Methods and compositions for treating hepatitis b virus-related conditions

Abstract

The present disclosure encompasses a lipid nanoparticle (LNP) comprising a polypeptide comprising a nucleic acid sequence encoding an engineered meganuclease that binds and cleaves a recognition sequence within a Hepatitis B virus (HBV) genome. Further, the disclosure encompasses pharmaceutical compositions comprising the LNPs, and the use of such compositions for inactivating a pol gene of an HBV genome or an HBV genome fragment in a cell and treating HBV infections or diseases associated with HBV infections.

Description

[0001] METHODS AND COMPOSITIONS FOR TREATING HEPATITIS B VIRUS-RELATED

[0002] CONDITIONS

[0003] FIELD OF THE INVENTION

[0004] The disclosure relates to the field of virology, molecular biology, and recombinant nucleic acid technology. In particular, the disclosure relates to optimized engineered meganucleases having specificity for a recognition sequence within the genome of the Hepatitis B virus (HBV). Such engineered meganucleases are useful in methods for treating HBV infections and diseases caused by HBV.

[0005] REFERENCE TO A SEQUENCE LISTING SUBMITTED ELECTRONIC ALLY AS AN XML FILE

[0006] The instant application contains a Sequence Listing which has been submitted in XML format via USPTO Patent Center and is hereby incorporated by reference in its entirety. Said XML copy, created on February 19, 2025, is named P89339 2080USP4 SL, and is 38.2 KB in size.

[0007] BACKGROUND OF THE INVENTION

[0008] Chronic hepatitis B (CHB) is a potentially life-threatening liver infection caused by HBV, which is highly infectious and easily transmissible through perinatal, percutaneous, and sexual exposure (World Health Organization, 2024). HBV has immunological characteristics that aid in the establishment of chronic infection that is difficult to eradicate. Chronic production of viral antigens may also lead to inflammation and necrosis, resulting in hepatitis, cirrhosis, hepatocellular carcinoma (HCC), and liver failure (Fanning, 2019). Patients with CHB are at a substantial health risk in the absence of effective therapy, with up to 40% ultimately at risk for the development of cirrhosis, hepatic decompensation, and / or HCC (Wright, 2006). Globally, an estimated 820,000 deaths from cirrhosis and HCC as a result of CHB infection were reported in 2019, and deaths due to HBV infections are expected to reach ~1 million by 2035 (Dusheiko, 2023; World Health Organization, 2024). As of 2019, an estimated 296 million persons worldwide were living with CHB, with 1.5 million new infections each year (World Health Organization, 2024).

[0009] Hepatitis B surface antigen (HBsAg) is the major viral component of the envelope for infectious HBV particles. HBsAg is secreted in excess into patients’ serum in the form of empty subviral particles, which have been implicated in impairment of the host immune response, thus maintaining chronic HBV infection (Yuen, 2023). In addition to cccDNA-derived HBsAg, HBsAg can also originate from host genome-integrated forms of HBV DNA; depending on the disease progression, integrated HBV DNA may be responsible for a large portion of secreted HBsAg

[0010] WBD (US) 4897-9494-7440vl 1 Atty Docket No. P89339 2080WO (01275) (Podlaha, 2019; Wooddell, 2017). Additionally, integrated HBV DNA is associated with the development of HCC, as an HBV DNA level of >2000 lU / mL is considered a strong prognostic indicator of cirrhosis and HCC (Dusheiko, 2023; Wooddell, 2017). Indeed, the loss of HBsAg in patients with CHB infection has been associated with improved outcomes, including reversal of cirrhosis, decreased risk of HCC, and prolonged survival (Benias & Min, 2011; Benvegnu, 1998; EASL, 2017; Fattovich, 1998; Kim, J. H., 2013; Liaw, 2012; Lok, 2013).

[0011] Current standard of care treatment for patients with CHB consists primarily of interferon (IFN)a and long-term treatment with NAs. Pegylated-IFNa (PEG-IFNa) is a pleotropic cytokine that inhibits HBV replication through degradation of cccDNA via epigenetic alterations (Zhao, 2022). In comparison to NAs, PEG-IFNa has a limited treatment duration, a higher rate of seroconversion of HBeAg and HBsAg, a higher probability of sustained off-treatment response, and lack of drug resistance. However, PEG-IFNa is not well tolerated and common adverse effects can potentially lead to significant morbidity or mortality (Woo, 2017). NAs are reverse transcriptase inhibitors that selectively target, bind, and inhibit HBV polymerase (Dusheiko, 2023). While NAs inhibit viral replication and spread, they are unable to eliminate cccDNA and do not directly target viral transcription or protein production (e.g., HBsAg) (Kwon, 2011; Liaw, 2012; Lok, 2013) which means that the treatment with NA products is indefinite.

[0012] Despite currently available treatment options, the current goal of treatment for CHB, functional cure (defined as undetectable serum HBV DNA and HBsAg, with or without anti-hepatitis B surface antibody seroconversion maintained for a minimum of 6 months after discontinuation of treatment), remains out of reach for the vast majority of patients (EASL, 2017, Liaw, 2012, Sarin, 2015, Terrault, 2018). As such, there is a significant unmet medical need for patients with CHB.

