when the fetus is inside a uterus of its carrier and the uterus is inside the body of a living carrier. In embodiments, the carrier may be a mammal. In certain embodiments, the mammal may be an animal. In additional embodiments, the mammal may be human. The living carrier may be the mother of the fetus or a surrogate.

In further embodiments, genome editing is performed in utero when the fetus is inside a uterus, and the uterus is outside the body of a carrier, e.g., not within the body of any living carrier, for example the uterus is in vitro or in an alternate embodiment, the uterus is ex vivo. In certain approaches the genome editors are administered as preassembled ribonucleoproteins or encoded in mRNA complexed to a carrier (e.g., vector or a LNP) and are administered directly into the umbilical vein. In other approaches, in utero transfusion is performed by fetal intrahepatic vein puncture. See Somerset et al., (2006).

In certain embodiments, the target codon is screened for a glutamine residue and a tryptophan residue, wherein the glutamine and tryptophan residues are within a base editing window of a protospacer adjacent motif (PAM) of the BE3, wherein the window is flanked by four proximal and four distal bases, wherein the proximal and distal bases match reference sequences.

In alternate embodiments, the CRISPR-mediated base editor is base editor 4 (BE4) rather than BE3.

In some embodiments, the method further comprises assessing C bases within the window for a change to another base. In embodiments, the gRNA is selected if the BE3 PAM sequence (NGG) is 13-17 nucleotides distal to the target cytosine base(s). In particular embodiments, the change is via a C to T on a sense strand, and the modified codon is changed to a nonsense codon. In additional embodiments, the change is via a G to A on an antisense strand, and the modified codon is changed to a nonsense codon. In further embodiments, the change is via a C to T on a sense strand, and the change is a missense variant. In additional embodiments, the change is via a G to A on an antisense strand, and the change is a missense variant. The base editing may occur prior to disease onset, wherein the disease is a phenotype resulting from a mutation in the therapeutic gene. In certain embodiments, the base editing decreases a risk of developing a disease.

In some embodiments, the therapeutic gene is a 4-hydroxyphenylpyruvate dioxygenase (HPD) gene. In certain embodiments, the gRNAs are selected from the sequences identified in Tables 2-4. In various embodiments, the introduction of a modified codon (e.g., a nonsense codon) in the HPD gene effects a permanent gene knockout of the Hpd gene. In embodiments, the permanent HPD gene knockout restores normal liver function in the subject prior to birth. In additional embodiments, the permanent HPD gene knockout treats hereditary tyrosinemia type I (HT1) disorder in the subject prior to birth.

The invention also provides a method for treating HT1 in a subject, the method comprising: identifying in vitro a target codon for base editing; generating the adenoviral vector by cloning BE3-encoding gene, a synthetic polyadenylation sequence from pCMV-BE3, CAG reporter from pCas9_GFP, and U6 promoter-driven gRNA cassette with a protospacer sequence into a dual -expression vector; administering to the fetal subject an adenoviral vector, the adenoviral vector comprising CRISPR-mediated base editor 3 (BE3) and a guide RNA (gRNA), and the gRNA targeting an enzyme in the tyrosine catabolic pathway; and introducing a modified codon in the therapeutic gene by base editing the therapeutic gene, wherein the base editing is performed by the adenoviral vector. In certain embodiments, the enzyme is HPD.

In alternate embodiments, the CRISPR-mediated base editor is base editor 4 (BE4) instead of BE3. In certain embodiments, the target codon is screened for a glutamine residue and a tryptophan residue, wherein the glutamine and tryptophan residues are within a base editing window of a protospacer adjacent motif (PAM) of the BE3, wherein the window is flanked by four proximal and four distal bases, wherein the proximal and distal bases match reference sequences.

In some embodiments, the method further comprises assessing C bases within the window for a change to another base. In further embodiments, the gRNA is selected if the BE3 PAM sequence (NGG) is 13-17 nucleotides distal to the target cytosine base(s). In various embodiments, the change is via a C to T on a sense strand, and the modified codon is changed to a nonsense codon. In other embodiments, the change is via a G to A on an antisense strand, and the modified codon is changed to a nonsense codon. In further embodiments, the change is via a C to T on a sense strand, and the change is a missense variant. In additional embodiments, the change is via a G to A on an antisense strand, and the change is a missense variant. In certain embodiments, the base editing occurs prior to disease onset, wherein the disease is a phenotype resulting from the mutation in the therapeutic gene. The base editing may decrease a risk of developing HT1.

In preferred approaches, the base editing compositions described herein are delivered using lipid nanoparticles. Several types of such particles are discussed above.

With respect to enhanced targeting therapeutic nucleic acid molecules to muscle cells, lipid nanoparticles including those using ionizable lipids SMI 02, Alc315 and C24 are modified to contain targeting moi eties to enhance uptake of the LNPs and mRNA delivery to myocytes. Specifically, the transferrin receptor 1 (CD71) is expressed on myocytes. LNPs have been modified to contain an antibody to transferrin receptor 1 on the surface such that systemic or local injection in a prenatal or postnatal recipient enhances LNP targeting of muscle cells including, but not limited to, the heart, diaphragm, and core and axial skeletal muscles. The attachment of the antibody to the transferrin 1 receptor to the LNP is performed via two different approaches.

