Haematopoietic stem and / or progenitor cell delivery method and compositions for use thereof

Abstract

The present disclosure provides a method for delivery of cargo to a haematopoietic stem or progenitor cell of a subject, the method comprising contacting a lipid nanoparticle encapsulating the cargo with the haematopoietic stem or progenitor cell ex vivo or in vivo, thereby causing cellular uptake of the cargo, the lipid nanoparticle having between 30 mol% and 70 mol% of a neutral or zwitterionic amphipathic lipid having a net-neutral charge at physiological pH, an ionizable cationic lipid, and a sterol, wherein the lipid nanoparticle is substantially uncharged at physiological pH and has an apparent pKa of between 6.0 and 7.5. Further provided are lipid nanoparticle compositions and uses thereof.

Description

HAEMATOPOIETIC STEM AND / OR PROGENITOR CELL DELIVERY METHOD AND COMPOSITIONS FOR USE THEREOFTechnical Field

[0001] The present disclosure relates to methods of delivery of mRNA and compositions for delivery thereof to stem and / or progenitor cells.Background

[0002] Lipid nanoparticle (LNP) formulations represent a revolution in the field of nucleic acid delivery. An early example of a lipid nanoparticle product approved for clinical use is Onpattro™. Onpattro™ is a lipid nanoparticle-based short interfering RNA (siRNA) drug formulation for the treatment of polyneuropathies induced by hereditary transthyretin amyloidosis. The success of this LNP delivery system paved the way for the clinical development of the leading LNP -based COVID-19 mRNA vaccines.

[0003] The Onpattro™ LNP formulation consists of four main lipid components, namely: ionizable amino lipid, distearoylphosphatidylcholine (DSPC), cholesterol, and polyethylene glycol conjugated lipids (PEG-lipids) at respective molar amounts of 50 / 10 / 38.5 / 1.5. Onpattro™ (herein “baseline”) is still considered the gold standard for comparison in studies of LNP -mediated efficacy and current approaches to LNP design make few deviations from the four-component system.

[0004] Of these four components, the ionizable lipid makes up the bulk of the Onpattro™ formulation (50 mol%) and is considered important for the in vitro and in vivo activity of the LNP. Accordingly, most work in the field has focused primarily on improving this lipid component. The ionizable lipid, which is typically an amino lipid, has been carefully designed so that it is charged at low pH and near-neutral at physiological pH. This allows for electrostatic interactions between the lipid and the negatively charged nucleic acid during initial formulation. Since the ionizable lipid is near neutral at physiological pH, toxicity and renal clearance is reduced. After cellular uptake by endocytosis, the acidic environment of the endosome leads to an increase in the net positive charge of the ionizable amino lipids, which promotes fusion with the anionic lipids of the endosomal membrane and subsequent membrane destabilization and release of the nucleic acidbased therapeutics into the cytoplasm to exert their effects.

[0005] With respect to the remaining three lipid components, the PEG-lipid is well known for improving circulation longevity of the LNP and cholesterol functions to stabilize the particle. Generally, however, comparatively less attention has been devoted to studying DSPC beyond its role as a structural lipid.

[0006] While strides have been made in research relating to LNP-mediated nucleic acid delivery, it is widely known that the Onpattro™ formulation largely accumulates in liver (hepatic) tissues. The ability of LNPs to accumulate in organs and tissues beyond the liver would greatly expand the clinical utility of these delivery systems. In order to improve the delivery of nucleic acid cargo to extrahepatic tissues, such as the bone marrow and spleen, the particles should exhibit enhanced circulation lifetimes. Traditional approaches to achieve this rely on optimizing the levels of PEG- lipid in the LNP, but the inclusion of PEG-lipids in LNPs often results in transfection potencies that are low or unfavorable immune responses.

[0007] Studies have been conducted in vivo to investigate the ability of four-component, Onpattro™-type formulations to deliver siRNA beyond the liver. In particular, siRNA gene silencing beyond the liver was investigated with Onpattro™-type LNPs (MC3 / Chol / DSPC / PEG- DMG) incorporating DSPC at 10 and 40 mol% (Ordobadi, Lipid Nanoparticles for Delivery of Bioactive Molecules, 2019, A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy, The University of British Columbia). It was shown that the 10 mol% DSPC Onpattro™-like formulations had similar liver accumulation and blood circulation lifetimes as 40 mol% DSPC formulations (MC3 / Chol / DSPC / PEG-DMG; 18.5 / 40 / 40 / 1.5 mol%). Further, the 40 mol% DSPC siRNA-LNP only performed comparably to 10 mol% DSPC formulations in bone marrow gene silencing. These previous studies thus showed no clear benefit resulting from adjusting the levels of DSPC to improve extrahepatic delivery of nucleic acid cargo.

[0008] The above studies investigating extrahepatic delivery were confined to LNPs having siRNA cargo. However, siRNA reduces mRNA production in a cell and thus its impacts are transitory. On the other hand, editing of a cell’s genetic material by editors, such as CRISPR / Cas, zinc finger nucleases, Transcription Activator-Like Effector Nucleases (TALENs), among others can be passed on to progeny cells, thereby creating opportunity to treat hereditary or acquired diseases or disorders. The nuclease can be encoded by mRNA and in the case of CRISPR / Cas, is translatedin vivo and complexes with guide RNA (gRNA) to form a ribonucleoprotein complex. It is desirable to target such gene editing systems to stem or progenitor cells that reside in the bone marrow, spleen, among other extrahepatic tissues as this could correct genetic mutations at the source and then effect downstream progeny cells. Nonetheless, this remains challenging until extrahepatic delivery of LNPs can be realized.

[0009] It has been suggested that targeting and improved cellular uptake beyond the liver, such as to haematopoietic stem or progenitor cells, could be improved by incorporating small-molecule ligands into LNP formulations that target specific tissue types, including surface receptors on stem cells. However, such an approach could lead to unwanted immune responses mounted against the targeting ligands.

[0010] There is thus a need in the art to improve the delivery of nucleic acid to haematopoietic stem and / or progenitor cells using lipid nanoparticles with improved targeting and / or uptake into such cells.Summary

[0011] The present disclosure addresses one or more of the foregoing problems in the prior art and / or provides useful alternatives to known compositions for the delivery of cargo to stem and / or progenitor cells.

[0012] The present disclosure is based on the finding that an LNP formulation (herein “IcLNP”) that includes elevated levels of neutral lipid relative to an Onpattro™ benchmark LNP (“baseline”) can improve the delivery of cargo to stem and / or progenitor cells. In some embodiments, the present disclosure provides an LNP formulation that has improved delivery of cargo in stem and / or progenitor cells over the baseline LNP, as measured in a sub-set of stem and progenitor cells in the bone marrow in an animal model at predetermined times after administration, such as at one hour, three hours or 24 hours post-administration.

[0013] According to one aspect of the disclosure, there is provided a method for delivery of cargo to a haematopoietic stem or progenitor cell of a subject, the method comprising contacting a lipid nanoparticle encapsulating the cargo with the haematopoietic stem or progenitor cell ex vivo or in vivo, thereby causing cellular uptake of the cargo, the lipid nanoparticle having between 30 mol%and 70 mol% of a neutral or zwitterionic amphipathic lipid having a net-neutral charge at physiological pH, an ionizable cationic lipid, and a sterol, wherein the lipid nanoparticle is substantially uncharged at physiological pH and has an apparent pKa of between 6.0 and 7.5.

[0014] According to one embodiment of the foregoing aspect, the neutral or zwitterionic lipid is a phospholipid having a choline head group and is selected from distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidylcholine (DMPC) and / or dipalmitoyl-phosphatidylcholine (DPPC).

[0015] According to another embodiment of the foregoing aspect or embodiment thereof, the contacting is in vivo and the haematopoietic stem or progenitor cell is in the spleen or bone marrow of the subject.

[0016] According to another embodiment of the foregoing aspect or embodiment thereof, the cargo comprises a nucleic acid that is part of a nucleic acid editor.

[0017] According to another embodiment of the foregoing aspect or embodiment thereof, the nucleic acid editor is a Cas-based editor, transcription activator-like effector nuclease (TALEN), megaTAL, zinc finger nuclease (ZFN), Adenosine Deaminase Acting on RNA (ADAR), prime editor, base editor, epigenetic editor, transposase, meganuclease, ARCUS gene editing system or a combination thereof.

[0018] According to another embodiment of the foregoing aspect or embodiment thereof, the nucleic acid editor is CRISPR and comprises a guide RNA and an mRNA or vector DNA coding for a Cas nuclease.

[0019] According to another embodiment of the foregoing aspect or embodiment thereof, the guide RNA is part of the nucleic acid editor and the method further comprises contacting the haematopoietic stem or progenitor cell in vivo or ex vivo with a second lipid nanoparticle comprising an mRNA coding for a Cas nuclease.

[0020] According to another embodiment of the foregoing aspect or embodiment thereof, the lipid nanoparticle is used to treat a disease or disorder that is a genetic disorder.

[0021] According to another embodiment of the foregoing aspect or embodiment thereof, the genetic disorder results from altered blood cell maturation and function.

[0022] According to another embodiment of the foregoing aspect or embodiment thereof, the genetic disorder resulting from the altered blood cell maturation and function is a disease of the innate and adaptive immune system, a red blood cell disorder, a platelet disorder or a bone marrow failure syndrome.

[0023] According to another embodiment of the foregoing aspect or embodiment thereof, the genetic disorder is a primary immunodeficiency.

[0024] According to another embodiment of the foregoing aspect or embodiment thereof, the primary immunodeficiency is selected from X-linked severe combined immunodeficiency (SCID), adenosine deaminase (ADA) deficiency SCID, Wiskott-Aldrich (WAS) syndrome and chronic granulomatous disease (CGD).

[0025] According to another embodiment of the foregoing aspect or embodiment thereof, the red blood cell disorder is an erythrocyte disorder.

[0026] According to another embodiment of the foregoing aspect or embodiment thereof, the erythrocyte disorder is a beta-Thalassaemia, a sickle cell disease or a pyruvate kinase deficiency.

[0027] According to another embodiment of the foregoing aspect or embodiment thereof, the lipid nanoparticle is used to treat a disease or disorder that is a cancer.

[0028] According to another embodiment of the foregoing aspect or embodiment thereof, the cancer is a haematological cancer.

[0029] According to a further aspect of the disclosure, there is provided a lipid nanoparticle encapsulating least a component of a nucleic acid editor for ex vivo or in vivo delivery to a haematopoietic stem or progenitor cell, the lipid nanoparticle having between 30 mol% and 70 mol% of neutral lipid or zwitterionic amphipathic lipid having a neutral or net-neutral charge at physiological pH, an ionizable cationic lipid, and a sterol, wherein the lipid nanoparticle is substantially uncharged at physiological pH and has an apparent pKa of between 6.0 and 7.5.

[0030] According to another embodiment of the foregoing aspect, the phospholipid having a choline head group is present at a content of at least 35 mol% and wherein the phospholipid is selected from distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidylcholine (DMPC) and / or dipalmitoyl-phosphatidylcholine (DPPC) and wherein the lipid nanoparticle has less than 40 mol% ionizable cationic lipid.

[0031] According to another embodiment of the foregoing aspect or embodiment thereof, the nucleic acid editor is a Cas-based editor, transcription activator-like effector nuclease (TALEN), megaTAL, zinc finger nuclease (ZFN), Adenosine Deaminase Acting on RNA (ADAR), prime editor, base editor, epigenetic editor, transposase, meganuclease, ARCUS gene editing system or a combination thereof.

[0032] According to another embodiment of the foregoing aspect or embodiment thereof, the nucleic acid editor is CRISPR and comprises a guide RNA and an mRNA coding for a Cas nuclease.

[0033] According to another embodiment of the foregoing aspect or embodiment thereof, the nucleic acid editor is used to treat a genetic disorder resulting from altered blood cell maturation and function.

[0034] According to another embodiment of the foregoing aspect or embodiment thereof, the genetic disorder resulting from the altered blood cell maturation and function is a disease of the innate and adaptive immune system, a red blood cell disorder, a platelet disorder or a bone marrow failure syndrome.

[0035] According to another embodiment of the foregoing aspect or embodiment thereof, the nucleic acid editor complex is used to treat a disorder that is an immunodeficiency, optionally a primary immunodeficiency.

[0036] According to another embodiment of the foregoing aspect or embodiment thereof, the primary immunodeficiency is selected from X-linked severe combined immunodeficiency (SCID), adenosine deaminase (ADA) deficiency SCID, Wiskott-Aldrich (WAS) syndrome and chronic granulomatous disease (CGD).

[0037] According to another embodiment of the foregoing aspect or embodiment thereof, the red blood cell disorder is an erythrocyte disorder.

[0038] According to another embodiment of the foregoing aspect or embodiment thereof, the erythrocyte disorder is a beta-Thalassaemia, a sickle cell disease or a pyruvate kinase deficiency.

[0039] According to another embodiment of the foregoing aspect or embodiment thereof, the lipid nanoparticle is used to treat a disease or disorder that is a cancer.

[0040] According to another embodiment of the foregoing aspect or embodiment thereof, the cancer is a haematological cancer.

[0041] According to a further aspect of the disclosure, there is provided use of a lipid nanoparticle to deliver and cause cellular uptake of a cargo to a haematopoietic stem or progenitor cell of a subject, the lipid nanoparticle having between 30 mol% and 70 mol% of a phospholipid having a choline head group, an ionizable cationic lipid, and a sterol, wherein the lipid nanoparticle is substantially uncharged at physiological pH and has an apparent pKa of between 6.0 and 7.5.

[0042] According to another embodiment of the foregoing aspect or embodiment thereof, the phospholipid having a choline head group is present at a content of at least 35 mol% and wherein the phospholipid is selected from distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidylcholine (DMPC) and / or dipalmitoyl-phosphatidylcholine (DPPC) and wherein the lipid nanoparticle has less than 40 mol% ionizable cationic lipid.

[0043] According to another embodiment of the foregoing aspect or embodiment thereof, the haematopoietic stem or progenitor cell is in the spleen or bone marrow of the subject.

[0044] According to another embodiment of the foregoing aspect or embodiment thereof, the cargo comprises a nucleic acid that is part of a nucleic acid editor complex.

[0045] According to another embodiment of the foregoing aspect or embodiment thereof, the nucleic acid editor is nucleic acid editor is a Cas-based editor, transcription activator-like effector nuclease (TALEN), megaTAL, zinc finger nuclease (ZFN), Adenosine Deaminase Acting on RNA (ADAR), prime editor, base editor, epigenetic editor, transposase, meganuclease, ARCUS gene editing system or a combination thereof.

[0046] According to another embodiment of the foregoing aspect or embodiment thereof, the nucleic acid editor is CRISPR and comprises a guide RNA and an mRNA or vector DNA coding for a Cas nuclease.

[0047] According to another embodiment of the foregoing aspect or embodiment thereof, nucleic acid is a guide RNA that is part of the nucleic acid editor and the method further comprises contacting the haematopoietic stem or progenitor cell in vivo or ex vivo with a second lipid nanoparticle comprising an mRNA coding for a Cas nuclease.

[0048] According to another embodiment of the foregoing aspect or embodiment thereof, the lipid nanoparticle is used to treat a disease or disorder that is a genetic disorder.

[0049] According to another embodiment of the foregoing aspect or embodiment thereof, the genetic disorder results from altered blood cell maturation and function.

[0050] According to another embodiment of the foregoing aspect or embodiment thereof, the genetic disorder resulting from the altered blood cell maturation and function is a disease of the innate and adaptive immune system, a red blood cell disorder, a platelet disorder or a bone marrow failure syndrome.

[0051] According to another embodiment of the foregoing aspect or embodiment thereof, the genetic disorder is an immunodeficiency, optionally a primary immunodeficiency.

[0052] According to another embodiment of the foregoing aspect or embodiment thereof, the primary immunodeficiency is selected from X-linked severe combined immunodeficiency (SCID), adenosine deaminase (ADA) deficiency SCID, Wiskott-Aldrich (WAS) syndrome and chronic granulomatous disease (CGD).

[0053] According to another embodiment of the foregoing aspect or embodiment thereof, the red blood cell disorder is an erythrocyte disorder.

[0054] According to another embodiment of the foregoing aspect or embodiment thereof, the erythrocyte disorder is a beta-Thalassaemia, a sickle cell disease or a pyruvate kinase deficiency.

[0055] According to another embodiment of the foregoing aspect or embodiment thereof, the lipid nanoparticle is used to treat a disease or disorder that is a cancer.