[0013] SUMMARY OF THE INVENTION

[0014] In one aspect, the disclosure provides a lipid nanoparticle (LNP) comprising a polynucleotide, wherein said polynucleotide comprises a nucleic acid sequence encoding an engineered meganuclease that binds and cleaves a recognition sequence comprising SEQ ID NO: 3 in a polymerase (pol) gene of a hepatitis B virus (HBV) genome or HBV genome fragment, and wherein said LNP comprises: a) Bis(2-butyloctyl) 10-(N-(3-(pyrrolidin-l- yl)propyl)nonanamido)nonadecanedioate (Compound of Structure 15 of Table 2); b) 2-[2-(o- methoxy (polyethyleneglycol2000) ethoxy]-N,N-ditetradecylacetamide (Compound of Structure Ila); c) l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); and d) cholesterol.

[0015] WBD (US) 4897-9494-7440vl 2 Atty Docket No. P89339 2080WO (01275) In some embodiments, the molar concentration of Bis(2 -butyloctyl) 10-(N-(3-(pyrrolidin-l- yl)propyl)nonanamido)nonadecanedioate in said LNP is between about 45% to about 50% of the total molar lipid concentration.

[0016] In some embodiments, the molar concentration of Bis(2 -butyloctyl) 10-(N-(3-(pyrrolidin-l- yl)propyl)nonanamido)nonadecanedioate in said LNP is about 47.5% of the total molar lipid concentration.

[0017] In some embodiments, the molar concentration of 2-[2-(o -methoxy (polyethyleneglycol2000) ethoxy]-N,N-ditetradecylacetamide in said LNP is between about 1.5% to about 3.5% of the total molar lipid concentration.

[0018] In some embodiments, the molar concentration of 2-[2-(o -methoxy (polyethyleneglycol2000) ethoxy]-N,N-ditetradecylacetamide in said LNP is about 2.5% of the total molar lipid concentration.

[0019] In some embodiments, the molar concentration of DSPC in said LNP is between about 7.5% to about 12.5% of the total molar lipid concentration.

[0020] In some embodiments, the molar concentration of DSPC in said LNP is about 10% of the total molar lipid concentration.

[0021] In some embodiments, the molar concentration of cholesterol in said LNP is between about 37.5% to about 42.5% of the total molar lipid concentration.

[0022] In some embodiments, the molar concentration of cholesterol in said LNP is about 40% of the total molar lipid concentration.

[0023] In some embodiments, the molar ratio of Bis(2 -butyloctyl) 10-(N-(3-(pyrrolidin-l- yl)propyl)nonanamido)nonadecanedioate:2-[2-(o -methoxy (polyethyleneglycol2000) ethoxy]-N,N- ditetradecylacetamide:DSPC:cholesterol in said LNP is about 47.5:2.5: 10:40.

[0024] In some embodiments, said LNP has a particle size of between about 55-75 nm.

[0025] In some embodiments, said LNP has a particle size of between about 62-73 nm.

[0026] In some embodiments, said LNP has a particle size of about 65 nm.

[0027] In some embodiments, said engineered meganuclease comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 5 or SEQ ID NO: 6.

[0028] In some embodiments, said engineered meganuclease comprises an amino acid sequence set forth in SEQ ID NO: 5 or SEQ ID NO: 6.

[0029] In some embodiments, said nucleic acid sequence encoding said engineered meganuclease comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at

[0030] WBD (US) 4897-9494-7440vl 3 Atty Docket No. P89339 2080WO (01275) least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 7 or SEQ ID NO: 8.

[0031] In some embodiments, said nucleic acid sequence encoding said engineered meganuclease comprises a sequence set forth in SEQ ID NO: 7 or SEQ ID NO: 8.

[0032] In some embodiments, said nucleic acid sequence encoding said engineered meganuclease comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 9 or SEQ ID NO: 10.

[0033] In some embodiments, said nucleic acid sequence encoding said engineered meganuclease comprises a sequence set forth in SEQ ID NO: 9 or SEQ ID NO: 10.

[0034] In some embodiments, said engineered meganuclease comprises a 5’ nuclear localization sequence (NLS) at its N-terminus.

[0035] In some embodiments, said 5’ NLS comprises an amino acid sequence having at least 80% or at least 90% sequence identity to SEQ ID NO: 11.

[0036] In some embodiments, said 5’ NLS comprises an amino acid sequence set forth in SEQ ID NO: 11.

[0037] In some embodiments, said engineered meganuclease comprises a 3’ nuclear localization sequence (NLS) at its C-terminus.

[0038] In some embodiments, said 3’ NLS comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 12.

[0039] In some embodiments, said 3’ NLS comprises an amino acid sequence set forth in SEQ ID NO: 12.

[0040] In some embodiments, said engineered meganuclease comprises a 5’ NLS at its N-terminus comprising an amino acid sequence having at least 80% or at least 90% sequence identity to SEQ ID NO: 11, and a 3’ NLS at its C-terminus comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 12.

[0041] In some embodiments, said engineered meganuclease comprises a 5’ NLS at its N-terminus comprising an amino acid sequence set forth in SEQ ID NO: 11, and a 3’ NLS at its C-terminus comprising an amino acid sequence set forth in SEQ ID NO: 12.

[0042] In some embodiments, said polynucleotide comprises a 5’ untranslated region (UTR) sequence.

[0043] In some embodiments, said 5’ UTR sequence comprises a nucleic acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 13.