In one approach, the antibody is first cut into F(ab)2 fragments using either IdeZ enzyme or ficin enzyme. Antibody fragments are then reduced via dithiothreitol (DTT) for 30 min at 25oC. DTT is removed using centrifugation through a 10 kDa filter, and the antibody product is resuspended in IX PBS. LNPs containing the anti-transferrin receptor F(ab)2 fragments are generated via reaction of LNPs to an excess of generated F(ab)2 fragments for 1 hour at room temperature followed by overnight incubation at 4oC. Free antibody fragments were removed through column filtration using Sephadex G-75 beads. F(ab)2 fragment bound LNPs are subsequently concentrated using 50 kDa Amicon Ultra-2 Centrifugal Filter Units. In an alternative approach SATA-maleimide chemistry is used to conjugate the LNPs to an antibody targeting the transferrin receptor. Specifically, LNPs are modified with maleimide functioning groups. The anti-transferrin receptor antibody is functionalized with N-succinimidyl S- acetylthioacetate (SATA) to introduce sulfhydryl groups. This process allows for a conjugation of the antibody to the LNPs via maleimide-sulfhydryl group reaction. Specifically, N-succinimidyl S-acetylthioacetate is deprotected using 0.5 M hydroxylamine followed by removal of unreacted components by Zeba spin desalting columns. The reactive sulfhydryl group on the antibody is then conjugated to maleimide moieties using thioether conjugation chemistry and the LNPs purified using Sepharose CL-4B gel filtration columns. This technique of conjugation using SATA-maleimide chemistry is previously described. See Parhiz et al. (2018) and Breda et al. (2023).

Several approaches for non-viral delivery of genome editors in vivo. In certain approaches cells to be targeted are isolated, treated ex vivo to introduce the genome editor complex and then reintroduced into the subject. This approach has been used successfully to treat a number of hematopoietic disorders. As we demonstrated in our previous filings genome editor complexes can be administered directly to the liver and successfully edit target genes providing therapeutic benefits. In certain embodiments, synthetic nanoparticle formulations such as those described herein can be administered systemically. Suitable genome editing formulations can be also comprise virus-like particles or extracellular vesicles. In certain approaches the VLPs, EVs or NLPs are studded with molecules that have affinity for receptor present on cells to be targeted. These can include receptors, membrane channel proteins, carbohydrates, antibodies or functional fragments thereof. See Tsuchida et al., (2023).

Any patent, patent application publication, or scientific publication, cited herein, is incorporated by reference herein in its entirety.

METHODS

LNP formulation

Commercially available SM102 and Alc315 ionizable lipids as well as the C24 ionizable lipids were mixed with helper lipids, cholesterol and PEG-DMA. The ratio of components constituting the lipid nanoparticles was 48% ionizable lipid, 9% DSPC, 3% DOPE, 38.5% cholesterol and 1.5% PEG-DMA. The N/P ratio was 4. ABE8.8 mRNA and gRNA (ratio of ABE8.8 mRNA:gRNA = 1 :1 ratio by mass), GFP mRNA, or Cre Recombinase mRNA was loaded in the LNPs and dosed as indicated below. The carrier for the LNP was phosphate buffered saline (PBS).

In vivo biodistribution studies in the mouse model

SMI 02, Alc315 and C24 LNPs were formulated as above carrying Cre recombinase mRNA (LNP.Cre_mRNA) and intravascularly injected at a dose of 1 to 3 mg/kg via vitelline vein (recapitulating umbilical vein injection in the human) to gestational day (E) 14 fetal or El 6 fetal SunlGFP mice, via facial vein to day of life 0 (P0) SunlGFP mice or via tail vein or retroorbital vein to 6-8 week old SunlGFP mice. The SunlGFP mice are a reporter mouse model such that successful expression of Cre recombinase results in GFP expression in the nuclear membrane. One month following injection, the heart, diaphragm, biceps and/or quadriceps, kidney, and lungs were harvested. Individual organs were disassociated into single cell suspensions and the cells lysed to access the nuclei. The nuclei were stained with antibodies to ERG (stains endothelial cells), PCM1 (stains myocytes), PAX7 (stains muscle satellite / stem cells) and Dapi (stains nuclei of all cells) and assessed by flow cytometry for GFP expression with or without the aforementioned co-stains. Finally muscles from SunlGFP mice injected with SM102 LNP.Cre mRNA was processed for immunohistochemistry and stained with antibodies for PAX7 (stains muscle satellite cells /stem cells) and Dapi (stains nuclei).

In vivo biodistribution studies in the NHP model SMI 02 and Alc315 LNPs were formulated as above carrying SunlGFP mRNA (LNP.SunlGFP_mRNA). Two mid-gestation Macaca fascicularis fetuses were used to assess the biodistribution of these LNPs following intravascular delivery. Specifically, one fetus was injected at gestational day 71 with Alc315 LNP carrying SunlGFP mRNA at a dose 5.5 mg/kg. A second fetus was injected at gestational day 98 with SMI 02 LNP carrying SunlGFP mRNA at a dose of 2.5 mg/kg. Both injections were performed using ultrasound guidance to identify the umbilical vein, and specifically the intrahepatic portion of the umbilical vein. The umbilical vein was cannulated with a 22-gauge spinal needle and blood was aspirated to confirm successful cannulation of the umbilical vein. Following cannulation, the injectate (consisting of the LNP carrying SunlGFP mRNA) was injected into the lumen of the umbilical vein in a total volume of 100 - 200 microliters over a period of 30 seconds to 60 seconds. The needle was withdrawn from the umbilical vein and maternal abdomen and the fetus and mom were monitored for fetal and maternal well-being. Twenty-four hours following injection of the LNP, fetuses were harvested by caesarian section and euthanized. The fetal heart was cannulated and the fetus perfused with sterile saline. Following perfusion, the organs, including the heart, diaphragm, biceps, quadriceps, kidney, and CD34 positive cells from the bone marrow, central liver and peripheral liver were harvested. Individual organs were processed into single cell suspensions and the cells lysed to obtain nuclei which were assessed by flow cytometry for GFP expression as well as other cell-specific co-stains including antibodies to CD34 (stains a hematopoietic progenitor cell), PCM1 (stains myocytes), and Dapi (stains nuclei).

Finally, heart specimens from the fetal NHP injected with SMI 02 LNP.SunlGFP_mRNA were processed for immunohistochemistry. They were stained with Dapi, anti GFP antibody and antibody to cardiac troponin for subsequent analyses.