[0056] According to another embodiment of the foregoing aspect or embodiment thereof, the cancer is a haematological cancer.Brief description of the drawings

[0057] Figure l is a diagram showing how haematopoietic stem and progenitor cells (HSPC) give rise to immune cells. The boxed region of the figure shows long term haematopoietic stem cells (LT-HSC), short term HSCs (ST-HSC) and multipotent progenitors (MPP). These cells are part of the Lineage'Scal+cKit+(LSK) population that give rise to lymphoid progenitors and myeloid progenitors. In some embodiments, lymphoid and myeloid progenitors then give rise to downstream mature immune cells, such as NK cells, T cells, B cells, monocytes, neutrophils, among others.

[0058] Figure 2A shows size (nm), polydispersity (PDI) and encapsulation% of lipid nanoparticles composed of 10:50:38.5: 1.5 mol / mol ionizable cationic lipid l / DSPC / cholesterol / PEG2ooo-DMG (Onpattro™) and IcLNP™ composed of 27.4:50:21.1 : 1.5 ionizable cationic lipid / DSPC / Chol:PEG2ooo-DMG. The ionizable cationic lipids used in the IcLNP™ compositions were 1, 2, 3, 4 and 5 as described in Table 1 of Example 1.

[0059] Figure 2B shows the percentage of enhanced green fluorescent protein (eGFP) positive cells (% eGFP) in bone marrow LK (Lineage'c-Kit+) and LSK (Lineage c-Kit Scal ) subpopulations as indicated after 24 hours post-injection of phosphate buffered saline (PBS); and eGFP-mRNA LNPs having 10 mol% DSPC (nMC3:DSPC:Chol:PEG2ooo-DMG-lipid (50:10:38.5: 1.5) (“Onpattro™”)); or 50 mol% DSPC (ionizable cationic lipid:DSPC:Chol:PEG- lipid (27.4:50:21.1 : 1.5 mohmol) (“IcLNP™”)) to C57B1 / 6 mice. The ionizable cationic lipids used in the IcLNP compositions were 1, 2, 3, 4 and 5 as described in Table 1 of Example 1.

[0060] Figure 2C shows the percentage of enhanced green fluorescent protein (eGFP) positive cells (% eGFP) in bone marrow multipotent progenitor (MPP; Lineage c-Kit Scal+CD34+) andlong term HSC (LT-HSC; Lineage cKit+Scal+CD34‘ CD135 ) sub-populations as indicated after 24 hours post-injection of phosphate buffered saline (PBS); and eGFP-mRNA LNPs having 10 mol% DSPC (nMC3:DSPC:Chol:PEG2ooo-DMG-lipid (50:10:38.5:1.5) (“Onpattro™”)); or 50 mol% DSPC (ionizable cationic lipid:DSPC:Chol:PEG-lipid (27.4:50:21.1 : 1.5 mol:mol) (“IcLNP™”)) to C57Bl / 6mice. The ionizable cationic lipids used in the IcLNP compositions were 1, 2, 3, 4 and 5 as described in Table 1 of Example 1.

[0061] Figure 2D shows the percentage of enhanced green fluorescent protein (eGFP) positive cells (% eGFP) in bone marrow long term HSC (LT-HSC; Lineage cKit Scal+CD34' CD135' CD48'CD150+) sub-populations as indicated after 24 hours post-injection of phosphate buffered saline (PBS); and eGFP-mRNA LNPs having 10 mol% DSPC (nMC3:DSPC:Chol:PEG2ooo-DMG- lipid (50: 10:38.5: 1.5) (“Onpattro™”)); or 50 mol% DSPC (ionizable cationic lipid:DSPC:Chol:PEG-lipid (27.4:50:21.1 : 1.5 mol:mol) (“IcLNP™”)) to mice. The ionizable cationic lipids used in the IcLNP™ compositions were 1, 2, 3, 4 and 5 as described in Table 1 of Example 1.

[0062] Figure 3A shows size (nm), poly dispersity (PDI) and encapsulation% of luciferase control lipid nanoparticle 1 (luciferase mRNA) composed of ionizable cationic lipid l :DSPC:Chol (40:40:20 mol:mol), eGFP-mRNA lipid nanoparticle 2 (Onpattro™) composed of ionizable cationic lipid l :DSPC:Chol:PEG2ooo-DMG (50:10:38.5: 1.5 moFmol) or eGFP-mRNA unshielded IcLNP™ (LNPs 3 and 4) composed of ionizable cationic lipid:DSPC:Chol (40:40:20 moFmol). Ionizable cationic lipid 1 was used in the luciferase mRNA and Onpattro™ LNP formulations 1 and 2 and ionizable lipids 3 and 6 were used in the IcLNP™ compositions 3 and 4 (described in Table 3 of Example 2).

[0063] Figure 3B shows the percentage of enhanced green fluorescent protein (eGFP) positive cells (% eGFP) in bone marrow LK (Lineage'c-Kit+) and LSK (Lineage c-Kit Scal ) subpopulations as indicated after 24 hours post-injection of LNP 1 (luciferase mRNA), LNP 2 eGFP- mRNA (Onpattro™) and LNPs 3 and 4 (IcLNP™) to C57B1 / 6 mice. The LNP formulations 1 to 4 are described in Figure 3 A above.

[0064] Figure 3C shows the percentage of enhanced green fluorescent protein (eGFP) positive cells (% eGFP) in bone marrow multipotent progenitor (MPP; Lineage c-Kit Scal+CD34+), shortterm HSC (ST-HSC; Lineage'c-Kit+Sca-1+CD34+) and long term HSC (LT-HSC; Lineage cKit+Scal+CD34' CD135 ) sub-populations as indicated after 24 hours post-injection of of LNP 1 (luciferase mRNA), LNP 2 eGFP-mRNA (Onpattro™) and LNPs 3 and 4 (IcLNP™) to C57B1 / 6 mice. The LNP formulations 1 to 4 are described in Figure 3 A above.

[0065] Figure 4A depicts an Ail4-Cre mouse model used to assess in vivo prime editing of LNPs in bone marrow. The mouse model has a tox -flanked STOP cassette preventing transcription of a CAG promoter-driven red fluorescent protein variant (tdTomato). The LNPs encapsulate mRNA encoding for Cre recombinase.

[0066] Figure 4B shows size (nm), poly dispersity (PDI) and encapsulation% of luciferase control lipid nanoparticle 1 (luciferase mRNA) composed of ionizable cationic lipid LDSPC:Chol:PEG2ooo-DMG (27.4:50:21.1 : 1.5 mol:mol), Cre recombinase-mRNA lipid nanoparticle 2 (Onpattro™) composed of ionizable cationic lipid l :DSPC:Chol:PEG2ooo-DMG (50:10:38.5: 1.5 mol:mol) or Cre recombinase-mRNA IcLNP™ 3 composed of ionizable cationic lipid l :DSPC:Chol:PEG2ooo-DMG (27.4:50:21.1: 1.5 mol:mol). Ionizable cationic lipid 1 was used in the luciferase mRNA and Onpattro™ LNP formulations 1 and 2 and ionizable lipid 3 was used in the IcLNP™ formulation 3 (see Table 5 of Example 3).

[0067] Figure 4C shows the percentage of TdTomato cells in the bone marrow of LK (Lineage' c-Kit+) and LSK (Lineage c-Kit Sca l ) sub-populations at a dose of 0.3 mg / kg as indicated after 24 hours post-injection of LNP 1 luciferase mRNA, LNP 2 Cre recombinase-mRNA (Onpattro™) and LNP 3 Cre recombinase-mRNA (IcLNP™) with elevated DSPC to the Ail4-Cre mouse model used to assess in vivo prime editing. The lipid nanoparticle formulations 1, 2 and 3 are as described in Figure 4B above.

[0068] Figure 4D shows the percentage of TdTomato cells in the bone marrow of multipotent progenitor (MPP; Lineage c-Kit Scal+CD34+), short term HSC (ST-HSC; Lineage'c-Kit4Sca- 1+CD34+) ) and long term HSC (LT-HSC; Lineage cKit Scal+CD34‘ CD135') sub-populations at a dose of 0.3 mg / kg as indicated after 24 hours post-injection of LNP 1 luciferase mRNA, LNP 2 Cre recombinase-mRNA (Onpattro™) and LNP 3 Cre recombinase-mRNA (IcLNP™) with elevated DSPC to the Ail4-Cre mouse model used to assess in vivo prime editing. The lipid nanoparticle formulations 1, 2 and 3 are as described in Figure 4B above.

[0069] Figure 4E shows the percentage of TdTomato+cells in the bone marrow of long term HSC (LT-HSC; Lineage cKit Scal+CD34' CD135'CD48'CD150+) sub-population at a dose of 0.3 mg / kg as indicated after 24 hours post-injection of LNP 1 luciferase mRNA, LNP 2 Cre recombinase-mRNA (Onpattro™) and LNP 3 Cre recombinase-mRNA (IcLNP™) with elevated DSPC to the Ail4-Cre mouse model used to assess in vivo prime editing. The lipid nanoparticle formulations 1, 2 and 3 are as described in Figure 4B above.

[0070]

[0071] Figure 5A shows size (nm), poly dispersity (PDI) and encapsulation% of luciferase control lipid nanoparticle 1 (luciferase mRNA) composed of ionizable lipid l :DSPC:Chol:PEG2ooo-DMG (27.4:50:21.1 : 1.5), and eGFP-mRNA lipid nanoparticles, including lipid nanoparticle 2 (Onpattro™) composed of ionizable lipid 1 :DSPC :Chol :PEG2ooo-DMG (50: 10:38.5: 1.5 mol : m ol ), lipid nanoparticle 3 composed of ionizable lipid 7:DSPC:Chol:PEG2ooo-DMG (27.4:50:21.1 : 1.5 mokmol) and ionizable lipid composed of 3:DSPC:Chol: PEG2000-DMG (27.4:50:21.1 : 1.5 mokmol). The ionizable cationic lipids 1, 3 and 7 used in the LNP compositions are described in Table 6 of Example 4. Figure 5B shows the percentage of enhanced green fluorescent protein (eGFP) positive cells (% eGFP) in bone marrow human CD34+cells as indicated at 48 hours postinjection of luciferase or eGFP-mRNA LNPs. The lipid nanoparticle formulations 1, 2, 3 and 4 examined are as described in Figure 5A (see also Table 6 of Example 4).

[0072] Figure 5C shows the percentage of enhanced green fluorescent protein (eGFP) positive cells (% eGFP) in bone marrow human-CD34+CD3810cells (enriched for HSPCs) as indicated at 48 hours post-injection of luciferase control and eGFP-mRNA LNPs. The lipid nanoparticle formulations 1, 2, 3 and 4 are as described in Figure 5A above (see also Table 6 of Example 4).

[0073] Figure 5D shows the percentage of enhanced green fluorescent protein (eGFP) positive cells (% eGFP) in bone marrow human CD34+CD38loCD90+(enriched for HSCs) as indicated at 48 hours post-injection of luciferase control and eGFP-mRNA LNPs. The lipid nanoparticle formulations 1, 2, 3 and 4 are as described in Figure 5A above (see also Table 6 of Example 4).

[0074] Figure 5E shows size (nm), poly dispersity (PDI) and encapsulation% of shows size (nm), poly dispersity (PDI) and encapsulation% of luciferase control lipid nanoparticle 1 (luciferasemRNA) composed of ionizable lipid l :DSPC:Chol:PEG2ooo-DMG (27.4:50:21.1 : 1.5), and GFP- mRNA lipid nanoparticles, including LNP group 2 composed of ionizable lipid 8:DSPC:Chol: PEG2000-DMG (27.4:50:21.1 : 1.5 mol:mol) at 1 mg / kg (N / P = 9), LNP group 3 composed of ionizable lipid 3:DSPC:Chol: PEG2000-DMG (27.4:50:21.1 : 1.5 mol:mol) at 1 mg / kg (N / P = 9), LNP group 4 composed of ionizable lipid 3:DSPC:Chol: PEG2000-DMG (27.4:50:21.1: 1.5) at 1 mg / kg (N / P =6) and LNP group 5 composed of ionizable lipid 3:DSPC:Chol: PEG2000-DMG (27.4:50:21.1 : 1.5) at 2.5 mg / kg (N / P = 9). The LNPs and ionizable cationic lipids 1, 3 and 8 used in the LNP compositions are described in Table 8 of Example 4.

[0075] Figure 5F shows the percentage of enhanced green fluorescent protein (eGFP) positive cells (% eGFP) in bone marrow human CD34+cells as indicated at 48 hours post-injection of luciferase mRNA and eGFP -mRNA LNPs. The lipid nanoparticle groups 1-5 are as described in Figure 5E above (see also Table 8 of Example 4).

[0076] Figure 5G shows the percentage of enhanced green fluorescent protein (eGFP) positive cells (% eGFP) in bone marrow human-CD34+CD3810cells (enriched for HSPCs) as indicated at 48 hours post-injection of luciferase mRNA and eGFP-mRNA LNPs. The lipid nanoparticle groups 1-5 are as described in Figure 5E above (see also Table 8 of Example 4).

[0077] Figure 5H shows the percentage of enhanced green fluorescent protein (eGFP) positive cells (% eGFP) in bone marrow human CD34+CD38loCD90+(enriched for HSCs) cells as indicated at 48 hours post-injection of luciferase mRNA and eGFP-mRNA LNPs. The lipid nanoparticle groups 1-5 are as described in Figure 5E above (see also Table 8 of Example 4).

[0078] Figure 51 shows size (nm), poly dispersity (PDI) and encapsulation% of shows size (nm), poly dispersity (PDI) and encapsulation% of lipid nanoparticle groups 1-6 are as described in Table 9 of Example 4).

[0079] Figure 5J shows the percentage of enhanced green fluorescent protein (eGFP) positive cells (% eGFP) in bone marrow human CD34+cells as indicated at 48 hours post-injection of luciferase mRNA and eGFP-mRNA LNPs. The lipid nanoparticle groups 1-6 are as described in Table 9 of Example 4.

[0080] Figure 5K shows the percentage of enhanced green fluorescent protein (eGFP) positive cells (% eGFP) in bone marrow human-CD34+CD3810cells (enriched for HSPCs) as indicated at 48 hours post-injection of luciferase mRNA and eGFP-mRNA LNPs. The lipid nanoparticle groups 1-6 are as described in Table 9 of Example 4.

[0081] Figure 5L shows the percentage of enhanced green fluorescent protein (eGFP) positive cells (% eGFP) in bone marrow human CD34+CD38loCD90+(enriched for HSCs) cells as indicated at 48 hours post-injection of luciferase mRNA and eGFP-mRNA LNPs. The lipid nanoparticle groups 1-6 are as described in Table 9 of Example 4.Detailed Description

[0082] The lipid nanoparticle described herein is an improvement over the conventional four- component LNP, referred to herein as a baseline composed of MC3 / DSPC / Chol / PEG-DMG at 50: 10:38.5: 1.5 mol %. In certain advantageous embodiments, the LNP of the disclosure (referred to as IcLNP™) comprises ionizable cationic lipid, a phosphatidylcholine lipid, cholesterol and optionally a hydrophilic polymer-lipid conjugate, and in which the phosphatidylcholine lipid is present at a mol% of at least 20 mol% or at least 30 mol% and in which the ionizable lipid is present at less than 40 mol%. As set forth herein, elevated phosphatidylcholine mol% beyond what is used in conventional formulations for nucleic acid delivery provides unexpected increases in cargo delivery to haematopoietic stem or progenitor cells (HSPCs) relative to the benchmark formulation.

[0083] Figure 1 is a diagram depicting HSPCs that are targeted by the LNPs of the disclosure. Without limitation, the IcLNP™ herein may target any one or a combination of the cell types within the boxed region of the figure (long term haematopoietic stem cells (LT-HSC), short term HSC (ST-HSC) and multipotent progenitors (MPP)). These cells are part of the Lineage- Scal+CKit+ (LSK) population. In another embodiment, the IcLNP™ may target lymphoid progenitors or myeloid progenitors, which are derived from the multipotent progenitor cells. As shown, lymphoid progenitor cells give rise to NK cells, T cells and B cells. The myeloid progenitor cells give rise to monocytes, megakaryocytes, neutrophils and red blood cells. Accordingly, any genetic mutation in the HSPC cells could be corrected by delivery of gene editing cargo via the IcLNP™ to the HSPC cells.

[0084] As used herein, the term “cargo,” includes any payload, such as nucleic acid (e.g., RNA, DNA or hybrids), protein, peptide or any fragment thereof, that is capable of encapsulation in the LNP herein.

[0085] In some embodiments, the nucleic acid does not encode for a reporter protein or peptide such as eGFP. In some embodiments, the LNP is part of a pharmaceutical composition that has a therapeutic, diagnostic or prophylactic effect to a subject.