[0044] WBD (US) 4897-9494-7440vl 4 Atty Docket No. P89339 2080WO (01275) In some embodiments, said 5’ UTR sequence comprises a nucleic acid sequence set forth in SEQ ID NO: 13.

[0045] In some embodiments, said 5’ UTR sequence comprises a nucleic acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 14.

[0046] In some embodiments, said 5’ UTR sequence comprises a nucleic acid sequence set forth in SEQ ID NO: 14.

[0047] In some embodiments, said polynucleotide comprises a 3’ untranslated region (UTR) sequence.

[0048] In some embodiments, said 3’ UTR sequence comprises a nucleic acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 15.

[0049] In some embodiments, said 3’ UTR sequence comprises a nucleic acid sequence set forth in SEQ ID NO: 15.

[0050] In some embodiments, said 3’ UTR sequence comprises a nucleic acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 16.

[0051] In some embodiments, said 3’ UTR sequence comprises a nucleic acid sequence set forth in SEQ ID NO: 16.

[0052] In some embodiments, said polynucleotide comprises a 5’ UTR sequence that comprises a nucleic acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 13, and a 3’ UTR sequence that comprises a nucleic acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 15.

[0053] In some embodiments, said polynucleotide comprises a 5’ UTR sequence that comprises a nucleic acid sequence set forth in SEQ ID NO: 13, and a 3’ UTR sequence that comprises a nucleic acid sequence set forth in SEQ ID NO: 15.

[0054] In some embodiments, said polynucleotide comprises a 5’ UTR sequence that comprises a nucleic acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 14, and a 3’ UTR sequence that comprises a nucleic acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 16.

[0055] WBD (US) 4897-9494-7440vl 5 Atty Docket No. P89339 2080WO (01275) In some embodiments, said polynucleotide comprises a 5’ UTR sequence that comprises a nucleic acid sequence set forth in SEQ ID NO: 14, and a 3’ UTR sequence that comprises a nucleic acid sequence set forth in SEQ ID NO: 16.

[0056] In some embodiments, said polynucleotide comprises a Kozak sequence comprising a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 17.

[0057] In some embodiments, said polynucleotide comprises a Kozak sequence comprising a nucleic acid sequence set forth in SEQ ID NO: 17.

[0058] In some embodiments, said polynucleotide comprises a Kozak sequence comprising a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 18.

[0059] In some embodiments, said polynucleotide comprises a Kozak sequence comprising a nucleic acid sequence set forth in SEQ ID NO: 18.

[0060] In some embodiments, said polynucleotide comprises a polyA sequence.

[0061] In some embodiments, said polyA sequence comprises a nucleic acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 19.

[0062] In some embodiments, said polyA sequence comprises a nucleic acid sequence set forth in SEQ ID NO: 19.

[0063] In some embodiments, said polynucleotide comprises a nucleic acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 20. In some embodiments, said polynucleotide comprises a nucleic acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 21.

[0064] In some embodiments, said polynucleotide comprises a nucleic acid sequence set forth in SEQ ID NO: 20. In some embodiments, said polynucleotide comprises a nucleic acid sequence set forth in SEQ ID NO: 21.

[0065] In some embodiments, said polynucleotide comprises a nucleic acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 22. In some embodiments, said polynucleotide comprises a nucleic acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 23.

[0066] In some embodiments, said polynucleotide comprises a nucleic acid sequence set forth in SEQ ID NO: 22. In some embodiments, said polynucleotide comprises a nucleic acid sequence set forth in SEQ ID NO: 23.

[0067] WBD (US) 4897-9494-7440vl 6 Aty Docket No. P89339 2080WO (01275) In some embodiments, said polynucleotide is a messenger RNA (mRNA).

[0068] In some embodiments, said mRNA comprises a 5’ cap structure.

[0069] In some embodiments, said 5’ cap structure comprises (m7G(5’)ppp(5’)(2’OMeA)pG).

[0070] In some embodiments, uridine bases in said mRNA are modified to pseudouridine, Nl- methyl-pseudouridine, 5-methoxyuridine, or 2-thiouridine. In some embodiments, uridine bases in said mRNA are modified to pseudouridine. In some embodiments, uridine bases in said mRNA are modified to Nl-methyl-pseudouridine. In some embodiments, uridine bases in said mRNA are modified to 5-methoxyuridine. In some embodiments, uridine bases in said mRNA are modified to 2-thiouridine.

[0071] In some embodiments, cytosine bases in said mRNA are modified to 5-methylcytidine, 2'- O-methylcytidine, or N4-acetyl-cytosine. In some embodiments, cytosine bases in said mRNA are modified to 5-methylcytidine. In some embodiments, cytosine bases in said mRNA are modified to 2'-O-methylcytidine. In some embodiments, cytosine bases in said mRNA are modified to N4- acetyl-cytosine.

[0072] In some embodiments, adenine bases in said mRNA are modified to N6-methyladenosine or Nl-methyl-adenine. In some embodiments, adenine bases in said mRNA are modified to N6- methyladenosine. In some embodiments, adenine bases in said mRNA are modified to Nl-methyl- adenine.

[0073] In some embodiments, said LNP has an N:P ratio of between about 4: 1-8: 1.

[0074] In some embodiments, said LNP has an N:P ratio of about 5: 1-7: 1.

[0075] In some embodiments, said LNP has an N:P ratio of about 6: 1.