In vivo base editing in the NHP model

In vivo CRISPR base editing of hepatocytes as well as other organs including heart, diaphragm, quadriceps, spleen, kidney, lung, gonad and hematopoietic cells was assessed following intravascular LNP delivery of an adenine base editor mRNA and HPD targeting gRNA in the Macaca fascicularis model at two different stages of development. Specifically, four midgestation Macaca fascicularis fetuses were injected via the intrahepatic portion of the umbilical vein with SMI 02 LNPs carrying ABE8.8 mRNA and HPD20 gRNA at the gestational age and doses indicated in table 1 . Two 3-year-old juvenile Macaca fascicularis NHPs were injected via the cephalic vein with SMI 02 LNPs carrying ABE8.8 mRNA and HPD20 gRNA at the doses indicated in table 1. On the day prior to LNP injection as well as 1-2 hours prior to LNP injection, the NHP mother or juvenile NHP was dosed with dexamethasone (Img/kg, intramuscular), famotidine (0.5 mg/kg, intramuscular), and diphenhydramine (5mg/kg, intramuscular). The LNP injection was performed following dilution in phosphate buffered saline to no more than a volume of 200 microliters for fetal recipients and 500 microliters for juvenile recipients. The injection was performed over 30 to 60 seconds in fetal recipients and over 1 hour in juvenile recipients. Injected animals were euthanized 1-2 weeks after injection and they were perfused with phosphate buffered saline. Following perfusion, individual organs were harvested. DNA from whole organs was extracted and next generation sequencing was performed to assess on-target A to G editing in the HPD gene.

In an additional set of NHP studies, 2 fetal Macaca fascicularis (gestational age El 14 and E123) and 4 juvenile Macaca fascicularis NHPs (2-3 years old) were injected via the intrahepatic umbilical vein (under ultrasound guidance) or the cephalic vein with LP01 LNPs carrying ABE8.8 mRNA and PCSK9 gRNA. The dose administered was 1.2-1.4 mg/kg of total RNA for fetal recipients and 2-2.5 mg/kg for juvenile recipients. The LNP was reconstituted in PBS and injected in the volume and over the injection time period noted above for the first set of NHP editing studies. Finally, the NHPs underwent the same premedication protocol as noted above. Two to three weeks after LNP injection, the injected fetal and juvenile NHPs were euthanized, perfused with heparinized saline (as noted above) and DNA from the organs harvested for next generation sequencing to assess on-target A to G editing at the PCSK9 locus.

To assess editing in hematopoietic cells of recipients, fetal liver and bone marrow mononuclear cells (MNCs) were isolated. DNA was isolated from an aliquot of the isolated MNCs and editing at the target HPD or PCSK9 site was assessed by next generation sequencing. Furthermore, the MNCs from the bone marrow and fetal liver were stained with an anti-CD34 antibody and CD34 positive cells isolated by flow cytometry sorting to a purity of greater than 98%. DNA was subsequently isolated from the sorted CD34 positive cells and assessed for on target editing by next generation sequencing. In vivo LNP mediated mRNA delivery in the sheep model

To assess biodistribution of LP01 LNP and mRNA expression of LP01 LNP packaged mRNA in fetal versus newborn recipients in a preclinical large animal model we formulated LP01 LNPs and packaged nuclear SunlGFP mRNA in the LNP. Two fetal sheep at E60 days, one fetal sheep at E90 days and a newborn sheep at day of life 3 were systemically injected with the LP01 LNP carrying SunlGFP mRNA via the umbilical vein and internal jugular vein respectively. The LNPs were reconstituted in PBS. For the fetal recipients, a 24 gauge angiocatheter was used to canulate the umbilical vein and a total volume of 250 microliters was injected over the course of approximately 60 seconds. For the day of life 3 recipients, a 22 gauge angiocatheter was used to canulate the internal jugular vein and a total volume of 20 milliliters was injected over the course of approximately 10 minutes. Twenty -four hours following injection, the organs were harvested and processed for flow cytometry to detect nuclear expression of GFP. To assess expression of GFP in hematopoietic progenitor cells, a single cell suspension from the fetal liver (for fetal recipients) and the bone marrow (for the newborn recipient) was costained with an anti-sheep CD34 antibody and subsequently assessed for coexpression of CD34 and GFP by flow cytometry. Prior to harvesting the organs, the recipients were perfused with heparanized saline to flush out the blood from the organs.

In vivo base editing of the dystrophin gene in muscle in the mouse model

We sought to assess the efficiency of in vivo adenine base editing of the exon 45 splice acceptor site of the mouse dystrophin gene based on the gestational age of systemic LNP delivery of a CRISPR editor mRNA and gRNA. As such, SM102 LNP was formulated with ABE8e mRNA and a gRNA targeting the exon 45 splice acceptor site of the mouse dystrophin gene. The LNP was injected into E14 or E16 fetal Balb/c mice via the vitelline vein (which is equivalent to the umbilical vein in large animal models and humans) in a total volume of 10-20 microliters or into 6-8 week old Balb/c mice via the retroorbital vein in a total volume of 100 microliters. One month after injection, mice were euthanized, perfused with heparinized saline, and DNA isolated from their organs including the liver, heart, diaphragm, quadriceps, and tibialis anterior. Next generation sequencing to assess on-target A to G editing at the exon 45 splice acceptor site was performed. Uninjected, age matched mice served as controls. Finally, in a subset of mice, nuclei from harvested muscle were stained with the antibody to PCM1 (a myocyte marker) and sorted by flow cytometry. Next generation sequencing was performed on PCM1+ sorted nuclei to assess in vivo editing in myocytes. Note, in addition to assessing in vivo editing of a muscle relevant gene, this approach is also clinically relevant because of the large number of Duchenne muscular dystrophy disease causing mutations in exon 45 of the dystrophin gene and the relevance of exon skipping of this and other exons that hardor disease-causing mutations as a viable therapeutic strategy.

The examples below are presented in order to more fully illustrate embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention.