[0086] As used herein, the term “encapsulation,” with reference to incorporating the cargo within a lipid nanoparticle refers to any association of the cargo with any lipid component or compartment of the lipid nanoparticle. In one example of the disclosure, the cargo is present in the core of the LNP. Without limitation, the lipid nanoparticle described herein may comprise a cargo that is a messenger RNA.

[0087] As used herein, the term “messenger RNA” or “mRNA”, refers to a polynucleotide that encodes and expresses at least one peptide, polypeptide or protein. The term is meant to include mRNA that is circular or linear as well as small activating RNA (saRNA) and trans-amplifying RNA (taRNA).

[0088] The concentration of mRNA in the LNP may be between 0.01 and 20 mg / mL or between 0.01 and 10 mg / mL or between 0.05 and 5 mg / mL or between 0. 075 and 4 mg / mL.

[0089] The mRNA as used herein encompasses both modified and unmodified mRNA. In one embodiment, the mRNA comprises one or more coding and non-coding regions. The mRNA can be purified from natural sources, produced using recombinant expression systems and optionally purified, or may be chemically synthesized.

[0090] In those embodiments in which an mRNA is chemically synthesized, the mRNA can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, and / or backbone modifications. In some embodiments, an mRNA is or comprises natural nucleosides (e.g., adenosine, guanosine, cytidine, uridine); nucleoside analogs (e.g., 2-aminoadenosine, 2- thiothymidine, inosine, pyrrol o-pyrimidine, 3 -methyl adenosine, 5 -methylcytidine, C-5 propynyl- cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5 -fluorouridine, C5- iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C 5 -methyl cytidine, 2-aminoadenosine,7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine, 2- thiocytidine, pseudouridine, and 5-methylcytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioates and 5'-N-phosphoramidite linkages).

[0091] The mRNAs of the disclosure may be synthesized according to any of a variety of known methods. For example, mRNAs in certain embodiments may be synthesized via in vitro transcription (IVT). Briefly, IVT is typically performed with a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may include DTT and magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7 or SP6 RNA polymerase), DNAse I, pyrophosphatase, and / or RNAse inhibitor.

[0092] In some embodiments, in vitro synthesized mRNA may be purified before encapsulation to remove undesirable impurities including various enzymes and other reagents used during mRNA synthesis.

[0093] The present disclosure may be used to formulate mRNAs of a variety of lengths. In some embodiments, the present disclosure may be used to formulate and encapsulate in vitro synthesized mRNA ranging from about 1-20 kb, about 1-20 kb, about 1-15 kb, about 1-10 kb, about 2-20 kb, about 2-15 kb, about 2-10 kb, about 5-20 kb, about 5-15 kb, about 5-12 kb, about 5-10 kb, about8-20 kb, or about 8-15 kb in length.

[0094] In those embodiments in which the mRNA is linear, the synthesis includes the addition of a “cap” on the 5' end, and a “tail” on the 3' end. The presence of the cap provides resistance to nucleases found in most eukaryotic cells. The presence of a “tail” serves to protect the mRNA from exonuclease degradation.

[0095] In some embodiments, mRNAs include a 5' and / or 3' untranslated region. In some embodiments, a 5' untranslated region includes one or more elements that affect an mRNA's stability or translation, for example, an iron responsive element. In some embodiments, a 5' untranslated region may be between about 50 and 500 nucleotides in length.

[0096] In some embodiments, a 3' untranslated region includes one or more of a polyadenylation signal, a binding site for proteins that affect an mRNA's stability of location in a cell, or one or more binding sites for miRNAs. In some embodiments, a 3' untranslated region may be between 50 and 500 nucleotides in length or longer.

[0097] In a further embodiment, the mRNA is circular. Advantageously, such mRNA lacks 5’ and 3’ ends and thus may be more stable in vivo due to its resistance to degradation by exonucleases. The circular mRNA may be prepared by any known method, including any one of the methods described in Deviatkin et al., 2023, “Cap-Independent Circular mRNA Translation Efficiency”, Vaccines, 11(2), 238, which is incorporated herein by reference. Translation of the circular mRNA is carried out by a cap-independent initiation mechanism.

[0098] While mRNA provided from in vitro transcription reactions may be desirable in certain embodiments, other sources of mRNA are contemplated, such as mRNA produced from bacteria, fungi, plants, and / or animals.

[0099] The mRNA sequence may comprise a reporter gene sequence, although the inclusion of a reporter gene sequence in pharmaceutical formulations for administration is optional. Such sequences are incorporated into mRNA for in vivo studies in animal models to assess biodistribution.

[0100] In another embodiment, the cargo is a DNA vector. The encapsulated DNA vector may be administered to a subject for the purpose of repairing, enhancing or blocking or reducing the expression of a cellular protein or peptide.

[0101] As will be appreciated by those of skill in the art, the vectors may encode promoter regions, operator regions or structural regions. The DNA vectors may contain double-stranded DNA or may be composed of a DNA-RNA hybrid. Non-limiting examples of double-stranded DNA include structural genes, genes including operator control and termination regions, and selfreplicating systems such as vector DNA.

[0102] Single-stranded nucleic acids include antisense oligonucleotides (complementary to DNA and RNA), ribozymes and triplex-forming oligonucleotides. In order to have prolonged activity, the single-stranded nucleic acids will most advantageously have some or all of the nucleotidelinkages substituted with stable, non-phosphodiester linkages, including, for example, phosphorothioate, phosphorodithioate, phophoroselenate, or O-alkyl phosphotriester linkages.

[0103] The DNA vectors may include nucleic acids in which modifications have been made in one or more sugar moieties and / or in one or more of the pyrimidine or purine bases. Such sugar modifications may include replacement of one or more hydroxyl groups with halogens, alkyl groups, amines, azido groups or functionalized as ethers or esters. In another embodiment, the entire sugar may be replaced with sterically and electronically similar structures, including azasugars and carbocyclic sugar analogs. Modifications in the purine or pyrimidine base moiety include, for example, alkylated purines and pyrimidines, acylated purines or pyrimidines, or other heterocyclic substitutes known to those of skill in the art.

[0104] The DNA vector may be modified in certain embodiments with a modifier molecule such as a peptide, protein, steroid or sugar moiety. Modification of a DNA vector with such molecule may facilitate delivery to a target site of interest. In some embodiments, such modification translocates the DNA vector across a nucleus of a target cell. By way of example, a modifier may be able to bind to a specific part of the DNA vector (typically not encoding of the gene-of-interest), but also has a peptide or other modifier that has nucleus-homing effects, such as a nuclear localization signal. A non-limiting example of a modifier is a steroid-peptide nucleic acid conjugate as described by Rebuffat et al., 2002, Faseb J. 16(11): 1426-8, which is incorporated herein by reference. The DNA vector may contain sequences encoding different proteins or peptides. Promoter, enhancer, stress or chemically -regulated promoters, antibiotic-sensitive or nutrient-sensitive regions, as well as therapeutic protein encoding sequences, may be included as required. Non-encoding sequences may be present as well in the DNA vector.

[0105] The nucleic acids used in the present disclosure can be isolated from natural sources, obtained from such sources as ATCC or GenBank libraries or prepared by synthetic methods. Synthetic nucleic acids can be prepared by a variety of solution or solid phase methods. Generally, solid phase synthesis is preferred. Known procedures for solid phase synthesis of nucleic acids by phosphite-triester, phosphotriester, and H-phosphonate chemistries are widely available.

[0106] In one embodiment, the DNA vector is double stranded DNA and comprises more than 700 base pairs, more than 800 base pairs or more than 900 base pairs or more than 1000 base pairs.

[0107] In another embodiment, the cargo is an siRNA. An siRNA becomes incorporated into endogenous cellular machineries to result in mRNA breakdown, thereby preventing transcription. Since RNA is easily degraded, its incorporation into a delivery vehicle can reduce or prevent such degradation, thereby facilitating delivery to a target site.

[0108] The siRNA encompassed by embodiments of the disclosure may be used to specifically inhibit expression of a wide variety of target polynucleotides. The siRNA molecules targeting specific polynucleotides for any therapeutic, prophylactic or diagnostic application may be readily prepared according to procedures known in the art. An siRNA target site may be selected and corresponding siRNAs may be chemically synthesized, created by in vitro transcription, or expressed from a vector or PCR product. The siRNA may be double-stranded RNA, or a hybrid molecule comprising both RNA and DNA, e.g., one RNA strand and one DNA strand. The siRNA may be of a variety of lengths, such as 1 to 30 nucleotides in length or 15 to 30 nucleotides in length or 20 to 25 nucleotides in length. In certain embodiments, the siRNA is double-stranded and has 3' overhangs or 5' overhangs. In certain embodiments, the overhangs are UU or dTdT 3'. In particular embodiments, the siRNA comprises a stem loop structure.

[0109] Without limitation, an example of an siRNA that can be targeted against haematopoietic stem or progenitor cells (HSPC) is siRNA against an HSPC retention factor, such as Sdfl. Silencing HSPC retention factors using siRNA may improve cell yield in stem cell transplantation in patients that are poor stem cell mobilizers (see Krohn-Grimberghe et al., 2020, Nat Biomed Eng., 4(11): 1076-1089, which is incorporated herein by reference).

[0110] In a further embodiment, the cargo molecule is a microRNA or small nuclear RNA. Micro RNAs (miRNAs) are short, noncoding RNA molecules that are transcribed from genomic DNA, but are not translated into protein. These RNA molecules are believed to play a role in regulation of gene expression by binding to regions of target mRNA. Binding of miRNA to target mRNA may downregulate gene expression, such as by inducing translational repression, deadenylation or degradation of target mRNA. Small nuclear RNA (snRNA) are typically longer noncoding RNA molecules that are involved in gene splicing. The snRNA molecules may have therapeutic or diagnostic importance in diseases that are an outcome of splicing defects.Editing cargo

[0111] In one embodiment, the LNP-encapsulated cargo edits a haematopoietic stem or progenitor cell to produce a desired modification to treat, prevent or ameliorate a disease or condition.

[0112] As used herein, the term “editing cargo” includes a protein and / or nucleic acid-based system that causes modification of a haematopoietic stem or progenitor cell at a specific locus or loci to produce a desired modification to treat, prevent or ameliorate a disease or condition.

[0113] As used herein, the term “nucleic acid editor” includes a protein and / or nucleic acid-based system that causes modification of any nucleic acid of a haematopoietic stem or progenitor cell at a specific locus or loci to produce a desired modification to treat, prevent or ameliorate a disease or condition.

[0114] The cargo may comprise a nucleic acid that encodes for a protein or peptide that forms part of a nucleic acid editing complex. A “nucleic acid editing complex” includes without limitation protein and / or nucleic acid based systems in which nucleic acid is inserted, deleted, modified (e.g., epigenetic editing) or replaced in the genetic material of an organism at a sitespecific location.

[0115] The nucleic acid editing complex may be used for ex vivo or in vivo genetic modification of a haematopoietic and / or progenitor cell and includes post-translational modifications.

[0116] Alternatively or additionally, the cargo comprises or encodes a peptide or protein that is part of an editor or forms an editing complex.

[0117] The nucleic acid editing complex includes, without limitation, Cas-based (e.g., CRISPR or non-CRISPR), transcription activator-like effector nuclease (TALEN), megaTALs, zinc finger nuclease (ZFN), Adenosine Deaminase Acting on RNA (ADAR), prime editors, base editors, epigenetic, transposase, meganuclease, ARCUS gene editing systems or any variant or combination thereof. These nucleic acid editing systems are exemplary and include any cargo that can modify genetic material (including RNA transcripts and non-coding regions) of a cell to treat, prevent or ameliorate a disorder or disease. Without limitation, the gene editing system mayinclude those that are designed by a process referred to as Directed Nuclease Editor (DNE), which is known to those of skill in the art.

[0118] Cas-based editing systems comprise CRISPR and non-CRISPR gene editing systems. In addition, the editing systems include those that cut DNA as well as epigenetic editing systems that modify nucleic acid markers, as discussed below.

[0119] The CRISPR gene editing cargo most advantageously comprises nucleic acid (e.g., mRNA) encoding for one or more of a Class II Cas nuclease family of proteins and a guide RNA. The nucleases encoded by the nucleic acid are enzymes with DNA endonuclease activity and can be directed to cleave a desired nucleic acid target by an appropriate guide RNA. The nuclease and guide RNA form a complex referred to as a ribonucleoprotein (RNP). In some embodiments, the nuclease is a Class II CRISPR enzyme, which is further subdivided into Types II, V and VI. According to one embodiment, the mRNA encodes for a Cas protein that is part of a Type II CRISPR / Cas system, such as a Cas9 protein or a Cpfl protein.

[0120] In another embodiment, the mRNA encodes for a Cas protein that is part of a Type V CRISPR / Cas system, such as Cas 12a. In another embodiment, the mRNA encodes for a Cas protein that is a Cas 13a, which is an RNA endonuclease and cleaves single-stranded RNA.

[0121] The guide RNA can direct the Cas nuclease to the target sequence on a target nucleic acid molecule, where the guide RNA hybridizes to the target sequence and the Cas nuclease cleaves or modulates the sequence. In some embodiments, the guide RNA binds to a class 2 nuclease, thereby providing specificity of cleavage.

[0122] Guide RNAs for the CRISPR / Cas9 nuclease system include CRISPR RNA (crRNA) or tracr RNA (tracr). In some embodiments, the crRNA can include a targeting sequence that is complementary to and hybridizes to a target sequence on a target nucleic acid molecule. The crRNA can also include a flagpole that is complementary to, and hybridize to, a portion of tracrRNA. In some embodiments, the crRNA can correspond to the structure of a naturally- occurring crRNA transcribed from a bacterial CRISPR locus, wherein the targeting sequence acts as a spacer for the CRISPR / Cas9 system. The flagpole corresponds to the part of the repetitive sequence adjacent to the spacer above the CRISPR locus.

[0123] The guide RNA of the RNP can target any sequence of interest through the targeting sequence of crRNA. In some embodiments, the targeting sequence of the guide RNA and the target sequence on the target nucleic acid molecule may be 100% complementary. In other embodiments, the targeting sequence of the guide RNA and the target sequence on the target nucleic acid molecule can comprise at least one mismatch.

[0124] The length of the targeting sequence may depend on the RNP system and components used. For example, different Cas proteins from different bacterial species have various optimal targeting sequence lengths. Thus, the targeting sequences are: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or more than 50 nucleotides in length can be included. In some embodiments, the targeting sequence can comprise a length of 18 to 24 nucleotides. In some embodiments, the targeting sequence can comprise 19-21 nucleotides in length. In some embodiments, the targeting sequence can comprise a length of 20 nucleotides.

[0125] In some embodiments, the editing system includes Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbll l, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, homologs thereof, or modified versions thereof.

[0126] As noted, non-CRISPR, Cas-based gene editing systems are encompassed by embodiments of the disclosure as well. A Cas-based editing system may include a Cas enzyme fused to deaminase (Luo et al., 2020, Microbial Cell Factories, 19(93), incorporated herein by reference). An example is a cytosine base editor or an adenine base editor produced by fusing endonuclease Cas to cytosine deaminase pmCDAl or heterodimer adenine deaminase TadA- TadA. A further non-limiting example is Cas fused to reverse transcriptase (Mohr et al., 2018, Mol Cell., 72(4):700-714, incorporated herein by reference).

[0127] Fanzor is a eukaryotic RNA-guided endonuclease that could function as a gene editor. (See Saito et al., 2023, Nature 620:660-668, which is incorporated herein by reference). In some embodiments, Fanzor proteins use RNA as a guide to target DNA precisely and can be modified to edit a haematopoietic and / or progenitor cell using LNPs described herein. In some examples,the compact Fanzor systems may have the ability to facilitate more improved delivery than CRISPR-Cas systems.

[0128] In those embodiments in which the cargo is a TALEN, the cargo comprises a nucleic acid encoding a peptide having a Transcription activator-like (TAL) effector DNA binding domain, a fragment or a variant thereof. In an embodiment, the system comprises a nucleic acid encoding a peptide having nuclease activity, e.g., endonuclease activity. In an embodiment, the peptide having nuclease activity is a type-II restriction 1 -like endonuclease, e.g., a Fokl endonuclease.

[0129] In those embodiments in which the cargo is a ZFN, the nucleic acid may encode a peptide having: a Zinc finger DNA binding domain, a fragment or a variant thereof; and / or nuclease activity, e.g., endonuclease activity. In an embodiment, the Zn finger binding domain comprises 1, 2, 3, 4, 5, 6, 7, 8 or more Zinc fingers. In an embodiment, the peptide having nuclease activity is a type-II restriction 1-like endonuclease, e.g., a Fokl endonuclease.