[0076] In some embodiments, said mRNA comprises a nucleic acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 20. In some embodiments, said mRNA comprises a nucleic acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 21.

[0077] In some embodiments, said mRNA comprises a nucleic acid sequence set forth in SEQ ID NO: 20. In some embodiments, said mRNA comprises a nucleic acid sequence set forth in SEQ ID NO: 21.

[0078] In one aspect, the present disclosure provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a plurality of LNP provided herein.

[0079] In some embodiments, said pharmaceutical composition comprises between about 15.7-19.2 mg / mL of Bis(2 -butyloctyl) 10-(N-(3-(pyrrolidin-l-yl)propyl)nonanamido)nonadecanedioate.

[0080] WBD (US) 4897-9494-7440vl 7 Atty Docket No. P89339 2080WO (01275) In some embodiments, said pharmaceutical composition comprises between about 17.4-18.5 mg / mL of Bis(2 -butyloctyl) 10-(N-(3-(pyrrolidin-l-yl)propyl)nonanamido)nonadecanedioate.

[0081] In some embodiments, said pharmaceutical composition comprises between about 2.2-3.0 mg / mL of 2-[2-(o -methoxy (polyethyleneglycol2000) ethoxy]-N,N-ditetradecylacetamide.

[0082] In some embodiments, said pharmaceutical composition comprises between about 2.6-2.8 mg / mL of 2-[2-(o -methoxy (polyethyleneglycol2000) ethoxy]-N,N-ditetradecylacetamide.

[0083] In some embodiments, said pharmaceutical composition comprises between about 2.5-3.4 mg / mL of DSPC.

[0084] In some embodiments, said pharmaceutical composition comprises between about 3.3. -3.4 mg / mL of DSPC.

[0085] In some embodiments, said pharmaceutical composition comprises between about 5.5-7.0 mg / mL of cholesterol.

[0086] In some embodiments, said pharmaceutical composition comprises between about 6.4-6.6 mg / mL of cholesterol.

[0087] In some embodiments, said pharmaceutical composition comprises between about 0.8-1.2 mg / mL of said polynucleotide.

[0088] In some embodiments, said pharmaceutical composition comprises between about 0.95-1.1 mg / mL of said polynucleotide.

[0089] In some embodiments, said pharmaceutical composition comprises about 1.0 mg / mL of said polynucleotide.

[0090] In some embodiments, the concentrations of Bis(2 -butyloctyl) 10-(N-(3 -(pyrrolidin- 1- yl)propyl)nonanamido) nonadecanedioate, 2- [2-(o -methoxy (polyethyleneglycol2000) ethoxy]- N,N-ditetradecylacetamide, DSPC, and cholesterol described above are relative to a concentration of about 1.0 mg / mL of said polynucleotide in said pharmaceutical composition, wherein the concentrations of Bis(2 -butyloctyl) 10-(N-(3 -(pyrrolidin- 1 -yl)propyl)nonanamido) nonadecanedi oate, 2-[2-(o -methoxy (polyethyleneglycol2000) ethoxy]-N,N-ditetradecylacetamide, DSPC, and cholesterol increase or decrease in said pharmaceutical composition proportionally to the change in concentration of the polynucleotide from 1.0 mg / mL.

[0091] In some embodiments, said pharmaceutical composition comprises: a) between about 15.7- 19.2 mg / mL of Bis(2 -butyloctyl) 10-(N-(3 -(pyrrolidin- l-yl)propyl)nonanamido) nonadecanedioate; b) between about 2.2-3.0 mg / mL of 2- [2-(o -methoxy (polyethyleneglycol2000) ethoxy]-N,N- ditetradecylacetamide; c) between about 2.5-3.4 mg / mL of DSPC; d) between about 5.5-7.0 mg / mL of cholesterol; and e) between about 0.8-1.2 mg / mL of said polynucleotide.

[0092] In some embodiments, said polynucleotide is an mRNA.

[0093] WBD (US) 4897-9494-7440vl 8 Atty Docket No. P89339 2080WO (01275) In some embodiments, said plurality of LNPs are formulated in phosphate-buffered saline and sucrose.

[0094] In some embodiments, the phosphate-buffered saline is Dulbecco’s phosphate-buffered saline.

[0095] In some embodiments, said sucrose is at a concentration of about 300 mM.

[0096] In some embodiments, said pharmaceutical composition has a pH between about 6.7-7.6.

[0097] In some embodiments, said pharmaceutical composition has a pH of about 6.92-7.42.

[0098] In some embodiments, said pharmaceutical composition has a pH of about 7.4-7.5.

[0099] In some embodiments, said pharmaceutical composition has a poly dispersity index equal to, or less than, 0.20.

[0100] In some embodiments, the osmolality of said pharmaceutical composition is between about 495-605 mOsm / kg.

[0101] In some embodiments, the osmolality of said pharmaceutical composition is about 564-603 mOsm / kg.

[0102] In some embodiments, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% of said plurality of LNPs comprise said polynucleotide (e.g., mRNA).

[0103] In one aspect, the present disclosure provides a method for inactivating a pol gene of an HBV genome or an HBV genome fragment. The method comprises introducing into a cell comprising said HBV genome or HBV genome fragment the LNP provided herein, wherein said engineered meganuclease is expressed in said cell and produces a cleavage site at a recognition sequence comprising SEQ ID NO: 3 in said pol gene.