EXAMPLE 1

Organ biodistribution of LNPs carrying mRNA delivered prenatally compared to postnatally in animal models

Three different LNPs, differing by the ionizable lipid used to formulate the LNP (SMI 02, Alc315 or C24), were formulated carrying Cre recombinase mRNA (LNP.Cre_mRNA) and delivered as described above. At the time of harvest, the mouse was systemically perfused by intracardiac puncture with phosphate buffered saline (PBS). Uninjected age matched mice served as negative controls. The heart, diaphragm, skeletal muscle (quadriceps and or biceps), kidney, and lung were harvested and individually morcellated to generate single cell suspensions and the nuclei were isolated for flow cytometric analyses for GFP as well as additional cell specific stains. Specifically, co-staining with Dapi was used to identify all nuclei, co-staining with PCM1 was used to identify myocytes, and co-staining with ERG was used to identify endothelial cells in specimens as indicated in Figure 1. Data represented is normalized for control background levels. Results from these studies demonstrated significantly improved LNP delivery with subsequent mRNA expression in myocytes as well as kidney cells in fetal mouse recipients compared to adult mouse recipients (Figure 1).

We next sought to determine if the ability to efficiently achieve exogenous mRNA expression in vivo in difficult to target organs via LNP delivery to fetal recipients was maintained in the more rigorous and clinically relevant NHP model. As described above, two mid-gestation Macaca fascicularis fetuses were injected with either an Alc315 LNP containing SunlGFP mRNA or a SM102 LNP containing SunlGFP. The fetal NHP recipient of the Alc315 LNP was injected at E71 at a dose of 5.5 mg/kg and the fetal NHP recipient of the SMI 02 LNP was injected at E98 at a dose of 2.5 mg/kg. Both fetuses were sacrificed at 24 hours post injection. Organs were processed as described above for the mouse studies and assessed by flow cytometry.

In addition to the organs described above for the mouse studies, bone marrow and liver were assessed for the percentage of CD34+ cells, a hematopoietic progenitor cell population, that were targeted in vivo by LNPs and expressed GFP. Finally, the heart tissue from the NHP fetal recipients was analyzed by immunohistochemistry to determine GFP expression in cardiomyocytes using cardiac troponin (cTnT) as a co-stain for cardiomyocytes. Negative controls for these NHP biodistribution studies consisted of organs processed the same way harvested from a fetal NHP recipient of the SM102 LNP containing ABE8.8 mRNA and a gRNA for the NHP HPD gene (HPD20 gRNA) (LNP ABE8.8 HPD20). This LNP does not contain an GFP mRNA. Data represented is normalized for control background levels.

As noted in Figure 2, these studies demonstrated efficient in vivo delivery of LNPs with subsequent mRNA expression in fetal cardiomyocytes, diaphragm, kidney, liver and bone marrow CD34+ cells, and some skeletal muscle cells. Histologic examination of hearts from experimental (recipients of the SMI 02 LNP containing Sun 1 GFP mRNA) and control (recipients of the SM102 LNP containing ABE8.8 mRNA and no GFP mRNA) fetal recipients confirms delivery of the LNP with mRNA expression in cardiomyocytes (Fig. 3A).

Ionizable lipid nanoparticle (LNP) mediated delivery of mRNA to muscle tissue including myocytes and satellite cells is dependent on gestational age at time of systemic injection in the mouse model.

Of note, the Sun 1 GFP mouse model contains a green fluorescent protein (GFP) around the nucleus that is expressed following Cre mediated excision of LoxP sites. Furthermore, antibodies specific for the nuclear transcription factor PCM1, which is highly expressed in myocytes, and PAX7, which is expressed in muscle satellite cell nuclei, were used to assess LNP mRNA expression in myocytes and muscle stem cells respectively. As such, we repeated the studies described in Figures 1A- 1G, now focusing on Alc315 and SM102 LNP delivery of Cre mRNA via systemic injection of the vitelline vein or retroorbital vein in E14 fetal, E16 fetal and 6-8 week old adult Sun 1 GFP mice. Injected mice were euthanized 1 month after LNP injection and the heart, diaphragm, and quadriceps of the mice were harvested. Prior to tissue harvesting, the mice were perfused with heparinized PBS. See Figs 1H to 10. Nuclei were isolated from the harvested organs and assessed by flow cytometry for GFP expression. Co-stains with antibodies to DAPI, PCM1 and PAX7 were used to identify all nuclei, myocytes and muscle satellite cells respectively. Of note, PAX7 does not stain satellite cells in the heart and thus costaining for this transcription factor was not performed in heart nuclei. These studies confirmed enhanced LNP mediated mRNA delivery to and expression in muscles cells following fetal injection that was not present following adult injection Finally, immunohistochemistry (IHC) was performed and demonstrated co-staining of GFP and PAX7 expression in the quadriceps and diaphragm of SunlGFP mice injected with LNPs as fetuses but not as adults (Fig. 3B). These studies suggest that a unique window of opportunity exists during fetal development that is not present in adults at which time LNP mediated delivery of mRNA to myocytes and muscle stem cells is possible independent of the LNP used.

In vivo base editing in difficult to target organs via LNP delivery of base editing mRNA to fetal NHPs

Encouraged that difficult to target organs/cells including the cardiomyocytes, diaphragm muscle cells, skeletal myocytes, kidney and CD34+ hematopoietic stem/progenitor cells could be targeted in fetal recipients in vivo via LNPs with exogenous mRNA expression in reporter mouse and NHP models, we next sought to determine if in vivo CRISPR-base editing could be accomplished in these organs/cells via LNP delivery in fetal NHP recipients. As such, midgestation Macaca fascicularis fetuses were injected via ultrasound guided cannulation of the umbilical vein where it enters the liver with SMI 02 LNP containing ABE8.8 mRNA and HPD20 gRNA. The LNP dose was between 1.8 and 2.5 mg/kg and the volume of injection was 150 microliters. Table 1 summarizes the fetal recipients of the LNP including the fetal ID number, dose of LNP, and gestational age at injection. Fetuses were sacrificed 1-2 weeks after injection and processed as described above. Specifically, the fetus was systemically perfused with PBS and then the organs harvested. Tissues of individual organs were subsequently processed to obtain genomic DNA from each organ / CD34+ cells. The genomic DNA was then analyzed by next generation sequencing to assess for adenine to guanine on-target base editing in the HPD gene. Controls for these studies consisted of fetal recipients of SM102 LNPs containing SunlGFP mRNA and no gene editing technology. The methods used for assessing on-target editing, including the primers and next generation sequencing protocol is the same as that used in the patent filing in 2022. Data represented is normalized for control background levels. As noted in Figure 4, on-target editing was noted in the fetal heart, diaphragm, kidney, skeletal muscle and CD34+ cells.