[0130] Adenosine Deaminase Acting on RNA (ADAR) is another editing cargo encompassed by embodiments of the disclosure that may be used for post-transcriptional modification of RNA. Examples include ADAR1 and ADAR2. ADAR1 may catalyze posttranscriptional deamination of C6 of adenosines in dsRNA, converting them to inosines (see Song et al., 2022, PMC, 13( 1) :el 665, incorporated herein by reference).

[0131] Meganucleases are enzymes in the endonuclease family that may induce homologous recombination, generate mutations and alter reading frames. The meganuclease includes homing endonucleases that are intron or intein endonucleases. In one embodiment, the meganuclease is from the LAGLID ADG family, a GIY-YIG endonuclease, an HNH endonuclease, a His-Cys box endonuclease or a PD-(D / E)XK endonuclease. Meganucleases may be combined with components of other gene editing system. In one embodiment, a DNA binding domain from a transcription activator-like (TAL) effector is combined with a meganuclease to produce a “megaTAL”. In another embodiment, a meganuclease may be fused to a DNA end-processing enzyme to promote an error-prone non-homologous end joining.

[0132] ARCUS nuclease is a gene editing system based on I-Crel, which is a kind of homing endonuclease that evolved in the algae Chlamydomonas reinhardtii. In some embodiments, thenuclease can deactivate itself after gene editing, thereby reducing off-targeting. ARCUS nucleases in some embodiments can generate a unique cleavage site that is a four-base-pair, 3’ overhang and may be able to carry out gene insertion, gene excision, gene repair or a combination thereof.

[0133] Epigenetic editing is also encompassed by examples of the disclosure. Such editing of genetic material does not cut nucleic acid but rather alters epigenomic marks “adorning” DNA. Changing the epigenic signature of a haematopoietic stem or progenitor cell can serve to modify an epigenetic signature of the cell and change its transcriptional profile. In some embodiments, the epigenetic editing system may target and edit one or more methylation sites of a nucleic acid sequence. In some embodiments, genome homing proteins with engineered or naturally occurring nuclease functions for gene editing, can be mutated and adapted to function as only delivery systems. In one embodiment, an epigenetic modifying enzyme or domain can be fused to the homing protein and local epigenetic modifications can be altered upon protein recruitment. A targeting protein that recognizes DNA sequences may be linked to an effector protein that alters epigenomic marks, such as methylation. Examples of targeting proteins include Transcription Activator-Like Effector (TALE), zinc finger proteins, and Cas systems, including but not limited to CRISPR-Cas. Non-limiting examples of effector proteins include TET1, which induces demethylation of cytosine at CpG sites; LSD1, which induces demethylation of H3K4mel / 2, which also causes an indirect effect of deacetylation on H3K27; and CIB1 / CRY2, which is a cryptochrome / blue light activated complex allowing chromatin to be modified upon illumination.

[0134] Further examples of effector proteins include DNA methyltransferase, a fragment (e.g., a biologically active fragment) or variant thereof (e.g, DNMT1, DNMT2 DNMT3A, DNMT3B, DNMT3L, or CpG methyltransferase (M. Sssl)); or a poly comb repressive complex or a component thereof, e.g,, PRC1 or PRC2, or PR-DUB, or a fragment (e.g,, biologically active fragment) or a variant thereof.

[0135] In an embodiment, the epigenetic editor comprises a molecule that modifies chromatin architecture and / or modifies a histone. In an embodiment, the epigenetic modulator is a molecule that modifies chromatin architecture, e.g., a SWI / SNF remodeling complex or a component thereof. In an embodiment, the epigenetic modulator is a molecule that modifies a histone, e.g,methylates and / or acetylates a histone, e.g, a histone modifying enzyme or a fragment (e.g,, biologically active fragment) or a variant thereof, e.g, HMT, HDM, HAT, or HD AC.Structural non-cationic lipid

[0136] The LNP generally includes one or more structural lipids, meaning an amphipathic lipid having one or more tails and that allows for the formation of particles and generally bears no net charge at physiological pH (7.4). The term includes neutral as well as zwitterionic lipids that impart substantially no charge at physiological pH to the LNP and includes phospholipids. In alternative embodiments, the structural lipid is a non-cationic lipid.

[0137] The term includes lipids with choline head groups (e.g., phosphatidylcholine), meaning an amphipathic lipid that has a choline head group and that bears no or substantially no net charge at physiological pH. The neutral lipid also includes phospholipids conjugated to sterols. In some embodiments, the lipid with a choline head group is a phosphatidylcholine lipid, such as a lipid that is selected from distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), l-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) and dipalmitoyl-phosphatidylcholine (DPPC) and sphingomyelin with a phosphocholine head group.

[0138] As used herein “head group”, means a moiety that imparts polarity to the lipid and comprises one or more electronegative atoms, such as nitrogen, oxygen and / or phosphorus. Generally, the electronegative atom or atoms are part of one or more functional groups. In some embodiments, the head group is zwitterionic. In some embodiments, the head group comprises a phosphate group and a nitrogen atom. In some embodiments, the head group is zwitterionic and has no net charge at physiological pH.

[0139] A lipid nanoparticle herein may have “substantially no charge”, which means a net surface charge of about zero, or near neutral at physiological pH, such as without limitation about -2.5 mV to about 2.5 mV.

[0140] In some embodiments, the structural lipid is a phosphatidylcholine lipid (PC-lipid) selected from distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), l-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) and dipalmitoyl-phosphatidylcholine (DPPC) and a sphingomyelin, include those with a phosphatidylcholine head group.

[0141] The structural, neutral, zwitterionic or non-cationic lipid content in some embodiments is greater than 20 mol%, greater than 25 mol%, greater than 30 mol%, greater than 32 mol%, greater than 34 mol%, greater than 36 mol%, greater than 38 mol%, greater than 40 mol%, greater than 42 mol%, greater than 44 mol%, greater than 46 mol%, greater than 48 mol% or greater than 50 mol%. In some embodiments, the upper limit of helper lipid content is 70 mol%, 65 mol%, 60 mol%, 55 mol%, 50 mol% or 45 mol%. The disclosure also encompasses sub-ranges of any combination of the foregoing numerical upper and lower limits.

[0142] For example, in certain embodiments, the phosphatidylcholine lipid content is from 20 mol% to 80 mol% or 25 mol% to 60 mol% or 30 mol% to 60 mol% or 35 mol% to 60 mol% or 40 mol% to 60 mol% or 42 mol% to 58 mol%, or 43 mol% to 57 mol% or 44 mol% to 56 mol% or45 mol% to 55 mol% of total lipid present in the lipid nanoparticle.

[0143] In some embodiments, the phosphatidylcholine lipid content is primarily composed of DSPC or DMPC or primarily DSPC. In such embodiments, the mixture may have a DSPC content of at least 20, 30, 35, 40 or 45 mol% based on the total lipid content of the lipid nanoparticle with the balance of the phosphatidylcholine lipid content being another phosphatidylcholine lipid(s). In another embodiment, the phosphatidylcholine content is made up of at least 40 or 50 mol% DSPC relative to the total phosphatidylcholine content of the lipid nanoparticle.

[0144] In certain embodiments, the DSPC lipid content is from 20 mol% to 80 mol% or 25 mol% to 60 mol% or 30 mol% to 60 mol% or 35 mol% to 60 mol% or 40 mol% to 60 mol% or 42 mol% to 58 mol%, or 43 mol% to 57 mol% or 44 mol% to 56 mol% or 45 mol% to 55 mol% of total lipid present in the lipid nanoparticle.

[0145] The LNP may comprise additional lipids besides a neutral or structural lipid. For example, the LNP may comprise structural lipids that have a net positive or negative charge atphysiological pH. Generally, as discussed below, such lipids may be present at less than 10 mol% or less than 5 mol%.

[0146] In alternative embodiments, the mixture may have a DMPC content of at least 20, 30, 35, 40 or 45 mol% based on the total lipid content of the lipid nanoparticle. In another embodiment, the phosphatidylcholine content is made up of at least 40 mol% DMPC relative to the total phosphatidylcholine content of the lipid nanoparticle.

[0147] In another embodiment the structural, neutral, zwitterionic or non-cationic lipid content of the lipid nanoparticle is composed of less than 20, 10, or 5 mol% of non-phosphatidylcholine lipids, such as DOPE (measured relative to total phosphatidylcholine, structural lipid or neutral lipid content).

[0148] In another embodiment the structural, neutral, zwitterionic or non-cationic lipid content of the lipid nanoparticle is composed of less than 20, 10, or 5 mol% of non-phosphatidylcholine lipids, such as POPC (measured relative to total phosphatidylcholine, structural lipid or neutral lipid content).

[0149] In some embodiments, the transition temperature of the structural, neutral, zwitterionic or non-cationic lipid, e.g., a phospholipid having a choline head group, is at least 20°C, 21 °C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C or 38°C. Without intending to be limited by any particular theory, it is believed that fusion and agglomeration of lipid nanoparticles with no hydrophilic polymer lipid conjugate (or low levels thereof) during particle formation using the mixing method described herein could be avoided by selecting a structural, neutral, zwitterionic or non-cationic lipid that is in the gel phase rather than in the disordered liquid crystalline phase at room temperature and above. The inclusion of such structural, neutral, zwitterionic or non-cationic lipid in the lipid nanoparticle may also improve blood stability after injection.

[0150] The structural, neutral, zwitterionic or non-cationic lipid content is determined based on the total amount of lipid in the lipid nanoparticle, including the sterol.Ionizable lipid

[0151] The LNP of the disclosure has an ionizable lipid. The ionizable lipid may be charged at low pH and have substantially no net charge at physiological pH. This allows for electrostatic interactions between the lipid and the negatively charged nucleic acid cargo during initial formulation. Since the ionizable lipid is near neutral at physiological pH, toxicity and renal clearance are reduced. After cellular uptake by endocytosis, the acidic environment of the endosome leads to an increase in the net positive charge of the ionizable amino lipids, which promotes fusion with the anionic lipids of the endosomal membrane and subsequent membrane destabilization and release of the nucleic acid-based therapeutics into the cytoplasm to exert their effects.

[0152] In some embodiments, the LNP has an apparent pKa of between 5.0 and 7.5, between 6.5 and 7.5 or between 6.6 and 7.3. The apparent pKa is measured using a 6-(p-Toluidino)-2- naphthalenesulfonic acid (TNS) assay adapted from previous studies from other groups (Shobaki et al., 2018, International Journal of Nanomedicine, 13:8395-8410; and Jayaraman et al., 2012, Angew. Chem Int. Ed., 51 :8529-8533, which are incorporated herein by reference for the purposes of determining apparent pKa). According to the method, a series of buffers are prepared spanning a pH range of 2-11 in 0.5 pH unit increments consisting of 130 mM NaCl, 10 mM ammonium acetate, 10 mM 2-(N-morpholino)ethanesulfonic acid (MES), and 10 mM HEPES. 0.15-0.2 mM of the LNP. A solution of 0.06 mM of TNS is subsequently mixed with 175 pL of the LNP at each buffered pH in triplicate in a black, polysterene 96-well plate, to yield a final concentration of 6.25 and 6 pM of lipid and TNS in each well, respectively. Fluorescence is subsequently measured using an SpectraMax™ M5 microplate reader at lex=321 nm, lem=445 nm. The fluorescence is then plotted against pH using a sigmoidal curve fit through Prism™, in which the pKa is determined to be the pH value with 50% of maximal fluorescent intensity.

[0153] In some embodiments, it is desirable to include less than 50 mol% ionizable lipid. That is, the ionizable lipid content may be less than 50 mol%, less than 45 mol%, less than 40 mol%, less than 35 mol%, less than 30 mol%, less than 25 mol%, less than 20 mol%, less than 15 mol%, less than 10 mol% or less than 5 mol%.

[0154] In certain embodiments, the ionizable lipid content is from 5 mol% to 50 mol% or 8 mol% to 47 mol% or 10 mol% to 50 mol% or 15 mol% to 45 mol% or 15 mol% to 35 mol% of total lipid present in the lipid nanoparticle.

[0155] As used herein, the term "ionizable cationic lipid" refers to a lipid that, at a given pH, such as physiological pH, is in an electrostatically neutral form and that accepts protons, thereby becoming electrostatically positively charged, and for which the electrostatically neutral form has a calculated logarithm of the partition coefficient between water and 1 -octanol (i.e., a cLogP) greater than 8. In some embodiments, the cationic lipid has an apparent pKa that is between 5.0 and 8.0, 5.0 and 7.5 or between 6.0 and 7.5. The apparent pKa is measured using a TNS assay as set forth hereinafter.

[0156] In one embodiment, the ionizable cationic lipid has one or more lipophilic chain comprising one or more biodegradable group as described hereinafter.

[0157] In some embodiments, the ionizable cationic lipid has an amino group. In another embodiment, the ionizable cationic lipid has a single amino group that is ionizable. In some embodiments, the ionizable cationic lipid comprises a protonatable tertiary amine (e.g., pH titratable) head group. Such lipids include, but are not limited to sulfur lipids, such as MF019 described herein and DODMA. Other lipids that may be used in the practice of the disclosure include MC3- and KC2-type lipids, which are well-known to those of skill in the art. In further embodiments, the ionizable lipid is selected from one or more lipids set forth in WO 2022 / 246555; WO 2022 / 246568; WO 2022 / 246571; WO 2023 / 147657; WO2022 / 155728; PCT / CA2023 / 050644 filed on May 11, 2023; PCT / CA2023 / 051272 filed on September 27, 2023; PCT / CA2023 / 051273 filed on September 27, 2023; U.S. provisional patent application No. 63 / 434,506 filed on December 22, 2022; PCT / CA2023 / 051274 filed on September 27, 2023; and US2024 / 0294462 filed on February 15, 2024, each incorporated herein by reference

[0158] In one embodiment, the ionizable cationic lipid has a protonatable amino head group; at least two lipophilic moieties, wherein the amino head group has a central nitrogen atom or carbon atom to which each of the two lipophilic moieties are directly bonded; each lipophilic chain has between 15 and 40 carbon atoms in total; and wherein the lipid has (i) an apparent pKaof between6 and 7.5 (when formulated in a lipid nanoparticle); and (ii) a ClogP of at least 11. The apparent pKa is measured using a TNS assay as set forth hereinafter.

[0159] In one embodiment, the lipid nanoparticle comprises a “sulfur-containing amino lipid”, which is an ionizable lipid having at least one ionizable nitrogen (tertiary nitrogen) and one or more sulfur atoms in at least one of its lipophilic chains. In one embodiment, the sulfur-containing amino lipid further comprises a biodegradable group such as an ester moiety in one or more of its lipophilic chains. Non-limiting examples of sulfur-containing lipids are provided in the foregoing co-owned PCT applications. The incorporation of the biodegradable group(s) into the lipid increases metabolism post-administration and improves clearance of the lipid from the body following delivery of the active agent to a target area. Generally, such lipids have decreased toxicity when compared to similar lipids without the biodegradable groups.

[0160] Sulfur-containing ionizable lipids and / or ionizable lipids with one or more biodegradable groups (e.g., esters, groups comprising an ester or a disulfide) in one or more of their lipophilic chains have been shown to be particularly efficacious when formulated in the unshielded LNPs as described in the Example section herein.

[0161] Non-limiting examples of atoms or substituents that may replace a carbon atom (interrupt the alkyl) in the lipophilic chains of the ionizable cationic lipid include cycloalkyl groups (mono or polycyclic); — O— ; — (C=O)O— ; — O(C=O)-; — C(=O); — O(C=O)O-; — S(O)X-; — S— ; — S— S— ; — C(=O)S-; — SC(=O)-; —NR'—; — NR'C(=O) — ; — C(=O)NR'— ; — NR'C(=O)NR'— ; — OC(=O)NR'— ; — NR'C(=O)OR’— ; — NR'S(O)XNR'— ; — NR'S(O)XR’— ; and — S(O)XNR' — , wherein R' at each occurrence is independently selected from H, C1-C15 alkyl or cycloalkyl, and x is 0, 1 or 2.

[0162] By the term “biodegradable group”, with reference to a group in a lipophilic chain of an ionizable lipid, includes a functional group with one or more electronegative atoms (e.g., O, N, S or P) that is metabolized in vivo by an enzyme, thereby increasing its metabolism relative to an otherwise identical ionizable lipid that does not contain such group. Non-limiting examples include— O-; — (C=O)O— ; — O(C=O)-; — C(=O); — O(C=O)O-; — S(O)X-; — S— ; — S— S— ; — C(=O)S-; — SC(=O)-; —NR'—; — NR'C(=O) — ; — C(=O)NR'— ; — NR'C(=O)NR'— ; — OC(=O)NR'— ; — NR'C(=O)OR’— ; — NR'S(O)XNR'— ; — NR'S(O)XR’— ; and — S(O)XNR'— ,wherein R' at each occurrence is independently selected from H, C1-C15 alkyl or cycloalkyl, and x is 0, 1 or 2.