[0104] In some embodiments, said pol gene is inactivated by introduction of an indel at said cleavage site, or wherein said pol gene is inactivated by elimination of said HBV genome or said HBV genome fragment.

[0105] In some embodiments, said HBV genome or said HBV genome fragment is comprised by covalently closed circular DNA (cccDNA).

[0106] In some embodiments, said cccDNA is eliminated following generation of said cleavage site.

[0107] In some embodiments, said pol gene is inactivated in said cccDNA by introduction of said indel at said cleavage site.

[0108] In some embodiments, said indel is introduced by non-homologous end joining (NHEJ).

[0109] In some embodiments, said inactivated pol gene does not encode an active and / or full- length HBV polymerase protein.

[0110] WBD (US) 4897-9494-7440vl 9 Atty Docket No. P89339 2080WO (01275) In some embodiments, said HBV genome or said HBV genome fragment is comprised in the genome of said target cell.

[0111] In some embodiments, said pol gene is inactivated in said HBV genome or said HBV genome fragment by introduction of an indel at said cleavage site.

[0112] In some embodiments, said genome is the nuclear genome.

[0113] In some embodiments, said genome is the mitochondrial genome.

[0114] In some embodiments, said indel is introduced by NHEJ.

[0115] In some embodiments, said inactivated pol gene does not encode an active and / or full- length HBV polymerase protein.

[0116] In some embodiments, said method inactivates an HBsAg gene in said HBV genome or said HBV genome fragment.

[0117] In some embodiments, said cell is a liver cell.

[0118] In some embodiments, said liver cell is a hepatocyte.

[0119] In some embodiments, said cell is in vitro.

[0120] In some embodiments, said cell is in vivo.

[0121] In some embodiments, said polynucleotide comprised by said LNP is an mRNA.

[0122] In some embodiments, the mRNA is an mRNA provided herein.

[0123] In one aspect, the present disclosure provides a method for inactivating a pol gene of an HBV genome or an HBV genome fragment in a target cell in a subject. The method comprises delivering to said target cell comprising said HBV genome or HBV genome fragment the LNP provided herein, wherein said engineered meganuclease is expressed in said target cell and produces a cleavage site at a recognition sequence comprising SEQ ID NO: 3 in said pol gene.

[0124] In some embodiments, said pol gene is inactivated by introduction of an indel at said cleavage site, or wherein said pol gene is inactivated by elimination of said HBV genome or said HBV genome fragment.

[0125] In some embodiments, said HBV genome or said HBV genome fragment is comprised by cccDNA.

[0126] In some embodiments, said cccDNA is eliminated following generation of said cleavage site.

[0127] In some embodiments, said pol gene is inactivated in said cccDNA by introduction of an indel at said cleavage site.

[0128] In some embodiments, said indel is introduced by NHEJ.

[0129] In some embodiments, said inactivated pol gene does not encode an active and / or full- length HBV polymerase protein.

[0130] WBD (US) 4897-9494-7440vl 10 Atty Docket No. P89339 2080WO (01275) In some embodiments, said HBV genome or said HBV genome fragment is comprised in the genome of said target cell.

[0131] In some embodiments, said pol gene is inactivated in said HBV genome or said HBV genome fragment by introduction of an indel at said cleavage site.

[0132] In some embodiments, said genome is the nuclear genome.

[0133] In some embodiments, said genome is the mitochondrial genome.

[0134] In some embodiments, said indel is introduced by NHEJ.

[0135] In some embodiments, said inactivated pol gene does not encode an active and / or full- length HBV polymerase protein.

[0136] In some embodiments, said method inactivates an HBsAg gene in said HBV genome or said HBV genome fragment.

[0137] In some embodiments, said target cell is a liver cell.

[0138] In some embodiments, said liver cell is a hepatocyte.

[0139] In some embodiments, said subject is administered a pharmaceutical composition provided herein.

[0140] In some embodiments, said polynucleotide comprised by said LNP is an mRNA.

[0141] In some embodiments, said mRNA is mRNA provided herein.

[0142] In one aspect, the present disclosure provides a method for treating a disease associated with hepatitis B virus infection. The method comprises administering to an HBV-infected subject a therapeutically effective dose of a pharmaceutical composition comprising a plurality of LNPs provided herein, wherein said plurality of LNPs are delivered to target cells in said subject that comprise an HBV genome or HBV genome fragment, wherein said engineered meganuclease is expressed in said target cells and produces a cleavage site at a recognition sequence comprising SEQ ID NO: 3 in a pol gene in said HBV genome or said HBV genome fragment.

[0143] In some embodiments, said pol gene is inactivated by introduction of an indel at said cleavage site, or wherein said pol gene is inactivated by elimination of said HBV genome or said HBV genome fragment.

[0144] In some embodiments, wherein said polynucleotide is an mRNA.

[0145] In some embodiments, wherein said mRNA is said mRNA provided herein.

[0146] In some embodiments, wherein said HBV genome or said HBV genome fragment is comprised by cccDNA.

[0147] In some embodiments, said cccDNA is eliminated following generation of said cleavage site.

[0148] In some embodiments, said pol gene is inactivated in said cccDNA by introduction of said indel at said cleavage site.

[0149] WBD (US) 4897-9494-7440vl 11 Aty Docket No. P89339 2080WO (01275) In some embodiments, said indel is introduced by NHEJ.