6324 2.5 mg/kg fetal E96 female

6033 2.4 fetal female

6338 1 .8 mg/kg fetal E85 male

Table 1. Fetal and juvenile NHP recipients of SM102 LNP containing ABE8.8 mRNA and HPD20 gRNA. E, gestational day; yr, years: ABE, adenine base editor.

Finally, we sought to confirm that the findings of editing of difficult to target organs via LNP delivery was unique to systemic injection (via the umbilical/intrahepatic vein) of the fetal NHP recipient, taking advantage of our mode and timing of delivery. As such, parallel studies were performed in 3 year old juvenile Macaca fascicularis recipients. Specifically, 2 juvenile NHPs were injected with SM102 LNPs containing ABE8.8 mRNA and HPD20 gRNA via cephalic vein (Table 1). Infusion was performed over 1 hour in a total volume of 250 microliters. The NHP recipients were sacrificed at 1 week post injection and processed as described above (systemic perfusion with PBS and organs individually harvested and processed for DNA isolation). Next generation sequencing was performed using the same protocol used for fetal recipients above. In contrast to fetal NHP recipients of SM102 LNP.ABE8.8 HPD20, juvenile recipients did not demonstrate any significant editing in the heart, diaphragm, kidney, or skeletal muscle (Figure 4). The liver of the juvenile recipients did demonstrate low-level on-target adenine to guanine editing of the HPD gene (Figure 5). This demonstrates that the LNPs were successfully injected. Notably, for diseases with a survival advantage of edited cells, such as hereditary tyrosinemia type 1, this level of on-target liver editing should be therapeutic. EXAMPLE 2

Gestational age and mode of delivery

Clinical application of in utero gene editing via LNP delivery of gene editing technology holds tremendous potential to mitigate genetic diseases in organs that are not readily accessible after birth and/or in which the disease process begins before or shortly after birth. In the current studies we have focused on base editing as an approach to in utero gene editing however, in utero editing via CRISPR mediated nonhomologous end joining as well as prime editing also hold significant therapeutic potential. See Newby et al. (2021). Clinical application of in utero gene editing via systemic delivery of an LNP (or other suitable vehicle) carrying gene editing technology could occur as early as 12 weeks of gestation up to including as late as the 40^th week of gestation. In certain embodiments, a single dose of the genome editing complex in a suitable carrier is administered. In other embodiments, multiple doses thoughout gestation can be administered to ensure adequate levels of editing are achieved. Suitable doses depend on several factors, e.g., the type of carrier or LNP employed and the gene to be targeted. Our data indicate that suitable doses can range from 0.1 mg/kg to 5 mg/kg; (e g., 0.1-0.2 mg/kg, 0.25-0.3 mg/kg, 0.5-0.75 mg/kg, 0.75-1 mg/kg, 1-1.5 mg/kg, 2-2.5 mg/kg, 3-3.5 mg/kg, 4-4.5 mg/kg and 5 mg/kg). In certain approaches, the safety profile is improved at lower doses.

As noted above, delivery of a therapeutic agent to a human fetal subject has been described (Somerset et al. (2006)). In approach, the treatment would be similar to that described above in the NHP fetus. Specifically, on the day prior to the LNP infusion and 1-2 hours prior to the LNP infusion the mother would be dosed with dexamethasone, famotidine, and diphenhydramine. With the mother under mild sedation and a local anesthetic applied to the abdominal wall, ultrasound guidance is used to access the umbilical vein either in the amniotic cavity or within the fetal liver (the intrahepatic portion of the umbilical vein) with a 20-gauge spinal needle. The therapeutic LNP gene editing formulation, diluted in phosphate buffered saline, is then delivered into the umbilical vein in a volume no larger than 500 microliters over the course of 30-60 seconds. The absolute volume of the injectate is dependent on the gestational age and thus size of the fetus and thus may range from 50 microliters to 500 microliters. The spinal needle is flushed with sterile PBS and the spinal needle is withdrawn. The fetus and mother are monitored for any untoward effects by ultrasound and assessment of vital signs including heart rate, blood pressure, and respiratory rate following the procedure.

EXAMPLE 3

LNP mediated delivery of adenine base editing mRNA and a guide RNA targeting the splice acceptor site of exon 45 of the dystrophin gene results in efficient base editing in fetal but not adult mice.

Duchenne muscular dystrophy (DMD) is the most common muscular dystrophy in children. It is an X-linked disease affecting 1 in 5000 boys and approximately 20,000 babies bom worldwide. Patients with DMD are usually wheelchair bound by teenage years and die by their 30s. It results from mutations in the dystrophin gene (DMD') with mutations in exons 44, 45, 50, 51 and 53 constituting 37% of disease-causing mutations. Naturally occurring mutations in the DMD gene resulting in a milder form of muscular dystrophy (Becker Muscular Dystrophy) suggest that editing strategies to “skip” exons containing DMD mutations represents a viable therapeutic approach. As such, we next sought to assess DMD exon 45 skipping via systemic LNP delivery of an adenine base editor to disrupt the exon 45 splice acceptor (SA) site in the mouse dystrophin gene in fetal and adult mouse recipients. In certain embodiments, the SM-102 ABE8e mRNALNPs was manufactured using the ionizable lipid SM-102, DSPC, cholesterol, and a PEG-DMG lipid at a final lipid molar ratio of 50:10:38.5: 1.5 and an N:P ratio of 6. LNP size and polydispersity were 80± 5nm (Z-average), and 0.1±0.05 SM102 LNP containing the ABE8e mRNA and the DMD exon 45 SA site targeting gRNA (at a mRNA to gRNA ratio of 1 : 1) was injected at a dose of 2 mg/kg into the vitelline vein or retroorbital vein of El 4, El 6, or 6-8 week old Balb/c mice. The gRNA sequence was 5’ GGCTTACAGGAACTCCAGGA 3’ (SEQ ID NO: 1) with a PAM sequence of TGG. The gRNA was modified as follows (SEQ ID NO: 2: mG*mG*mC*rUrUrArCrArGrGrArArCrUrCrCrArGrGrArGrUrUrUrUrArGrAmGmCm

UmAmGmAmAmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUr UrArUrCrAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUm CmGmGmUmGmCmU*mU*mU*U where m_* represents Phosphorothioated 2'-O-methyl RNA bases (3' and 5' end modifications), m_ represents 2' O-methyl RNA bases (scaffold base pairing; terminal Uridine modification) and r represents RNA bases. One month after injection, DNA from the liver, heart, diaphragm, quadricep and tibialis anterior of injected and noninjected control mice was harvested and assessed by next generation sequencing for on-target adenine to guanine editing.

As discussed above, fetal and adult recipients both demonstrate efficient liver editing. However, editing of muscle was significantly higher in fetal recipients with E14 recipients demonstrating greater muscle editing than E16 recipients and adult recipients demonstrating minimal editing above noninjected control mice. We also assessed muscle tissue editing in E14 fetal recipients at an LNP RNA dose of 2 mg/kg and 3 mg/kg compared to adult recipients that received an LNP RNA dose of 2 mg/kg. These studies confirmed improved muscle editing via LNP delivery in fetal recipients compared to adult recipients (Figures 6A -6D). Finally, we used flow cytometry to sort PCM1+ nuclei from the diaphragm and quadriceps of E14 Balb/c fetal mice injected with SMI 02 LNP containing ABE8e mRNA and the DMD exon 45 SA targeting gRNA. Next generation sequencing of DNA from the sorted nuclei compared to whole tissue confirmed that editing was occurring in myocytes (Figure 6E).

Myotonic dystrophy type 1

Myotonic dystrophy type 1 (DM1) has an incidence of 1/2,100 births in the United States. It results from an expansion of CTG repeats in the 3’ untranslated region (UTR) of the DMPK (dystrophia myotonica protein kinase) gene located at 19ql 3.32. Increasing numbers of CTG repeats correlates with greater severity and earlier onset of the disease. The most severe form of the disease, congenital DM1, has greater than 1000 CTG repeats and can present before birth. Specifically, congenital DM1 often presents before birth as polyhydramnios and reduced fetal movement (signs and symptoms identifiable on prenatal ultrasound and via a maternal history). Furthermore, there is a high risk of congenital DM1 if the mother has mild or classic DM1 due to increased repeat expansion on the maternal allele that is passed to the fetus. This allows for directed prenatal screening in pregnancies with a family history. An estimated 1/50,000 births in the United States have the congenital form of myotonic dystrophy type 1 (DM1). These severe cases of DM1 can be fatal because of respiratory insufficiency and/or cardiac failure. Specifically, mechanical ventilation is required for 70%-80% or more of congenital DM1 patients and mortality rate approaches 40% in severely affected infants. Gene editing approaches including dual nuclease nonhomologous end-joining or prime editing to remove the DMPK CTG repeats in the heart, diaphragm and other skeletal muscles as well as other affected organs including the central nervous system, pancreas and eye represent a therapeutic approach for this disease. One study in the mouse model of DM1 suggests that less than 10% editing in muscle is adequate to reduce pathologic RNA foci within myonuclei (Lo Scrudato et al., Molecular Therapy 2019). Furthermore, in this study, mice were treated after disease onset had occurred. Prenatal editing, prior to significant disease onset, may result is improved outcomes.

EXAMPLE 4

LP01 LNP FORMULATION FOR TARGETED GENE EDITING IN MULTIPLE FETAL ORGANS IN NHPs

Systemic injection of LNPs made of the ionizable lipid LP01 carrying adenine base editing mRNA and &PCSK9 targeting guide RNA result in efficient editing of muscle cells in fetal but not adult nonhuman primate recipients.

LNPs made with the ionizable lipid LP01, an amine lipid, DSPC, cholesterol, and a PEG- DMG lipid at an N:P ratio of 6 were combined at a final ratio of 51.68:9.0:36.32:3.0. LNP size and poly dispersity were 65nm (Z-average), and 0.05 respectively. ABE8.8 mRNA and a gRNA targeting the nonhuman primate (NHP) PCSK9 gene were packaged in the LNP at a ratio of 1 : 1. The LNPs were then systemically delivered to juvenile NHPs (2-3 years old) or fetal NHPs at E105 (0.68G) and E96 (0.62G). The dose of the juvenile recipients was 2.0-2.5 mg/kg and the dose of the fetal recipients was 1.22-1.39 mg/kg. The LNPs were administered after premedication of the recipients as noted above (for recipients of the SM102 LNP containing ABE8.8 mRNA and HPD targeting gRNA). Similarly, the LNPs were resuspended in PBS and the volume of LNP administered as well as the time period over which the LNPs were given was the same as noted above. Organs were harvested at 2-3 weeks post injection and assessed by next generation sequencing for on-target adenine to guanine base editing. At the time of sacrifice, the fetal and juvenile NHPs were perfused with heparinized saline prior to harvesting the organs for subsequent analyses. Both fetal and juvenile recipients demonstrated robust on-target editing in DNA isolated from the liver (Fig. 7A). In contrast, DNA isolated from muscle tissues including the heart, diaphragm, biceps, and quadriceps demonstrated significant on-target editing in fetal but not juvenile recipients (Figs 7B). This data in the NHP, together with that presented above in the mouse studies, suggest the nonobvious finding that LNP mediated delivery of gene editing technology can successfully edit muscle tissue in fetal but not postnatal recipients. Systemic injection of LNPs made of the ionizable lipid LP01 carrying adenine base editing mRNA and a PCSK9 targeting guide RNA result in efficient editing of hematopoietic progenitor cells in fetal nonhuman primate recipients.