[0163] Non-limiting examples of atoms or substituents that may replace a hydrogen atom in the ionizable cationic lipid include halogen; deuterium, an alkyl group; a cycloalkyl group (mono or polycyclic); an oxo group (=0); a hydroxyl group (-OH); — (C=O)OR'; — O(C=O)R'; — C(=O)R'; O(C=O)OR'-; —OR'; — S(O)XR'; —SR', — S— SR'; — C(=O)SR'; — SC(=O)R'; — NR'R'; — NR'C(=O)R'; — C(=O)NR'R'; — NR'C(=O)NR'R'; — OC(=O)NR'R'; — NR'C(=O)OR'; — NR'S(O)XNR'R'; — NR'S(O)XR'; and — S(O)XNR'R', wherein R' at each occurrence is independently selected from H, C1-C15 alkyl or cycloalkyl, and x is 0, 1 or 2.

[0164] In one embodiment, the ionizable amino lipid is a “non-furan ring-containing ionizable lipid”, which is an ionizable lipid that lacks a furan ring, such as an oxolane in the head group moiety. In one embodiment, the ionizable lipid is a “non-MC3 type lipid”, which is an ionizable lipid that lacks in its head group a terminal ionizable amino group that is a methyl 4- (dimethylamino)butanoate. In another embodiment, the ionizable lipid is a non-furan ringcontaining ionizable lipid that is also a non-MC3 type lipid. In one embodiment, the ionizable amino lipid is a “non-cyclopentane ring-containing ionizable lipid”, which is an ionizable lipid that lacks a cyclopentane moiety in the head group moiety.

[0165] Optionally, at least one of the lipophilic moieties bonded to the head group has a biodegradable group. In one non-limiting example, at least one of the lipophilic moieties has an ester group in any orientation and a sulfur atom. In one embodiment, the ionizable cationic lipid has a lipophilic moiety of the formula:

[0166] In one embodiment, R1and R2are, independently, linear, cyclic or branched optionally substituted C3-C20 alkyl and optionally with varying degrees of unsaturation; and n is 4 to 8.

[0167] In some embodiments, it is desirable to include less than 50 mol% ionizable cationic lipid in the LNP. That is, the ionizable cationic lipid content may be less than 50 mol%, less than 45mol%, less than 40 mol%, less than 35 mol%, less than 30 mol%, less than 25 mol%, less than 20 mol%, less than 15 mol%, less than 10 mol% or less than 5 mol%.

[0168] In certain embodiments, the ionizable cationic lipid content is from 5 mol% to 50 mol% or 8 mol% to 47 mol% or 10 mol% to 50 mol% or 15 mol% to 45 mol% or 15 mol% to 35 mol% of total lipid present in the lipid nanoparticle.

[0169] The ionizable lipid component may include an ionizable anionic lipid as part of the ionizable lipid content. An example of such a lipid is cholesteryl hemisuccinate (CHEMS). Further examples of ionizable anionic lipids are described in co-pending and co-owned PCT / CA2024 / 050347 filed on March 22, 2024, which is incorporated herein by reference in its entirety.

[0170] In some embodiments, the ionizable cationic lipid is not a lipidoid structure, including but not limited to C12-200 (see Khare et al., 2022, AAPS Journal, 24:8, incorporated by reference) and related structures known to those of skill in the art.Sterol

[0171] The LNP further includes a sterol in some embodiments. The term “sterol” refers to a naturally-occurring or synthetic compound having a gonane skeleton and that has a hydroxyl moiety attached to one of its rings, typically the A-ring.

[0172] Examples of sterols include cholesterol, or a cholesterol derivative, the latter referring to a cholesterol molecule having a gonane structure and one or more additional functional groups.

[0173] The cholesterol derivative includes P-sitosterol, 3-sitosterol, campesterol, stigmasterol, fucosterol, or stigmastanol, dihydrocholesterol, ent-cholesterol, epi-cholesterol, desmosterol, cholestanol, cholestanone, cholestenone, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'- hydroxybutyl ether, 3P[N-(N'N'-dimethylaminoethyl)carbamoyl cholesterol (DC-Chol), 24(S)- hydroxycholesterol, 25 -hydroxy cholesterol, 25(R)-27-hydroxycholesterol, 22-oxacholesterol, 23- oxacholesterol, 24-oxacholesterol, cycloartenol, 22-ketosterol, 20-hydroxysterol, 7- hydroxy cholesterol, 19-hydroxy cholesterol, 22-hydroxy cholesterol, 25-hydroxy cholesterol, 7- dehydrocholesterol, 5a-cholest-7-en-3P-ol, 3,6,9-trioxaoctan-l-ol-cholesteryl-3e-ol,dehydroergosterol, dehydroepiandrosterone, lanosterol, dihydrolanosterol, lanostenol, lumisterol, sitocalciferol, calcipotriol, coprostanol, cholecalciferol, lupeol, ergocalciferol, 22- dihydroegocalciferol, ergosterol, brassicasterol, tomatidine, tomatine, ursolic acid, cholic acid, chenodeoxycholic acid, zymosterol, diosgenin, fucosterol, fecosterol or a salt or ester thereof.

[0174] In one embodiment, the sterol is present at from 15 mol% to 50 mol%, 18 mol% to 45 mol%, 20 mol% to 45 mol%, 25 mol% to 45 mol% or 30 mol% to 45 mol% based on the total lipid present in the lipid nanoparticle.

[0175] In another embodiment, the sterol is cholesterol and is present at from 15 mol% to 50 mol%, 18 mol% to 45 mol%, 20 mol% to 45 mol%, 25 mol% to 45 mol% or 30 mol% to 45 mol% based on the total lipid present in the lipid nanoparticle.

[0176] In another embodiment, the sterol is a cholesterol derivative and is present at from 15 mol% to 50 mol%, 18 mol% to 45 mol%, 20 mol% to 45 mol%, 25 mol% to 45 mol% or 30 mol% to 45 mol% based on the total lipid present in the lipid nanoparticle.

[0177] In one embodiment, the combined (i) sterol content (e.g., cholesterol or cholesterol derivative thereof); and (ii) neutral lipid content is at least 50 mol%; at least 55 mol%, at least 60 mol%, at least 65 mol%, at least 70 mol%, at least 75 mol%, at least 80 mol% or at least 85 mol% based on the total lipid present in the lipid nanoparticle.Hydrophilic polymer-lipid conjugate

[0178] In one embodiment, the lipid nanoparticle comprises a hydrophilic-polymer lipid conjugate capable of incorporation into the LNP. The conjugate includes a vesicle- forming lipid having a polar head group, and covalently attached to the head group, a polymer chain that is hydrophilic. Examples of hydrophilic polymers include polyethyleneglycol (PEG), polyvinylpyrrolidone, polyvinylmethylether, polyhydroxypropyl methacrylate, polyhydroxypropylmethacrylamide, polyhydroxyethyl acrylate, polymethacrylamide, polydimethylacrylamide, polymethyloxazoline, polyethyloxazoline, polyhydroxyethyloxazoline, polyhydroxypropyloxazoline, polysarcosine and polyaspartamide. In one embodiment, the hydrophilic-polymer lipid conjugate is a PEG-lipid conjugate. The hydrophilic polymer lipid conjugate may also be a naturally-occurring or synthesized oligosaccharide-containing molecule,such as monosialoganglioside (GMI). The ability of a given hydrophilic-polymer lipid conjugate to enhance the circulation longevity of the LNPs herein could be readily determined by those of skill in the art using known methodologies.

[0179] The hydrophilic polymer lipid conjugate may be present in the nanoparticle at 0.5 mol% to 5 mol%, or at 0.5 mol% to 3 mol%, or at 0.5 mol% to 2.5 mol% or at 0.5 mol% to 2.0 mol% or at 0.5 mol% to 1.8 mol% of total lipid. In certain embodiments, the hydrophilic polymer lipid conjugate may be present in the nanoparticle at 0 mol% to 5 mol%, or at 0 mol% to 3 mol%, or at 0 mol% to 2.5 mol% or at 0 mol% to 2.0 mol% or at 0 mol% to 1.8 mol% of total lipid.

[0180] In another embodiment, the PEG-lipid conjugate is present in the nanoparticle at 0.5 mol% to 5 mol%, or at 0.5 mol% to 3 mol% or at 0.5 mol% to 2.5 mol% or at 0.5 mol% to 2.0 mol% or at 0.5 mol% to 1.8 mol% of total lipid. In certain embodiments, the PEG-lipid conjugate may be present in the nanoparticle at 0 mol% to 5 mol%, or at 0 mol% to 3 mol%, or at 0 mol% to2.5 mol% or at 0 mol% to 2.0 mol% or at 0 mol% to 1.8 mol% of total lipid.

[0181] In one embodiment, the lipid nanoparticle has “substantially no hydrophilic polymer- conjugate” or is “unshielded”, “non-sterically stabilized” or “uncoated”, meaning the lipid nanoparticle has less than 0.8 mol% total hydrophilic-polymer conjugate content (e.g., a hydrophilic-polymer-lipid conjugate) as measured based on the total lipid content of the nanoparticle. The hydrophilic polymer conjugate includes a lipophilic portion that resides in the lipid nanoparticle lipid layer (e.g., bilayer or monolayer) and a polymer chain that is hydrophilic and extends into the surrounding solution. In some embodiments, the hydrophilic-polymer lipid conjugate content is less than 0.75, 0.70, 0.65, 0.60, 0.55, 0.50, 0.45, 0.40, 0.35, 0.30, 0.25, 0.20, 0.15, 0.10 mol% as measured based on the total lipid content of the nanoparticle. In further embodiments, the hydrophilic-polymer lipid conjugate mol% content is between 0 and 0.75 mol%, 0 and 0.70 mol%, 0 and 0.65 mol%, 0 and 0.60 mol%, 0 and 0.55 mol%, 0 and 0.50 mol%, 0 and 0.45 mol%, 0 and 0.40 mol%, 0 and 0.35 mol%, 0 and 0.30 mol%, 0 and 0.25 mol%, 0 and 0.20 mol%, 0 and 0.15 mol% or 0 and 0.10 mol%.

[0182] In another embodiment, the lipid nanoparticle is “PEG-less”, meaning that the lipid nanoparticle has no detectable amounts of polyethylene-glycol lipid conjugate.

[0183] In some embodiments, the amphipathic polymer content is less than 0.75, 0.70, 0.65, 0.60, 0.55, 0.50, 0.45, 0.40, 0.35, 0.30, 0.25, 0.20, 0.15, 0.10 mol% as measured based on the total lipid content of the nanoparticle. In further embodiments, the amphipathic polymer mol% content is between 0 and 0.75 mol%, 0 and 0.70 mol%, 0 and 0.65 mol%, 0 and 0.60 mol%, 0 and 0.55 mol%, 0 and 0.50 mol%, 0 and 0.45 mol%, 0 and 0.40 mol%, 0 and 0.35 mol%, 0 and 0.30 mol%, 0 and 0.25 mol%, 0 and 0.20 mol%, 0 and 0.15 mol%, 0 and 0.10 mol% or 0 and 0.05 mol%. Examples of amphipathic polymers are provided in US 2021 / 0046192, which is incorporated herein by reference.

[0184] In some embodiments, the poloxamer content is less than 0.50, 0.45, 0.40, 0.35, 0.30, 0.25, 0.20, 0.15, 0.10 mol% as measured based on the total lipid content of the nanoparticle. In further embodiments, the poloxamer mol% content is between 0 and 0.50 mol%, 0 and 0.45 mol%, 0 and 0.40 mol%, 0 and 0.35 mol%, 0 and 0.30 mol%, 0 and 0.25 mol%, 0 and 0.20 mol%, 0 and 0.15 mol%, 0 and 0.10 mol% or 0 and 0.05 mol%.

[0185] In further embodiments, the LNP lacks a surface stabilizer that is a protein, referred to as a protein stabilizer. This includes an apolipoprotein stabilizer, derivative or mimetic thereof (see e.g., WO 2023 / 233042, which is incorporated herein by reference). Such apolipoprotein may be selected from one or a combination of apo Al, apo Al - Milano, apo A2, apo A4, apo A5, apo B48, apo Bl 00, apo C-l, apo C-l I, apo C-lll, apo C-IV, apo D, apo E, apo F, apo H, apo L and apo M. In some embodiments, the protein stabilizer content is less than 0.75, 0.70, 0.65, 0.60, 0.55, 0.50, 0.45, 0.40, 0.35, 0.30, 0.25, 0.20, 0.15 or 0.10 mol% as measured based on the total lipid content of the nanoparticle. In some embodiments, the protein stabilizer content is less than 0.45, 0.40, 0.35, 0.30, 0.25, 0.20, 0.15 or 0.10 mol% as measured based on the total lipid content of the nanoparticle. In further embodiments, the protein stabilizer content mol% content is between 0 and 0.75 mol%, 0 and 0.70 mol%, 0 and 0.65 mol%, 0 and 0.60 mol%, 0 and 0.55 mol%, 0 and 0.50 mol%, 0 and 0.45 mol%, 0 and 0.40 mol%, 0 and 0.35 mol%, 0 and 0.30 mol%, 0 and 0.25 mol%, 0 and 0.20 mol%, 0 and 0.15 mol% or 0 and 0.10 mol%. Since lipid nanoparticles can adsorb proteins after administration, the protein content is measured in vitro prior to administration.

[0186] Alternatively, in some embodiments a lipid nanoparticle preparation lacks or has low levels thereof of one or more stabilizing agents, which includes a surface stabilizer as described above and a cryoprotectant. In some embodiments, the lipid nanoparticle preparation has less than 2 w / v, 1.75 w / v, 1.50 w / v, 1.25 w / v, 1.00 w / v, 0.75 w / v, 0.50 w / v, 0.25, 0.10 or 0.05 w / v of one or more cryoprotectants in the preparation. In some embodiments, a lipid nanoparticle preparation having a plurality of LNPs has low levels or lacks glycerol and / or propylene glycol as a cryoprotectant, such as at concentration levels less than 2 w / v, 1.75 w / v, 1.50 w / v, 1.25 w / v, 1.00 w / v, 0.75 w / v, 0.50 w / v, 0.25, 0.10 or 0.05 w / v in the preparation.

[0187] In another embodiment, the lipid nanoparticle is “PEG-less”, meaning that the lipid nanoparticle has no detectable amounts of polyethylene-glycol lipid conjugate. In further embodiments, the lipid nanoparticle is “PEGless”, meaning that the LNP lacks a surface stabilizer comprising a hydrophilic polymer with a polyethylene oxide repeating unit or includes low levels thereof, such as less than 0.75, 0.70, 0.65, 0.60, 0.55, 0.50, 0.45, 0.40, 0.35, 0.30, 0.25, 0.20, 0.15, 0.10 mol% as measured based on the total lipid content of the lipid nanoparticle. In further embodiments, surface stabilizer comprising a hydrophilic polymer has a mol% content that is between 0 and 0.75 mol%, 0 and 0.70 mol%, 0 and 0.65 mol%, 0 and 0.60 mol%, 0 and 0.55 mol%, 0 and 0.50 mol%, 0 and 0.45 mol%, 0 and 0.40 mol%, 0 and 0.35 mol%, 0 and 0.30 mol%, 0 and 0.25 mol%, 0 and 0.20 mol%, 0 and 0.15 mol% or 0 and 0.10 mol%.

[0188] Examples of lipid nanoparticles with low levels or no hydrophilic polymer lipid conjugate that can be used in the practice of the disclosure are described in co-owned and co-pending U.S. provisional patent application titled “Method of delivery of DNA or RNA Cargo using Uncoated Lipid Nanoparticles and Compositions Thereof’ filed in September 2023.Additional lipid components

[0189] The LNP may comprise additional lipid components besides those described above (neutral lipid, cholesterol, ionizable cationic lipid and the optional hydrophilic polymer-lipid conjugate). Without limitation, such additional lipid components may be present at less than 10 mol%, 9 mol%, 8 mol%, 7 mol%, 6 mol%, 5 mol%, 4 mol%, 3 mol%, 2 mol%, 1 mol% or 0.5 mol% (relative to total lipid in the LNP). Such additional lipids include lipids comprising a targeting moiety, charged lipid (cationic or anionic lipid that is charged at physiological pH) orother lipid components such as vitamins (e.g., tocopherol). In some embodiments, the LNP consists essentially of neutral lipid, cholesterol, ionizable cationic lipid and the optional hydrophilic polymer-lipid conjugate, meaning any additional lipid is present at less than 5 mol% measured relative to total lipid in the LNP.