[0150] In some embodiments, said inactivated pol gene does not encode an active and / or full- length HB V polymerase protein.

[0151] In some embodiments, said HBV genome or said HBV genome fragment is comprised in the genome of said target cells.

[0152] In some embodiments, said pol gene is inactivated in said HBV genome or said HBV genome fragment by introduction of an indel at said cleavage site.

[0153] In some embodiments, said genome is the nuclear genome.

[0154] In some embodiments, said genome is the mitochondrial genome.

[0155] In some embodiments, said indel is introduced by NHEJ.

[0156] In some embodiments, said inactivated pol gene does not encode an active and / or full- length HBV polymerase protein.

[0157] In some embodiments, said method inactivates an HBsAg gene in said HBV genome or said HBV genome fragment.

[0158] In some embodiments, said target cells are liver cells.

[0159] In some embodiments, said liver cells are hepatocytes.

[0160] In some embodiments, said pharmaceutical composition is said pharmaceutical composition provided herein.

[0161] In some embodiments, said pharmaceutical composition is administered to said subject by intravenous (IV) administration.

[0162] In some embodiments, said pharmaceutical composition is administered by IV administration over about 2 hours.

[0163] In some embodiments, said pharmaceutical composition is administered at a dose of between about 0.01-2.0 mg / kg of said polynucleotide (e.g., mRNA).

[0164] In some embodiments, said pharmaceutical composition is administered at a dose of between about 0.1-1.5 mg / kg of said polynucleotide (e.g., mRNA).

[0165] In some embodiments, said pharmaceutical composition is administered at a dose of about 0.1 mg / kg of said polynucleotide (e.g., mRNA).

[0166] In some embodiments, said pharmaceutical composition is administered at a dose of about 0.2 mg / kg of said polynucleotide (e.g., mRNA).

[0167] In some embodiments, said pharmaceutical composition is administered at a dose of about 0.3 mg / kg of said polynucleotide (e.g., mRNA).

[0168] In some embodiments, said pharmaceutical composition is administered at a dose of about 0.4 mg / kg of said polynucleotide (e.g., mRNA).

[0169] WBD (US) 4897-9494-7440vl 12 Atty Docket No. P89339 2080WO (01275) In some embodiments, said pharmaceutical composition is administered at a dose of about 0.5 mg / kg of said polynucleotide (e.g., mRNA).

[0170] In some embodiments, said pharmaceutical composition is administered at a dose of about 0.6 mg / kg of said polynucleotide (e.g., mRNA).

[0171] In some embodiments, said pharmaceutical composition is administered at a dose of about 0.7 mg / kg of said polynucleotide (e.g., mRNA).

[0172] In some embodiments, said pharmaceutical composition is administered at a dose of about 0.8 mg / kg of said polynucleotide (e.g., mRNA).

[0173] In some embodiments, said pharmaceutical composition is administered at a dose of about 0.9 mg / kg of said polynucleotide (e.g., mRNA).

[0174] In some embodiments, said pharmaceutical composition is administered at a dose of about 1.0 mg / kg of said polynucleotide (e.g., mRNA).

[0175] In some embodiments, said subject is administered a single dose of said pharmaceutical composition on day 0.

[0176] In some embodiments, said subject is further administered a second dose of said pharmaceutical composition following administration of said first dose.

[0177] In some embodiments, said subject is further administered a second dose of said pharmaceutical composition about 8 weeks following administration of said first dose. In some embodiments, said subject is further administered a second dose of said pharmaceutical composition about 4 weeks following administration of said first dose. In some embodiments, said subject is further administered a second dose of said pharmaceutical composition about 5 weeks following administration of said first dose. In some embodiments, said subject is further administered a second dose of said pharmaceutical composition about 6 weeks following administration of said first dose. In some embodiments, said subject is further administered a second dose of said pharmaceutical composition about 7 weeks following administration of said first dose. In some embodiments, said subject is further administered a second dose of said pharmaceutical composition about 9 weeks following administration of said first dose. In some embodiments, said subject is further administered a second dose of said pharmaceutical composition about 10 weeks following administration of said first dose. In some embodiments, said subject is further administered a second dose of said pharmaceutical composition about 11 weeks following administration of said first dose. In some embodiments, said subject is further administered a second dose of said pharmaceutical composition about 12 weeks following administration of said first dose. In some embodiments, said subject is further administered a second dose of said pharmaceutical composition up to about 52 weeks following administration of said first dose.

[0178] WBD (US) 4897-9494-7440vl 13 Aty Docket No. P89339 2080WO (01275) In some embodiments, said first dose and said second dose are equivalent.

[0179] In some embodiments, said second dose is higher than said first dose.

[0180] In some embodiments, said second dose is lower than said first dose.

[0181] In some embodiments, said second dose is between about 0.01-2.0 mg / kg of said polynucleotide (e.g., mRNA).

[0182] In some embodiments, said second dose is between about 0.1-1.5 mg / kg of said polynucleotide (e.g., mRNA).

[0183] In some embodiments, said second dose is about 0.1 mg / kg of said polynucleotide (e.g., mRNA).

[0184] In some embodiments, said second dose is about 0.2 mg / kg of said polynucleotide (e.g., mRNA).

[0185] In some embodiments, said second dose is about 0.3 mg / kg of said polynucleotide (e.g., mRNA).