It has been difficult to target hematopoietic stem cells for CRSIPR gene editing in vivo in NHPs via LNP delivery in postnatal recipients. We hypothesized that fetal development may also provide a favorable environment for in vivo CRISPR gene editing of hematopoietic stem cells (HSCs) via LNP delivery of CRISPR mRNA and a relevant gRNA. As such, we assessed for in vivo editing of CD34+ cells, a marker of a hematopoietic progenitor cell, and CD34- cells isolated from the fetal liver and bone marrow of NHPs injected with the above LP01 LNP containing ABE8.8 mRNA and the PCSK9 targeting gRNA. We found significant on-target editing of CD34+ cells, [from ~4 to ~18%] especially those isolated from the fetal liver, supporting the nonobvious finding that the fetal environment is conducive to in vivo HSC editing via LNP delivery of CRISPR editing technology (Fig. 7C).

EXAMPLE 5

LP01 LNP mediated delivery and expression of mRNA in fetal versus newborn sheep further evidences that the prenatal environment provides advantages for targeting muscle and hematopoietic stem cells.

To assess biodistribution of LP01 LNP and mRNA expression of LP01 LNP packaged mRNA in fetal versus newborn recipients in a preclinical large animal model we formulated LP01 LNPs according to the parameters noted above and packaged nuclear SunlGFP mRNA in the LNP. Two fetal sheep at gestational age 60 days, one fetal sheep at gestational age 90 days and a newborn sheep at day of life 3 were systemically injected with the LP01 LNP carrying SunlGFP mRNA via the umbilical vein and internal jugular vein respectively. The LNPs were reconstituted in PBS. For the fetal recipients, a 24 gauge angiocatheter was used to canulate the umbilical vein and a total volume of 250 microliters was injected over the course of approximately 60 seconds. For the day of life 3 recipients, a 22 gauge angiocatheter was used to canulate the internal jugular vein and a total volume of 20 milliliters was injected over the course of approximately 10 minutes. Twenty -four hours following injection, the organs were harvested and processed for flow cytometry to detect nuclear expression of GFP Prior to harvesting the organs, the recipients were perfused with heparanized saline to flush out the blood from the organs. Results from these studies again confirmed enhanced LNP delivery and expression of GFP in the liver, CD34+ hematopoietic cells, and muscle, including the heart, diaphragm, biceps and quadriceps, in fetal compared to newborn recipients (Figure 8). These fetal/neonatal sheep studies, together with the above NHP and mouse studies, support the nonobvious finding that the developmental properties of the fetus together with our LNPs allow for LNP mediated delivery of RNA, including therapeutic mRNAfor gene editing and replacement therapies as well as siRNA, to organs not readily accessible for LNP mediated RNA delivery after birth.

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While certain of the preferred embodiments of the present invention have been described and specifically exemplified above, it is not intended that the invention be limited to such embodiments. Various modifications may be made thereto without departing from the scope and spirit of the present invention, as set forth in the following claims.

Claims

What is claimed is:

1. A sterile injectable composition for systemic injection into a vein selected from an umbilical vein, an intrahepatic umbilical vein, or a cephalic vein of a fetal subject in need thereof, comprising lipid nanoparticles (LNP) containing ionizable lipid and an RNA of interest which forms a ribonucleoprotein complex upon expression in a target tissue or organ in a fetal subject in need thereof for correction of a genetic mutation associated with disease, said composition effecting correction in a fetal target tissue or fetal organ in utero selected from one or more of heart, diaphragm, skeletal muscle, kidney, and CD34+ hematopoietic progenitor cells in the absence of a viral vector.

2. The sterile injectable composition of claim 1, wherein said ionizable lipid is selected from LP01, SM102, Alc315.

3. The sterile injectable composition of claim 1, said LNP comprising ionizable lipid LP01, an amine lipid, DSPC, cholesterol, and a PEG-DMG lipid at an N:P ratio of 6 combined at a final ratio of 51.68:9.0:36.32:3.0, said LNP having a size and poly dispersity of 65nm, and 0.05 (Z- average), respectively.

4. The sterile injectable composition of claim 1, said LNP comprising ionizable lipid SM102, DSPC, cholesterol and PEG-DMG at the percentages of 50: 10:38.5: 1.5 and an N:P ratio of 5.67, said LNP having a size and poly dispersity of 80nm (Z-average), and 0.12 respectively.

5. The composition of any one of claim 1 to claim 4, wherein said ribonucleoprotein complex formed is an adenine base editor or a cytosine base editor.

6. The composition of one of claim 1 to claim 4, wherein said ribonucleoprotein complex comprises a guide strand for CRISPR -Cas directed editing.

7. The composition of claim 1, wherein said disease is selected from hereditary tyrosinemia type 1, Duchenne muscular dystrophy, familial hypercholesterolemia and said genetic mutation is in a gene selected from HPD, dystrophin or PCSK9 respectively.

8. A method for delivery of an RNA of interest as claimed in any one of claims 1 to 4 to a target tissue or organ in a fetal subject in need thereof for correction of a genetic mutation associated with disease, comprising accessing the umbilical cord of a fetus or fetal intrahepatic vein and administering one or more doses of an effective amount of a pharmaceutical formulation comprising a carrier operably linked to a nucleic acid encoding a gene editing ribonucleoprotein complex under conditions where said formulation enters one or more fetal tissues or organs selected from heart, diaphragm, skeletal muscle, kidney, and CD34+ hematopoietic progenitor cells, wherein said ribonucleoprotein complex editing corrects said mutation, thereby treating said disease in the absence of a viral vector.

9. The method of claim 8, wherein said administration occurs after 12 weeks and before 40 weeks of gestation and said disease is selected from hereditary tyrosinemia type 1, Duchenne muscular dystrophy, familial hypercholesterolemia, and said genetic mutation is in a gene selected from HPD, dystrophin or PCSK9 respectively.