[0190] The LNP may comprise a targeting moiety for targeting the lipid nanoparticle to stem and progenitor cells, such as HSPCs. An antibody conjugated LNP may be targeted to receptors present on stem and progenitor cells, such as HSPCs. Examples are CD117, CD49d, CD44 and IL-6R receptors expressed on HSPCs. Thus, the LNP may include a ligand-lipid conjugate in which the ligand is an anti- CD49d, CD44 and IL-6R antibody. The targeting moiety may be conjugated directly to a lipophilic moiety that resides in the LNP membrane or may be conjugated to the distal end of a hydrophilic polymer, if present. Examples of LNPs with targeting moi eties are described in co-owned and co-pending U.S. provisional patent application No. 63 / 431,17, filed on December 8, 2022, and which is incorporated herein by reference.

[0191] In one embodiment, the LNP lacks a ligand-lipid conjugate for targeting to stem or progenitor cells. In such embodiments, the ligand-lipid conjugate is undesirable as it may induce an immune response. Instead, targeting may be achieved by the inherent long circulating properties of the IcLNP™ due to elevated phosphatidylcholine content. Thus, in some embodiments the ligand-lipid conjugate is present at less than 1 mol%, less than 0.5 mol% or is 0 mol%.

[0192] The additional component may include an anionic phospholipid, such as phosphatidylserine, and / or an ionizable anionic lipid. An example of such a lipid is cholesteryl hemisuccinate (CHEMS). Further examples of ionizable anionic lipids are described in copending and co-owned PCT / CA2024 / 050347 filed on March 22, 2024, which is incorporated herein by reference in its entirety.

[0193] Alternatively or additionally, the additional lipid component may include permanently charge cationic lipid, including lipids with a quarternary ammonium cation (e.g., DOTMA, DOSPA, DDAB and DOTAP) or permanently charged anionic lipid, such as phosphatidylserine. Such permanently charged lipids, in some examples, are most advantageously present at less than10 mol%, 9 mol%, 8 mol%, 7 mol%, 6 mol%, 5 mol%, 4 mol%, 3 mol%, 2 mol%, 1 mol%, 0.5 mol% or 0.25 mol% relative to total lipid content.Nanoparticle preparation and morphology

[0194] Delivery vehicles incorporating the cargo can be prepared using a variety of suitable methods, such as a rapid mixing / ethanol dilution process. Examples of preparation methods are described in Jeffs, L.B., et al., Pharm Res, 2005, 22(3):362-72; and Leung, A.K., et al., The Journal of Physical Chemistry. C, Nanomaterials and Interfaces, 2012, 116(34): 18440-18450, each of which is incorporated herein by reference in its entirety.

[0195] Without being bound by theory, the mechanism whereby a lipid nanoparticle comprising encapsulated cargo can be formed using the rapid mixing / ethanol dilution process can be hypothesized as beginning with formation of a dense region of hydrophobic mRNA-ionizable lipid core at low pH (e.g., pH 4) surrounded by a monolayer of helper lipid / cholesterol that fuses with smaller empty vesicles as the pH is raised due to the conversion of the ionizable cationic lipid to the neutral form. As the proportion of bilayer helper lipid increases, the bilayer lipid progressively forms blebs and the ionizable lipid migrates to the interior hydrophobic core. At high enough helper lipid contents, the exterior bilayer preferring helper lipid can form a complete lipid layer, such as a continuous or discontinuous bilayer, around the interior trapped volume.

[0196] The LNP may comprise a “core” region. It has been observed that the LNP core is non- homogeneous in that it includes both an electron dense region and an aqueous portion or compartment as visualized by cryo-EM microscopy. In some embodiments, the core may be characterized as non-solid. Without being limiting, the electron dense region within the core may be partially surrounded by the aqueous portion or compartment within the enclosed space as observed by cryo-TEM. The aqueous portion may form a distinct aqueous region or compartment within the lipid nanoparticle. In other words, it is believed that the aqueous portion or compartment is not merely a hydration layer. Such non-solid core particles are described in co-owned and copending WO 2022 / 251959, the contents of which are incorporated herein by reference.

[0197] In one embodiment, at least one about fifth of the core (trapped volume) contains the aqueous portion or compartment, and in which the electron dense region within the core is partiallycontiguous with the lipid layer comprising the bilayer, as determined qualitatively by cryo-EM. In another embodiment, at least one about quarter of the core contains the aqueous portion or compartment, and in which the electron dense core is either partially contiguous with the lipid layer comprising the bilayer, as determined qualitatively by cryo-EM. In a further embodiment, at least one about one third of the core contains the aqueous portion or compartment, and in which the electron dense region is either partially contiguous with the lipid layer comprising the bilayer, as determined qualitatively by cryo-EM. In another embodiment, at least one about one half of the core contains the aqueous portion or compartment, and in which the electron dense core is partially contiguous with the lipid layer comprising the bilayer, as determined qualitatively by cryo-EM.

[0198] In another embodiment, the electron dense region of the LNP unexpectedly appears to be completely surrounded by the aqueous portion of the core as visualized by cryo-TEM microscopy. This morphology is observed in a single plane and a portion of the electron dense region as observed is contiguous with the lipid layer (e.g., bilayer) but cannot be seen since this portion is not within the plane that can be visualized.

[0199] In one embodiment, the electron dense region is generally spherical in shape. In another embodiment, the electron dense region is hydrophobic.

[0200] The lipid nanoparticles herein may exhibit particularly high trapping efficiencies of mRNA. Thus, in one embodiment, the trapping efficiency is at least 50, 55, 60, 65, 70, 75, 80, 85 or 90%.

[0201] In another embodiment, the cargo and cationic ionizable lipid are present in the electron dense region. In a further embodiment, the helper lipid is present in the lipid layer comprising the bilayer.

[0202] The lipid nanoparticle may comprise a single bilayer or may be a combination of a bilayer and a monolayer in some embodiments. In one embodiment, the lipid layer is a continuous bilayer that surrounds the core.

[0203] In certain embodiments the electron dense region of the core is separated from the lipid layer comprising the bilayer by the aqueous portion or compartment. For example, the disclosureprovides a lipid nanoparticle preparation comprising a plurality of lipid nanoparticles in which at least 20%, 30%, 40%, 50%, 60% or 70% of the particles as determined by cryo-EM microscopy have a core with an electron dense region and an aqueous portion or compartment and in which the aqueous portion or compartment is partially surrounded by the lipid layer comprising the bilayer as visualized by cryo-EM microscopy.

[0204] In another embodiment, and without being limiting, the disclosure provides a lipid nanoparticle preparation comprising a plurality of lipid nanoparticles in which generally at least 10%, 20%, 30%, 40%, 50%, 60% or 70% of the particles have an elongate shape (e.g., generally oval-shaped) as determined qualitatively by cryo-EM microscopy. In this latter embodiment, the electron dense region of the core may be partially surrounded the aqueous space as visualized by cryo-EM microscopy.

[0205] In one embodiment, the lipid nanoparticle is part of a preparation of lipid nanoparticles, and wherein the electron dense region of at least 20% of the lipid nanoparticles are either (i) enveloped by the aqueous portion, or (ii) is partially surrounded by the aqueous portion and wherein a portion of a periphery of the electron dense region is contiguous with the lipid layer, as visualized by cryo-EM microscopy in a single plane.

[0206] In certain embodiments, the disclosure provides a lipid nanoparticle preparation comprising a plurality of lipid nanoparticles in which generally at least 10%, 20%, 30%, 40%, 50%, 60% or 70% of the particles as determined by cryo-EM microscopy have a core with an electron dense region that is contiguous with the lipid layer comprising the bilayer as visualized by cryo-EM microscopy.

[0207] In another embodiment, and without being limiting, the disclosure provides a lipid nanoparticle preparation comprising a plurality of lipid nanoparticles in which generally at least 10%, 20%, 30%, 40%, 50%, 60% or 70% of the particles have a core comprising an electron dense region that appears to be surrounded or enveloped by a continuous aqueous space disposed between the lipid layer (e.g., bilayer) and the electron dense region, as visualized in one plane by cryo-EM microscopy.

[0208] LNPs are visualized by cryo-TEM as described in co-owned and co-pending WO 2022 / 251959, incorporated herein by reference.

[0209] In another embodiment, the poly dispersity index (PDI) of the LNP preparation is less than 0.50, 0.45, 0.40, 0.35, 0.30, 0.25, 0.2, 0.15, 0.12 or 0.10.

[0210] In another embodiment, the particle size distribution is such that at least 90% of the particles in the LNP preparation of the disclosure have a diameter of between 40 and 150 nm or between 40 and 140 nm or between 45 and 150 nm or between 50 and 150 nm or between 50 and 120 nm or between 50 and 140 nm.

[0211] The lipid nanoparticles herein may exhibit particularly high encapsulation efficiencies of nucleic acid. As used herein, the term “encapsulation,” with reference to incorporating the cargo (e.g., nucleic acid) within a lipid nanoparticle refers to any association of the cargo with any lipid component or compartment of the lipid nanoparticle, including a lipophilic or the aqueous portion. In one embodiment, the cargo is present at least in the core of the LNP.

[0212] In one embodiment, the encapsulation efficiency is at least 50, 55, 60, 65, 70, 75, 80, 85, 90% or 92%. The encapsulation efficiency of the cargo is determined as set forth in the Materials and Methods section in the Examples herein.

[0213] Embodiments of the present disclosure also provide lipid nanoparticles described according to the molar ratio between the positively charged amine groups of the amine lipid (N) and the negatively charged phosphate groups (P) of the oligonucleotide to be encapsulated. This may be mathematically represented by the equation N / P. In one embodiment, the N / P ratio of the lipid nanoparticle is between 2 and 15, between 3 and 15, between 4 and 15 or between 4.5 and 10 or between 5 and 10 or between 5.5 and 8.

[0214] In one embodiment, the N / P ratio of the lipid nanoparticle is at least 2, 3, 4, 4.25, 4.50, 4.75, 5.0, 5.25, 5.5, 5.75, 6.0 or 6.25. The upper limit may be 15, 14, 13, 12, 11, 10, 9 or 8. The disclosure also encompasses a combination of any two of the upper and lower limits.

[0215] In one embodiment, the lipid nanoparticle has a weight nucleic acid / micromole of total lipid that is 0.05: 1 to 1 : 1. In one embodiment, the lower limit is 0.06: 1, 0.08:1, 0.10: 1, 0.12: 1,0.14: 1, 0.16: 1, 0.18: 1, 0.20: 1, 0.22: 1, 0.24: 1, 0.26: 1, 0.28:1, 0.30: 1, 0.32: 1, 0.34: 1, 0.36: 1, 0.38:1 or 0.40: 1 weight nucleic acid / micromole of total lipid. In another embodiment, the upper limit is 0.80: 1, 0.82: 1, 0.84: 1, 0.86: 1, 0.88: 1, 0.90: 1, 0.92: 1, 0.94: 1, 0.96:1 or 0.98: 1 weight nucleic acid / micromole of total lipid. The disclosure also encompasses a combination of any two of the upper and lower limits.

[0216] In one embodiment, the mRNA copy number / LNP is 1-10 or 4-8.Improved delivery to haematopoietic stem or progenitor cells

[0217] The lipid nanoparticle compositions have improved delivery to a haematopoietic stem and / or progenitor cells in vivo. In an embodiment, the cell is a haematopoietic stem or progenitor cell (HSPC). In one embodiment, the cell is a long term haematopoietic stem cell, a short-term haematopoietic stem cell or a multipotent progenitor cell. In a further embodiment, the cell is a common myeloid progenitor cell. In another embodiment, the cell is a common lymphoid progenitor cell.

[0218] The lipid nanoparticle causes a modification of a haematopoietic stem or progenitor cell by delivery of the cargo to the intracellular compartment of the haematopoietic stem or progenitor cell. The modification includes, without limitation, editing of nucleic acid by a nucleic acid editor, such as a gene editing complex; silencing of a gene; and / or replacing gene function through expression of protein or peptide encoded by mRNA or pDNA.

[0219] As used herein, “expression” of nucleic acid (e.g., mRNA or pDNA) refers to translation of the nucleic acid into a peptide (e.g., an antigen), polypeptide, or protein (e.g., an enzyme) and also can include, as indicated by context, the post-translational modification of the peptide, polypeptide or fully assembled protein (e.g., enzyme). The nucleic acid (e.g., mRNA or pDNA) may encode for a protein or peptide in a gene editing complex.

[0220] In one embodiment, the lipid nanoparticle exhibits at least a 2, 5 or 10% increase in gene expression of a cargo nucleic acid in vivo as measured in an HSPC relative to an Onpattro™ control.

[0221] As described in Example 1, the LNP of the disclosure may provide improved nucleic acid delivery to an HSPC than previous baseline (Onpattro™) formulations for nucleic acid delivery. Whether or not a lipid particle exhibits such enhanced delivery to an HSPC sub -population can be determined by biodistribution studies in an in vivo mouse model. In such embodiments, enhanced green fluorescent protein (eGFP) may be used to detect nucleic acid expression in a given HSPC population. In particular, according to such embodiments, LNP mRNA systems are prepared encapsulating mRNA coding for eGFP and biodistribution and GFP expression in cell populations in HSPCs are evaluated using flow cytometry following systemic administration. It will be understood that measurement of GFP expression is used as a proxy to assess whether a given LNP falls within the scope of the invention. Any clinical formulations meeting these expression criteria will typically not contain a reporter such as GFP, although animal studies based on GFP expression as described herein can be used to determine whether or not a lipid nanoparticle possesses the improved HSPC targeting. In other words, the test using an animal model to assess improved delivery to an HSPC relative to baseline, will typically involve obtaining a clinical LNP formulation with a cargo having a therapeutic or prophylactic effect on a subject (typically human) and conducting tests on an LNP having the same lipid components to determine if the LNP formulation itself exhibits improved HSPC targeting. For example, to determine if a given clinical LNP formulation exhibits the improved HSPC delivery, the cargo could be replaced with mRNA coding for eGFP and expression in vivo compared to baseline.

[0222] To assess whether a given lipid nanoparticle exhibits an increase in gene expression in a relevant HSPC at 4 hours, 12 hours, 24 hours, 48 hours or 3 days post-injection, the mRNA-LNP of the disclosure is compared to the baseline formulation of Example 1. The two LNPs being compared are subjected to the same experimental methods and materials to determine in vivo expression as set forth in Example 1.

[0223] In one embodiment, the lipid nanoparticle exhibits at least a 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290% or 300% increase in gene expression of an encapsulated mRNA encoding enhanced GFP (eGFP) as measured in vivo in an HSPC cell population sub-set at 4, 24 hours and / or 3 days post-injection as compared to a lipid nanoparticle encapsulating eGFP-mRNA with a baseline formulation of nor-MC3 / DSPC7cholesterol / PEG-lipid at 50 / 10 / 38.5 / 1.5; mokmol, wherein the protein expression from the nucleic acid is measured in a mouse model by detection of the eGFP translated from the mRNA. The measurement is carried out using flow cytometry and protein expression from the nucleic acid as determined by quantifying % positive cell counts in a given HSPC cell subset as set forth in Example 1. The percentage increase is determined by comparing the percentage positive cells (i.e., detection of eGFP) in a given HSPC cell type in the bone marrow and comparing this percentage to the percentage of positive cells resulting from injecting the baseline formulation using otherwise identical materials and methods.

[0224] In one embodiment, the HSPC cell sub-sets are selected from one or any combination of LK (lineage'cKit+), LSK (lineage'Scal+cKit+), multipotent progenitor (MPP), shortterm HSC (ST- HSC) and long-term HSC (LT-HSC). In one embodiment, the HSPC cell sub-sets quantified for eGFP expression are from the bone marrow and the increased expression is measured by determining positive cell counts (% positive for eGFP) by flow cytometry at 4, 24 hours or 3 days post-injection.Clinical and non-clinical uses of the LNP herein

[0225] In some embodiments, the lipid nanoparticle comprising nucleic acid is part of a pharmaceutical composition and is administered to treat and / or prevent a disease condition. The treatment may provide a prophylactic (preventive), ameliorative or a therapeutic benefit. The pharmaceutical composition will be administered at any suitable dosage.

[0226] A wide range of nucleic acid modifications to a haematopoietic stem and / or progenitor cells is encompassed by the disclosure. This includes an insertion, deletion, mutation (e.g., a missense mutation, a silent mutation or a nonsense mutation), a duplication, an inversion, epigenetic modifications (e.g., modification of methylation, chromatin architecture or a histone), or any combination thereof.