[0186] In some embodiments, said second dose is about 0.4 mg / kg of said polynucleotide (e.g., mRNA).

[0187] In some embodiments, said second dose is about 0.5 mg / kg of said polynucleotide (e.g., mRNA).

[0188] In some embodiments, said second dose is about 0.6 mg / kg of said polynucleotide (e.g., mRNA).

[0189] In some embodiments, said second dose is about 0.7 mg / kg of said polynucleotide (e.g., mRNA).

[0190] In some embodiments, said second dose is about 0.8 mg / kg of said polynucleotide (e.g., mRNA).

[0191] In some embodiments, said second dose is about 0.9 mg / kg of said polynucleotide (e.g., mRNA).

[0192] In some embodiments, said second dose is about 1.0 mg / kg of said polynucleotide (e.g., mRNA).

[0193] In some embodiments, said first dose is about 0.1 mg / kg of said polynucleotide (e.g., mRNA) and said second dose is about 0.1 mg / kg of said polynucleotide (e.g., mRNA).

[0194] In some embodiments, said first dose is about 0.2 mg / kg of said polynucleotide (e.g., mRNA) and said second dose is about 0.2 mg / kg of said polynucleotide (e.g., mRNA).

[0195] In some embodiments, said first dose is about 0.3 mg / kg of said polynucleotide (e.g., mRNA) and said second dose is about 0.3 mg / kg of said polynucleotide (e.g., mRNA).

[0196] In some embodiments, said first dose is about 0.4 mg / kg of said polynucleotide (e.g., mRNA) and said second dose is about 0.4 mg / kg of said polynucleotide (e.g., mRNA).

[0197] WBD (US) 4897-9494-7440vl 14 Aty Docket No. P89339 2080WO (01275) In some embodiments, said first dose is about 0.5 mg / kg of said polynucleotide (e.g., mRNA) and said second dose is about 0.5 mg / kg of said polynucleotide (e.g., mRNA).

[0198] In some embodiments, said first dose is about 0.6 mg / kg of said polynucleotide (e.g., mRNA) and said second dose is about 0.6 mg / kg of said polynucleotide (e.g., mRNA).

[0199] In some embodiments, said first dose is about 0.7 mg / kg of said polynucleotide (e.g., mRNA) and said second dose is about 0.7 mg / kg of said polynucleotide (e.g., mRNA).

[0200] In some embodiments, said first dose is about 0.8 mg / kg of said polynucleotide (e.g., mRNA) and said second dose is about 0.8 mg / kg of said polynucleotide (e.g., mRNA).

[0201] In some embodiments, said first dose is about 0.9 mg / kg of said polynucleotide (e.g., mRNA) and said second dose is about 0.9 mg / kg of said polynucleotide (e.g., mRNA).

[0202] In some embodiments, said first dose is about 1.0 mg / kg of said polynucleotide (e.g., mRNA) and said second dose is about 1.0 mg / kg of said polynucleotide (e.g., mRNA).

[0203] In some embodiments, said subject is further administered a third dose of said pharmaceutical composition following administration of said second dose.

[0204] In some embodiments, said subject is further administered a third dose of said pharmaceutical composition about 8 weeks following administration of said second dose. In some embodiments, said subject is further administered a third dose of said pharmaceutical composition about 4 weeks following administration of said second dose. In some embodiments, said subject is further administered a third dose of said pharmaceutical composition about 5 weeks following administration of said second dose. In some embodiments, said subject is further administered a third dose of said pharmaceutical composition about 6 weeks following administration of said second dose. In some embodiments, said subject is further administered a third dose of said pharmaceutical composition about 7 weeks following administration of said second dose. In some embodiments, said subject is further administered a third dose of said pharmaceutical composition about 9 weeks following administration of said second dose. In some embodiments, said subject is further administered a third dose of said pharmaceutical composition about 10 weeks following administration of said second dose. In some embodiments, said subject is further administered a third dose of said pharmaceutical composition about 11 weeks following administration of said second dose. In some embodiments, said subject is further administered a third dose of said pharmaceutical composition about 12 weeks following administration of said second dose. In some embodiments, said subject is further administered a third dose of said pharmaceutical composition up to about 52 weeks following administration of said second dose.

[0205] In some embodiments, said first dose, said second dose, and said third dose are equivalent.

[0206] In some embodiments, said third dose is higher than said second dose. In some embodiments, said third dose is lower than said second dose. WBD (US) 4897-9494-7440vl 15 Aty Docket No. P89339 2080WO (01275) In some embodiments, said third dose is between about 0.01-2.0 mg / kg of said polynucleotide (e.g., mRNA).

[0207] In some embodiments, said third dose is between about 0.1-1.5 mg / kg of said polynucleotide (e.g., mRNA).

[0208] In some embodiments, said third dose is about 0.1 mg / kg of said polynucleotide (e.g., mRNA).

[0209] In some embodiments, said third dose is about 0.2 mg / kg of said polynucleotide (e.g., mRNA).

[0210] In some embodiments, said third dose is about 0.3 mg / kg of said polynucleotide (e.g., mRNA).

[0211] In some embodiments, said third dose is about 0.4 mg / kg of said polynucleotide (e.g., mRNA).

[0212] In some embodiments, said third dose is about 0.5 mg / kg of said polynucleotide (e.g., mRNA).