10. The method of claim 8, wherein multiple doses are administered after 12 weeks of gestation.

11. The method of claim 8, wherein said carrier is modified to contain a targeting molecule which binds to a tissue or organ of interest.

12. The method of claim 8, wherein 2 or 3 doses are administered at least 2 weeks apart.

13. The method of claim 8, wherein a ribonucleoprotein complex forms upon expression of said RNA effective for adenine base editing or cytosine base editing.

14. The method of claim 8, wherein said ribonucleoprotein complex comprises a guide strand for CRISPR-Cas directed editing.

15. The method of claim 13, wherein, the ribonucleoprotein is an adenine base or cytosine base editor having an editor mRNA and gRNA ratio of 1 : 1 by mass.

16. The method of claim 8 or 9, wherein said formulation is administered at a dose ranging between 0.1 to 5 mg/kg.

17. The method of claims 8 to 15, wherein said dose is selected from 0.1-0.2 mg/kg, 0.25- 0.30 mg/kg, 0.50-0.75 mg/kg, 0.75-1 mg/kg, 1-1.5 mg/kg, 2-2.5 mg/kg, 3-3.5 mg/kg, 4-4.5 mg/kg and 5 mg/kg.

18. A sterile injectable composition for treatment of hereditary tyrosinemia type 1 formulated for systemic injection into the intrahepatic umbilical vein or the cephalic vein of a subject in need thereof, comprising i) lipid nanoparticles (LNP) containing 48% ionizable lipid, 9% DSPC, 3% DOPE, 38.5% cholesterol and 1.5% PEG-DMA, an N:/P ratio of 4, an adenine base editor ABE8.8 mRNA and a gRNA targeting an HPD gene, or ii) LNP containing ionizable lipid LP01, an amine lipid, DSPC, cholesterol, and a PEG-DMG lipid at an N:P ratio of 6 combined at a final ratio of 51.68:9.0:36.32:3.0, said LNP having a size and poly dispersity of 65nm, and 0.05 respectively, an adenine base editor ABE8.8 mRNA and a gRNA targeting an HPD gene; said an editor mRNA and gRNA ratio of i) or ii) being 1 : 1 by mass, said composition effecting base editing in a target tissue or organ selected from one or more of heart, diaphragm, skeletal muscle, kidney, and CD34+ hematopoietic progenitor cells.

19. The composition of claim 18, which is administered at a dose selected from .0.1 -0.2 mg/kg, 0.25-0.3 mg/kg, 0.5-0.75 mg/kg, 0.75-1 mg/kg, 1-1.5 mg/kg, 2-2.5 mg/kg, 3-3.5 mg/kg, 4-4.5 mg/kg and 5 mg/kg25-.3O mg/kg, ,50-.75mg/kg, ,75-lmg/kg, l-1.5mg/kg, 2-25mg/kg, 3- 3.5mg/kg, 4-4.5mg/kg and 5mg/kg.

20. The composition of claim 18 or 19 formulated for a single administration.

21. The composition of claim 18 or 19 formulated for multiple administrations.

22. The method of claim 8, wherein said mutation is present in an exon in a dystrophin gene selected from exons 44, 45, 50, 51 or 53, said target tissue is selected from one or more of muscle, heart, and diaphragm, said disease is Duchenne muscular dystrophy and said mutation is edited to correct the mutation or edited to effect exon skipping of the mutation-containing exon.

23. The method of claim 8, wherein the mutation comprises an expansion of CTG repeats in a 3’ untranslated region (UTR) of a dystrophia myotonica protein kinase (DMPK) gene located at 19q 13.32, said target tissue is muscle selected from heart, skeletal muscle and diaphragm, said disease is Myotonic dystrophy type 1 and said mutation is edited via nuclease mediated excision of CTG repeats or insertion of a termination signal prior to the CTG repeats via prime editing.

24. The method of claim 8, wherein the mutation is present in a gene selected from PTPN11, SOS1, RAFI, and RIT1, said disease of Noonan syndrome, said target tissue is selected from muscle, and heart and the editing strategy is the correction of the disease causing mutation.

25. The method of claim 8, wherein the mutation is duplication of a chromosome selected from chromosome 21, 13, or 18, the target tissue selected from muscle, and heart, said disease is trisomy 21, trisomy 13 or trisomy 18 respectively, and the editing approach involves nuclease mediated degradation of one of the three copies of the chromosome via targeting of alleles specific to that copy of chromosome 21, 13 or 18, or via epigenetic silencing of one of the three copies of chromosome 21, 13, or 18, via induction of Barr body formation.

26. The method of claim 8, wherein the mutation is C.2560OT in the GAA gene, the target tissue is liver and heart, the disease is Pompe disease and said mutation is corrected via adenine base editing or prime editing.

27. The method of claim 8, wherein the mutation is a GAA triplet repeat in the frataxin (FXN) gene, the target tissue is heart and the editing occurs via nuclease editing which excises the triplet repeats or prime editing for insertion of a termination signal prior to the GAA repeats.

28. The method of claim 8, wherein the mutation is a single point mutation associated with i) a beta7^Glu>Val substitution in the beta globin gene, and said disease is sickle cell disease; ii) a mutation in a beta globin gene causative of beta thalassemia; or iii) a mutation in an alpha globin gene associated with alpha thalassemia, wherein said target tissue is CD34+ cells and editing is performed via prime editing or base editing to correct the underlying mutation, or base editing to change the mutation to a less severe disease causing mutation, or base editing or nuclease editing to upregulate fetal gamma globin or zeta globin expression.

29. The method of claim 8, wherein the mutation is present in a PKD1 or PKD2 gene, the said disease is autosomal dominant polycystic kidney disease, said target tissue is kidney and the editing occurs via at a method selected from base editing, prime editing or nuclease editing to disrupt the PDK1 or PDK2 3’UTR miR-17 binding element in the PKD1 or PKD2 gene.

30. A kit for practicing the method of claim 8.