[0227] The LNPs described herein may be used to treat and / or prevent any disease, disorder or condition in a mammalian subject. In an embodiment of any of the methods, compositions, or cells disclosed herein, the disease or disorder is selected from the group consisting of a hemoglobinopathy, a clotting factor disorder, a blood cell disorder, and an immune cell disorder,such as an immunodeficiency. Examples of diseases or disorders that could be treatable or preventable by the LNPs herein include those described in Ferrari et al, 2021, Nature Reviews, “Gene therapy using haematopoietic stem and progenitor cells” 22:216-234, which is incorporated herein by reference. In one embodiment, the lipid nanoparticle is used to treat a disease or disorder that is a genetic disorder. For example, and without limitation, the genetic disorder may result from altered blood cell maturation and function. In one embodiment, the genetic disorder resulting from the altered blood cell maturation and function is a disease of the innate and adaptive immune system, a red blood cell disorder, a platelet disorder or a bone marrow failure syndrome.

[0228] In one embodiment, the genetic disorder is a primary immunodeficiency. For example, the primary immunodeficiency may be selected from X-linked severe combined immunodeficiency (SCID), adenosine deaminase (ADA) deficiency SCID, Wiskott-Aldrich (WAS) syndrome and chronic granulomatous disease (CGD).

[0229] In another embodiment, the red blood cell disorder is an erythrocyte disorder. For example, the erythrocyte disorder may be beta-Thalassaemia, sickle cell disease or a pyruvate kinase deficiency.

[0230] Further, a HSPC can be genetically modified with nucleic acid to express therapeutic proteins or peptides and thereby increase protein or peptide levels in the blood or other tissues such as bone, CNS, kidney, eye, liver, heart joint, skeletal and peripheral nervous system. Examples of diseases or genetic conditions that are treatable by this approach include cystinosis, Fabry disease, metachromatic leukodystophy (MLD), mucopolysaccharidosis (MPSI), including but not limited to type I, II or III, cystinosis, Fabry disease and Pompe disease.

[0231] In some embodiments, a stem and / or progenitor cell modified by an LNP of the disclosure can function as a delivery vehicle itself. For example, HSPCs and blood cells of myeloid lineage are able to cross the blood-brain barrier and penetrate into the central nervous system (CNS). HSPCs modified by LNPs may be able to replace macrophages and microglia-like cells that have been damaged. Such an approach could be used to treat peroxisomal and lysosomal storage diseases, since damage to microglia has been implicated in these diseases.

[0232] In some embodiments, the lipid nanoparticle is used to treat a disease or disorder that is an acquired disease, such as cancer, acquired immune disease (e.g., HIV / AIDS), chronic infections or neurodegenerative disorders such as multiple sclerosis. In one non-limiting example, an HSPC could be modified by the LNP of the disclosure to produce cytokines. In one embodiment, an HSPC could be modified to produce cytokines, which have antibacterial or anti-viral functions. In another embodiment, an HSPC may be modified to produce surface receptors that modulate an immune response. For example, an immune checkpoint molecule, such as PDL1, which inhibits autoimmune responses, could be expressed in an HSPC to treat type 1 diabetes.

[0233] In another embodiment, the lipid nanoparticle could be used to treat cancer. For example, an HSPC could be modified by LNPs herein to achieve expression of a therapeutic protein or peptide in a target cancerous tissue. In one non-limiting example, HSPCs could be modified by LNPs herein to express protein to block tumour cell growth or cause immune recognition of cancer. For example, HSPC-mediated interferon gene therapy may provide protective immunity against tumour antigens (see Escobar et al., 2018, Natu. Commun., 9:2896, which is incorporated herein by reference).

[0234] In one embodiment, the cancer is leukemia. Leukemia develops from blood forming cells in the bone marrow and is caused by BCR / ABL1 oncogene, which is generated by the Philadelphia chromosome (Ph) translocation (fusion of BCR gene to the ABL gene). The LNP of the disclosure with siRNA or antisense oligonucleotide cargo against the oncogene could be used for targeted gene silencing. (See Jyotsana et al., 2019, Ann Hematol., 98(8): 1905-1918, incorporated herein by reference).

[0235] In an alternative embodiment, the LNP of the disclosure could be used to treat a lymphoma (e.g., non-Hodgkin B cell lymphoma). In one non-limiting example, the LNP of the disclosure encapsulating siRNA or antisense oligonucleotide cargo could be used to silence expression of genes implicated in lymphoma, such as the cell cycle regulator, cyclin DI or the antiapoptotic proteins, B-cell lymphoma 2 (Bcl-2) or myeloid cell leukemia (Mcl-1). (See Knapp et al., 2018, Bioeng Transl Med., 3(2): 138-147, incorporated herein by reference).

[0236] In an alternative embodiment, the LNP of the disclosure could be used to treat multiple myeloma. For example, LNPs of the disclosure with siRNA or antisense oligonucleotide cargocould be used to silence CKAP5. (See Tarab-Ravski et al., 2023, Advanced Science, 10:2301377, which is incorporated herein by reference).

[0237] In another embodiment, LNPs of the disclosure are used to improve mobilization of stem cells from the bone marrow. Such mobilization facilitates migration of the cells from the bone marrow to the peripheral blood. Improving stem cell yield by mobilizing cells into peripheral circulation can be facilitated with an LNP encapsulating cargo such as siRNA in combination with one or more mobilizing agents (see below). The cells so mobilized can be collected for ex vivo treatment (using LNPs herein or another therapy) or used directly for bone marrow transplantation to a patient in need of a bone marrow transplant.Administration of LNPs

[0238] In one embodiment, the LNPs herein are injected intravenously to a subject and are targeted to the bone marrow. The inventors have found that LNPs with elevated levels of a neutral or zwitterionic lipid (e.g., a phosphatidylcholine lipid or other structural lipid) have improved biodistribution to the bone marrow. In particular, flow cytometry has revealed that haematopoietic stem or progenitor cells have increased expression of mRNA relative to a baseline formulation at 4 days post-administration (Figures 2B-D). Advantageously, in some embodiments, such targeting to bone marrow haematopoietic stem or progenitor cells can be achieved without a targeting ligand (thereby avoiding an unwanted immune response against the ligand).

[0239] In another embodiment, haematopoietic stem or progenitor cells are mobilized into peripheral blood of a subject using a mobilizing agent. As noted, this may be achieved using one or more of granulocyte colony stimulating factor, AMD3100, Me6, EP80031, ML141, FG-4497, IL-33, ARL67156, meloxicam, desipramine and reboxetine. The peripheral haematopoietic stem or progenitor cells may be modified by the lipid nanoparticles of the disclosure upon intravenous administration. The peripheral haematopoietic stem or progenitor cells so modified may subsequently regraft in the bone marrow of the subject.

[0240] In another embodiment, the haematopoietic stem or progenitor cells of a subject are directly modified by intraosseous injection (IO) of a subject.

[0241] The LNP compositions herein can alternatively or additionally be used to contact a stem or progenitor cell ex vivo after isolation of the stem or progenitor cell from a subject and subsequently reintroduced back to the subject. Bone marrow is harvested from the subject, such as through multiple aspirations from the iliac crests or leukapheresis following the administration of mobilizing agents. A non-limiting example of a mobilizing agent is granulocyte colonystimulating factor, which is optionally used in combination with CXCR4 agonist Plerixafor™. The stems or progenitor cells are extracted from the blood, and the collected stem cells in some examples are subsequently enriched for CD34+cells. The enriched cells are treated with LNPs disclosed herein to modify them (e.g., to express a therapeutic protein or correct a genetic defect) and then reintroduced back to a subject.

[0242] Thus, in some embodiments of the disclosure, the LNP is administered intravenously to a subject to (a) target haematopoietic stem or progenitor cells either with or without prior mobilization of haematopoietic stem or progenitor cells in a subject to the peripheral blood; or administered by direct injection to the bone marrow (intraosseous injection (IO)). In some advantageous embodiments, the LNP of the disclosure is administered intravenously to the subject to target haematopoietic stem or progenitor cells without prior mobilization of haematopoietic stem or progenitor cells in a subject to the peripheral blood.

[0243] In one embodiment, the LNP is part of a pharmaceutical composition administered parenterally, i.e., intra-arterially, intravenously, subcutaneously or intramuscularly. In yet a further embodiment, the pharmaceutical compositions are for intraosseous injection (IO).

[0244] The pharmaceutical composition comprises pharmaceutically acceptable salts and / or excipients.

[0245] The composition described herein may be administered to a subject such as a patient. The term subject as used herein includes a human or a non-human subject.

[0246] The examples below are intended to illustrate the preparation of specific lipid nanoparticle nucleic acid preparations and properties thereof but are in no way intended to limit the scope of the invention.EXAMPLESMaterials and MethodsLNP preparation

[0247] The LNPs were prepared by dissolving mRNA in 25 mM sodium acetate, pH 4.0, while the lipid components at the mole % specified were dissolved in absolute ethanol. The lipids in ethanol and the eGFP mRNA in buffer were combined in a 1 :3 volume by volume ratio using a t- junction with dual-syringe pumps. The solutions were pushed through the t-junction at a combined flow rate of 20 mL / min (5 mL / minute for the lipid-containing syringe, 15 mL / minute for the mRNA-containing syringe). The mixture was subsequently dialyzed overnight against at least -100 volumes of l x phosphate buffered saline, pH 7.4 using Spectro / Por dialysis membranes (molecular weight cut-off 12000-14000 Da). The LNPs were concentrated as required with an Amicon Ultra™ 10000 MWCO (molecular weight cut-off), regenerated cellulose concentrator.

[0248] Encapsulation efficiency was calculated by determining unencapsulated mRNA content by measuring the fluorescence upon the addition of RiboGreen™ to the mRNA-LNP (F) and comparing this value to the total mRNA content that is obtained upon lysis of the LNP by 2% Triton X-100 (Ft): % encapsulation = (Ft - Fi) / Ftx 100.

[0249] The particle size and poly dispersity index (PDI) were characterized using a Zetasizer Nano ZS™.Flow cytometry in vivo studies

[0250] The LNPs at the eGFP mRNA concentration of 1.0 mg / mL were injected intravenously (i.v.) in mice at a volume using the formula weight of the mouse (in grams) * 10 pL. Bone marrow was harvested at 24 hours after the LNP injections.

[0251] The bone marrow was harvested and processed into a single cell suspension. In particular, the mice were anesthetized with 5% isoflurane until reflex was lost and then exposed to CO2 with 1% air. The marrow was isolated from the femur by centrifugation of the bone for 30 s at 3,810 g and resuspended in FACS buffer (lx sterile PBS (pH 7.4), 2.5 mM ethylenediaminetetraacetic acid (EDTA), 0.05% (w / v) sodium azide (NaNs), 2% (v / v) heat-inactivated fetal bovine serum (EH- FBS)).

[0252] After isolation, the bone marrow cells were stained. Eight to ten million cells were added to a well of 96-well round bottom plates and the volume in each well was increased to 150 uL using FACS buffer. Cells were centrifuged at 484 g at 4°C for 5 minutes and the liquid was discarded. Subsequently, cells were incubated with a solution containing 5% rat serum and then a solution containing staining antibodies for 45 minutes. Cells were centrifuged at 484 g at 4°C for5 minutes and the liquid was discarded. The volume was increased to 150 pL and cells were centrifuged at 484 g at 4°C for 5 minutes and the liquid was discarded twice. A volume of 150 pL of propidium iodide (PI) was added at a 1 :5,000 dilution (1 mg / mL stock) and the stained, single cells were introduced to a flow cytometer (Cytoflex™, Beckman Coulter™). Single colour setups were used to generate the compensation matrix which was applied to all the samples.

[0253] The flow cytometry data was analyzed using FlowJo™ version 10 (Becton Dickension™6 Company (BD)). Corresponding HSPC cell sub-sets were identified based on the gating scheme shown in Table 2. The bar graphs for the percentage of eGFP positive (eGFP+) cells were generated using Prism™ version 8 (GraphPad™) software.Al 14-Cre mouse model prime editing studiesThe Ai 14-Cre mouse model is depicted in Figure 4A. The LNPs at the Cre mRNA concentration of 0.3 mg / mL were injected intravenously (i.v.) in Ail4 mice at a volume using the formula weight of the mouse (in grams) * 10 pL. Bone marrow was harvested at 24 hours after the LNP inj ections.

[0254] The bone marrow was harvested and processed into a single cell suspension. In particular, the mice were anesthetized with 5% isoflurane until reflex was lost and then exposed to CO2 with 1% air. The marrow was isolated from the femur by centrifugation of the bone for 30 s at 3,810 g and resuspended in FACS buffer (lx sterile PBS (pH 7.4), 2.5 mM ethylenediaminetetraacetic acid (EDTA), 0.05% (w / v) sodium azide (NaNs), 2% (v / v) heat-inactivated fetal bovine serum (EH- FBS)).

[0255] After isolation, the bone marrow cells were stained. Eight to ten million cells were added to a well of 96-well round bottom plates and the volume in each well was increased to 150 uL using FACS buffer. Cells were centrifuged at 484 g at 4°C for 5 minutes and the liquid was discarded. Subsequently, cells were incubated with a solution containing Fc block and then asolution containing staining antibodies for 45 minutes. Cells were centrifuged at 484 g at 4°C for5 minutes and the liquid was discarded. The volume was increased to 150 pL and cells were centrifuged at 484 g at 4°C for 5 minutes and the liquid was discarded twice. A volume of 150 pL of DAPI was added at a 1 :2,000 dilution and the stained, single cells were introduced to a flow cytometer (Cytoflex™, Beckman Coulter™). Single colour set-ups were used to generate the compensation matrix which was applied to all the samples.

[0256] The flow cytometry data was analyzed using Flow Jo™ version 10 (Becton Dickension™6 Company (BD)). Corresponding HSPC cell sub-sets were identified based on the gating scheme shown in Table 2. The bar graphs for the percentage of TdTomato positive (TdTomato+) cells were generated using Prism™ version 8 (GraphPad™) software.Humanised eGFP mouse studiesNBSGW neonates aged 1 to 3 days were intrahepatically injected with 30 000 to 50 000 human umbilical cord blood (UCB)-derived CD34+HSPCs per animal in 30 pL of injection media (filtered RPMI 1640, 1 pM EDTA). Starting at the eighth week after injection, human chimerism was measured as the percentage of human CD45+cells among the mouse and human CD45+in the peripheral blood. Blood analysis was performed every other week until necropsy.

[0257] The LNPs at the eGFP mRNA concentration of 1.0 mg / mL were injected intravenously (i.v.) in humanized NBSGW mice at a volume using the formula weight of the mouse (in grams) * 10 pL. Bone marrow was harvested at 48 hours after the LNP injections.

[0258] BM tissue was harvested following euthanasia by crushing bone tissue in a 30 mm plate and washing the crushed tissue with a wash buffer (PBS + 2% fetal bovine serum). Erythrocytes were lysed using ammonium-chloride-potassium (ACK) buffer. After lysis, the remaining cells were stained with appropriate antibodies for 15 minutes at room temperature, spun down at 800g for 5 minutes, and resuspended in PBS. Data were acquired with FACSDiva™ software on a FACSymphony™ cell analyzer and analyzed in FlowJo™ v. 10.2 and higher.Example 1: Bone marrow mRNA gene expression in HSPC bone marrow cell subsets increases significantly using high DSPC LNPs

[0259] The effect of increasing the amount of DSPC from 10 mol% to 50 mol% in an LNP containing enhanced green fluorescent protein mRNA (“eGFP mRNA”) cargo was evaluated in bone marrow haematopoietic stem and progenitor cells (HSPCs) in vivo.

[0260] In particular, the following eGFP mRNA formulations (reported in mol%) comprising ionizable lipid (as indicated), DSPC, cholesterol and PEG-lipid were compared in this example.Table 1: Formulations examined in vivo containing eGFP mRNA

[0261] The baseline formulation and the IcLNP™ formulations in Table 1 above had favourable physiochemical characteristics (Figure 2A).

[0262] Flow cytometry was used to investigate the expression of eGFP mRNA in haematopoietic stem and progenitor cell subsets of bone marrow of C57B1 / 6 mice at various time points postinjection. The flow cytometry gating scheme of the bone marrow HSPCs is shown in Table 2 below.Table 2: Gating scheme on live haematopoietic stem and progenitor (HSPC) cells

[0263] The results for the mRNA expression detected in the bone marrow sub-cell populations are presented in Figure 2B-2C. The data was subjected to a 2-way Anova statistical analysis to the group B control. Figure 2D was subjected to a 1-way Anova statistical analysis to the group B control.

[0264] The results in Figures 2B-D show that at 24 hours post-administration, the haematopoietic stem or progenitor cell sub-populations in the bone marrow of mice treated with the GFP mRNA- containing IcLNP™ having 50 mol% DSPC and ionizable lipid 3 as the cationic lipid (Group D in Table 1) exhibited an increase of 5% or more of cells positive for GFP over the baseline (Group B in Table 1) formulation with only 10 mol% DSPC (Figures 2B-D). For the LSK (Lineage c-Kit Scal+) group, GFP mRNA-containing IcLNP™ having 50 mol% DSPC and lipids 2, 3, 4 and 5 as the cationic ionizable lipids (Groups C-F in Table 1) all had improved % eGFP+ cells relative to the baseline (Figure 2B). Similar results for IcLNP™ formulations (Groups C-F) were observed with IcLNP™ for MPP (Lineage c-Kit Scal+CD34+) and LT-HSC (Lineage ckit+Scal+CD34' CD135'CD48'CD150+) as shown in Figure 2C (left group of bars) and Figure 2D.Example 2: Expression of mRNA in HSPC bone marrow cell subsets using unshielded high DSPC LNPs

[0265] The effect of eliminating the hydrophilic polymer-conjugate, PEG2000-DMG, on eGFP expression in LNPs was evaluated in bone marrow haematopoietic stem and progenitor cells (HSPCs) in vivo. Unexpectedly, such unshielded IcLNP™ formulations exhibited improved eGFP relative to a baseline formulation comprising PEG-lipid.

[0266] In particular, the following luciferase control and eGFP mRNA LNP formulations comprising ionizable lipid (as indicated), DSPC and cholesterol were compared in this example (Table 3, LNPs 1, 3 and 4). The PEG-containing control was Onpattro™ baseline (LNP 2). The luciferase and IcLNP™ formulations lacking PEG were prepared by a modified method in which, after T-mixing, buffer was added to the formulation mixture in increasing ratios of LNP: IX buffer prior to dialysis and gently mixed, followed by transfer to dialysis in 1 X PBS followed by processing as usual.Table 3: Unshielded formulations (IcLNP™) examined in vivo containing eGFP mRNA

[0267] The luciferase control, Onpattro™ benchmark and the IcLNP™ formulations in Table 3 above had favourable physiochemical characteristics (Figure 3 A).

[0268] Flow cytometry was used to investigate the expression of eGFP mRNA in haematopoietic stem and progenitor cell subsets of bone marrow of C57B1 / 6 mice at 24 hours post-injection. The flow cytometry gating scheme of the bone marrow HSPCs is shown in Table 4 below.Table 4: Gating scheme on live haematopoietic stem and progenitor (HSPC) cells for uLNP studies

[0269] The results in Figures 3B-C show that at 24 hours post-administration, the hematopoietic stem or progenitor cell sub-populations in the bone marrow of mice treated with the GFP mRNA- unshielded LNPs containing 40 mol% DSPC (LNPs 3 and 4 in Table 3) exhibited an increase of cells positive for GFP over the Onpattro™ benchmark (LNP 2 in Table 3). This was observed for all sub-populations of bone marrow HSPC analyzed.Example 3: Prime editing in HSPC bone marrow cell subsets using high DSPC LNPs

[0270] Prime editing of HSPC bone marrow cells in vivo was next investigated with high DSPC LNPs. The formulations tested are set out in Table 5 below.Table 5: Formulations examined in vivo for prime editing

[0271] As shown in Figure 4B, the physiochemical properties of the foregoing LNPs were all found to be within acceptable ranges (PDI, encapsulation efficiency and average particle size).

[0272] Prime editing was examined for LNPs 1, 2 and 3 (Table 5) at a dose of 0.3 mg / kg using the Ail4-Cre mouse model. The mouse model has a tox -flanked STOP cassette preventing transcription of a CAG promoter-driven red fluorescent protein variant (TdTomato). The LNPs encapsulated mRNA encoding for Cre recombinase. The mice express TdTomato fluorescence upon LNP delivery and expression of mRNA encoding Cre recombinase. A diagram depicting the mechanism is shown in Figure 4A. The TdTomato fluorescence was examined 24 hours after administration.

[0273] The gating scheme for flow cytometry was as described in Table 4 above.

[0274] Figures 4C-F show the TdTomato expression in the bone marrow in various HSC subpopulations. For the true LT-HSC sub-population, the TdTomato expression was significantly higher than the Onpattro™ benchmark (Figure 4F).Example 4: Expression of mRNA in HSPC bone marrow cell subsets of humanized mice using high DSPC LNPs

[0275] Expression of GFP in HSPC bone marrow cells in a humanized mouse model was next investigated with high DSPC LNPs. The formulations tested are set out in Table 6 below.Table 6: Formulations examined in humanized mice in study 1

[0276] As shown in Figure 5A, the physiochemical properties of the foregoing LNPs were all found to be within acceptable ranges (PDI, encapsulation efficiency and average particle size).

[0277] In a first study, flow cytometry was used to investigate the expression of eGFP mRNA in LNPs 1-4 in Table 6 at 48 hours post-injection at a dose of 1 mg / kg. The gating scheme is set forth in Table 7 below.Table 7: Gating scheme for humanized mouse study

[0278] The LNP formulation 4 (Table 6) having ionizable lipid 3:DSPC:Chol: PEG2000-DMG (27.4:50:21.1 : 1.5 mol:mol), demonstrated 30-40% GFP+cells in the enriched HSC population (Figure 5D).

[0279] A second study was conducted in humanized mice in which dosages and N / P were varied. The LNPs examined in study 2 and dosages are summarized in Table 8 below. An alternate LNP was also used in the study, namely LNP 2 (Table 8) with ionizable lipid 8 at a dose of 1 mg / kg (N / P = 9). As indicated in Table 8, IcLNP™ with ionizable lipid 3 was injected at a dose of 1 mg / kg (regular dose) at an N / P = 6 (LNP group 3), at a regular dose of 1.0 mg / kg at an N / P = 9 (LNP 4) and at a high dose of 2.5 mg / kg at an N / P = 9 (LNP group 5).Table 8: Formulations examined in humanized mice in study 2

[0280] The physiochemical properties of the LNPs of study 2 were all within acceptable ranges (Figure 5E).

[0281] As can be seen in Figures 5F-H, LNP groups 3 and 4 at the regular dose result in similar eGFP expression in human HSPC populations and the results demonstrate about 20% GFP+cells in enriched HSC populations. Increasing the dose of to 2.5 mg / kg (N / P of 9) in the group 5 LNPs lead to a 2-fold increase in GFP+cells in enriched HSC populations (Figures 5F-H, right bar).

[0282] A third study was conducted in humanized mice in which a PEGless LNP (unshielded, IcLNP™) was examined for eGFP expression in HSPC populations, as well as intra-osseus injection. The LNPs examined are set forth in Table 9 below. The ionizable lipid was lipid 3 (apart from the LNP 2 control).Table 9: Formulations examined in humanized mice in study 3

[0283] The results in Figures 5 J, 5K and 5L show that intra-osseus injection (LNP 6) resulted in comparable eGFP expression as IcLNP™ (LNPs 4-6) in the cell populations indicated as the results with intravenous injection. The IcLNP™ 5 sample with no PEG exhibited enhanced eGFP expression relative to the controls.

[0284] The examples are intended to illustrate the preparation of specific lipid nanoparticle mRNA preparations and properties thereof but are in no way intended to limit the scope of the invention.

[0285] The article "a" or "an" as used herein is meant to include both singular and plural, unless otherwise indicated.

Claims

CLAIMS:

1. A method for delivery of cargo to a haematopoietic stem or progenitor cell of a subject, the method comprising contacting a lipid nanoparticle encapsulating the cargo with the haematopoietic stem or progenitor cell ex vivo or in vivo, thereby causing cellular uptake of the cargo, the lipid nanoparticle having between 30 mol% and 70 mol% of a neutral or zwitterionic amphipathic lipid having a net-neutral charge at physiological pH, an ionizable cationic lipid, and a sterol, wherein the lipid nanoparticle is substantially uncharged at physiological pH and has an apparent pKa of between 6.0 and 7.5.

2. The method of claim 1, wherein the neutral or zwitterionic lipid is a phospholipid having a choline head group and is selected from distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidylcholine (DMPC) and / or dipalmitoyl-phosphatidylcholine (DPPC).

3. The method of claim 1 or 2, wherein the contacting is in vivo and the haematopoietic stem or progenitor cell is in the spleen or bone marrow of the subject.

4. The method of any one of claims 1 to 3, wherein the cargo comprises a nucleic acid that is part of a nucleic acid editor.

5. The method of claim 4, wherein the nucleic acid editor is a Cas-based editor, transcription activator-like effector nuclease (TALEN), megaTAL, zinc finger nuclease (ZFN), Adenosine Deaminase Acting on RNA (ADAR), prime editor, base editor, epigenetic editor, transposase, meganuclease, ARCUS gene editing system or a combination thereof.

6. The method of claim 5, wherein the nucleic acid editor is CRISPR and comprises a guide RNA and an mRNA or vector DNA coding for a Cas nuclease.

7. The method of claim 6, wherein the guide RNA is part of the nucleic acid editor and the method further comprises contacting the haematopoietic stem or progenitor cell in vivo or ex vivo with a second lipid nanoparticle comprising an mRNA coding for a Cas nuclease.

8. The method of any one of claims 1 to 7, wherein the lipid nanoparticle is used to treat a disease or disorder that is a genetic disorder.

9. The method of claim 8, wherein the genetic disorder results from altered blood cell maturation and function.

10. The method of claim 9, wherein the genetic disorder resulting from the altered blood cell maturation and function is a disease of the innate and adaptive immune system, a red blood cell disorder, a platelet disorder or a bone marrow failure syndrome.

11. The method of claim 10, wherein the genetic disorder is a primary immunodeficiency.

12. The method of claim 11, wherein the primary immunodeficiency is selected from X-linked severe combined immunodeficiency (SCID), adenosine deaminase (ADA) deficiency SCID, Wiskott-Aldrich (WAS) syndrome and chronic granulomatous disease (CGD).

13. The method of claim 10, wherein the red blood cell disorder is an erythrocyte disorder.

14. The method of claim 13, wherein the erythrocyte disorder is a beta-Thalassaemia, a sickle cell disease or a pyruvate kinase deficiency.

15. The method of any one of claims 1 to 7, wherein the lipid nanoparticle is used to treat a disease or disorder that is a cancer.

16. The method of claim 15, wherein the cancer is a haematological cancer.

17. A lipid nanoparticle encapsulating least a component of a nucleic acid editor for ex vivo or in vivo delivery to a haematopoietic stem or progenitor cell, the lipid nanoparticle having between 30 mol% and 70 mol% of neutral lipid or zwitterionic amphipathic lipid having a neutral or net- neutral charge at physiological pH, an ionizable cationic lipid, and a sterol, wherein the lipid nanoparticle is substantially uncharged at physiological pH and has an apparent pKa of between 6.0 and 7.5.

18. The lipid nanoparticle of claim 17, wherein the phospholipid having a choline head group is present at a content of at least 35 mol% and wherein the phospholipid is selected from distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidylcholine (DMPC) and / or dipalmitoyl-phosphatidylcholine (DPPC) and wherein the lipid nanoparticle has less than 40 mol% ionizable cationic lipid.

19. The lipid nanoparticle of claim 17 or 18, wherein the nucleic acid editor is a Cas-based editor, transcription activator-like effector nuclease (TALEN), megaTAL, zinc finger nuclease (ZFN), Adenosine Deaminase Acting on RNA (ADAR), prime editor, base editor, epigenetic editor, transposase, meganuclease, ARCUS gene editing system or a combination thereof.

20. The lipid nanoparticle of claim 19, wherein the nucleic acid editor is CRISPR and comprises a guide RNA and an mRNA coding for a Cas nuclease.

21. The lipid nanoparticle of any one of claim 17 to 20, wherein the nucleic acid editor is used to treat a genetic disorder resulting from altered blood cell maturation and function.

22. The lipid nanoparticle of claim 21, wherein the genetic disorder resulting from the altered blood cell maturation and function is a disease of the innate and adaptive immune system, a red blood cell disorder, a platelet disorder or a bone marrow failure syndrome.

23. The lipid nanoparticle of claim 21, wherein the nucleic acid editor complex is used to treat a disorder that is an immunodeficiency, optionally a primary immunodeficiency.

24. The lipid nanoparticle of claim 23, wherein the primary immunodeficiency is selected from X-linked severe combined immunodeficiency (SCID), adenosine deaminase (ADA) deficiency SCID, Wiskott-Aldrich (WAS) syndrome and chronic granulomatous disease (CGD).

25. The lipid nanoparticle of claim 22, wherein the red blood cell disorder is an erythrocyte disorder.

26. The lipid nanoparticle of claim 25, wherein the erythrocyte disorder is a beta-Thalassaemia, a sickle cell disease or a pyruvate kinase deficiency.

27. The lipid nanoparticle of any one of claims 17 to 20, wherein the lipid nanoparticle is used to treat a disease or disorder that is a cancer.

28. The lipid nanoparticle of claim 27, wherein the cancer is a haematological cancer.

29. Use of a lipid nanoparticle to deliver and cause cellular uptake of a cargo to a haematopoietic stem or progenitor cell of a subject, the lipid nanoparticle having between 30 mol% and 70 mol% of a phospholipid having a choline head group, an ionizable cationic lipid, and asterol, wherein the lipid nanoparticle is substantially uncharged at physiological pH and has an apparent pKa of between 6.0 and 7.5.

30. Use of the lipid nanoparticle of claim 29, wherein the phospholipid having a choline head group is present at a content of at least 35 mol% and wherein the phospholipid is selected from distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidylcholine (DMPC) and / or dipalmitoyl-phosphatidylcholine (DPPC) and wherein the lipid nanoparticle has less than 40 mol% ionizable cationic lipid.

31. Use of the lipid nanoparticle of claim 29 or 30, wherein the haematopoietic stem or progenitor cell is in the spleen or bone marrow of the subject.

32. Use of the lipid nanoparticle of claim 29, 30 or 31, wherein the cargo comprises a nucleic acid that is part of a nucleic acid editor complex.

33. Use of the lipid nanoparticle of claim 32, wherein the nucleic acid editor is nucleic acid editor is a Cas-based editor, transcription activator-like effector nuclease (TALEN), megaTAL, zinc finger nuclease (ZFN), Adenosine Deaminase Acting on RNA (ADAR), prime editor, base editor, epigenetic editor, transposase, meganuclease, ARCUS gene editing system or a combination thereof.

34. Use of the lipid nanoparticle of claim 33, wherein the nucleic acid editor is CRISPR and comprises a guide RNA and an mRNA or vector DNA coding for a Cas nuclease.

35. Use of the lipid nanoparticle of claim 32, wherein the nucleic acid is a guide RNA that is part of the nucleic acid editor and the method further comprises contacting the haematopoietic stem or progenitor cell in vivo or ex vivo with a second lipid nanoparticle comprising an mRNA coding for a Cas nuclease.

36. Use of the lipid nanoparticle of any one of claims 29 to 35, wherein the lipid nanoparticle is used to treat a disease or disorder that is a genetic disorder.

37. Use of the lipid nanoparticle of claim 36, wherein the genetic disorder results from altered blood cell maturation and function.

38. Use of the lipid nanoparticle of claim 37, wherein the genetic disorder resulting from the altered blood cell maturation and function is a disease of the innate and adaptive immune system, a red blood cell disorder, a platelet disorder or a bone marrow failure syndrome.

39. Use of the lipid nanoparticle of claim 36, wherein the genetic disorder is an immunodeficiency, optionally a primary immunodeficiency.

40. Use of the lipid nanoparticle of claim 39, wherein the primary immunodeficiency is selected from X-linked severe combined immunodeficiency (SCID), adenosine deaminase (ADA) deficiency SCID, Wiskott-Aldrich (WAS) syndrome and chronic granulomatous disease (CGD).

41. Use of the lipid nanoparticle of claim 38, wherein the red blood cell disorder is an erythrocyte disorder.

42. Use of the lipid nanoparticle of claim 41, wherein the erythrocyte disorder is a beta- Thalassaemia, a sickle cell disease or a pyruvate kinase deficiency.

43. Use of the lipid nanoparticle of any one of claims 29 to 35, wherein the lipid nanoparticle is used to treat a disease or disorder that is a cancer.

44. Use of the lipid nanoparticle of claim 43, wherein the cancer is a haematological cancer.

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