[0213] In some embodiments, said third dose is about 0.6 mg / kg of said polynucleotide (e.g., mRNA).

[0214] In some embodiments, said third dose is about 0.7 mg / kg of said polynucleotide (e.g., mRNA).

[0215] In some embodiments, said third dose is about 0.8 mg / kg of said polynucleotide (e.g., mRNA).

[0216] In some embodiments, said third dose is about 0.9 mg / kg of said polynucleotide (e.g., mRNA).

[0217] In some embodiments, said third dose is about 1.0 mg / kg of said polynucleotide (e.g., mRNA).

[0218] In some embodiments, said third dose is about 1.1 mg / kg of said polynucleotide (e.g., mRNA).

[0219] In some embodiments, said third dose is about 1.2 mg / kg of said polynucleotide (e.g., mRNA).

[0220] In some embodiments, said third dose is about 1.3 mg / kg of said polynucleotide (e.g., mRNA).

[0221] In some embodiments, said third dose is about 1.4 mg / kg of said polynucleotide (e.g., mRNA).

[0222] In some embodiments, said third dose is about 1.5 mg / kg of said polynucleotide (e.g., mRNA).

[0223] WBD (US) 4897-9494-7440vl 16 Atty Docket No. P89339 2080WO (01275) In some embodiments, said first dose is about 0.1 mg / kg of said polynucleotide (e.g., mRNA), said second dose is about 0.1 mg / kg of said polynucleotide (e.g., mRNA), and said third dose is about 0.1 mg / kg of said polynucleotide (e.g., mRNA).

[0224] In some embodiments, said first dose is about 0.2 mg / kg of said polynucleotide (e.g., mRNA), said second dose is about 0.2 mg / kg of said polynucleotide (e.g., mRNA), and said third dose is about 0.2 mg / kg of said polynucleotide (e.g., mRNA).

[0225] In some embodiments, said first dose is about 0.3 mg / kg of said polynucleotide (e.g., mRNA), said second dose is about 0.3 mg / kg of said polynucleotide (e.g., mRNA), and said third dose is about 0.3 mg / kg of said polynucleotide (e.g., mRNA).

[0226] In some embodiments, said first dose is about 0.4 mg / kg of said polynucleotide (e.g., mRNA), said second dose is about 0.4 mg / kg of said polynucleotide (e.g., mRNA), and said third dose is about 0.4 mg / kg of said polynucleotide (e.g., mRNA).

[0227] In some embodiments, said first dose is about 0.5 mg / kg of said polynucleotide (e.g., mRNA), said second dose is about 0.5 mg / kg of said polynucleotide (e.g., mRNA), and said third dose is about 0.5 mg / kg of said polynucleotide (e.g., mRNA).

[0228] In some embodiments, said first dose is about 0.6 mg / kg of said polynucleotide (e.g., mRNA), said second dose is about 0.6 mg / kg of said polynucleotide (e.g., mRNA), and said third dose is about 0.6 mg / kg of said polynucleotide (e.g., mRNA).

[0229] In some embodiments, said first dose is about 0.7 mg / kg of said polynucleotide (e.g., mRNA), said second dose is about 0.7 mg / kg of said polynucleotide (e.g., mRNA), and said third dose is about 0.7 mg / kg of said polynucleotide (e.g., mRNA).

[0230] In some embodiments, said first dose is about 0.8 mg / kg of said polynucleotide (e.g., mRNA), said second dose is about 0.8 mg / kg of said polynucleotide (e.g., mRNA), and said third dose is about 0.8 mg / kg of said polynucleotide (e.g., mRNA).

[0231] In some embodiments, said first dose is about 0.9 mg / kg of said polynucleotide (e.g., mRNA), said second dose is about 0.9 mg / kg of said polynucleotide (e.g., mRNA), and said third dose is about 0.9 mg / kg of said polynucleotide (e.g., mRNA).

[0232] In some embodiments, said first dose is about 1.0 mg / kg of said polynucleotide (e.g., mRNA), said second dose is about 1.0 mg / kg of said polynucleotide (e.g., mRNA), and said third dose is about 1.0 mg / kg of said polynucleotide (e.g., mRNA).

[0233] In some embodiments, said first dose is about 1.1 mg / kg of said polynucleotide (e.g., mRNA), said second dose is about 1.1 mg / kg of said polynucleotide (e.g., mRNA), and said third dose is about 1.1 mg / kg of said polynucleotide (e.g., mRNA).

[0234] WBD (US) 4897-9494-7440vl 17 Atty Docket No. P89339 2080WO (01275) In some embodiments, said first dose is about 1.2 mg / kg of said polynucleotide (e.g., mRNA), said second dose is about 1.2 mg / kg of said polynucleotide (e.g., mRNA), and said third dose is about 1.2 mg / kg of said polynucleotide (e.g., mRNA).

[0235] In some embodiments, said first dose is about 1.3 mg / kg of said polynucleotide (e.g., mRNA), said second dose is about 1.3 mg / kg of said polynucleotide (e.g., mRNA), and said third dose is about 1.3 mg / kg of said polynucleotide (e.g., mRNA).

[0236] In some embodiments, said first dose is about 1.4 mg / kg of said polynucleotide (e.g., mRNA), said second dose is about 1.4 mg / kg of said polynucleotide (e.g., mRNA), and said third dose is about 1.4 mg / kg of said polynucleotide (e.g., mRNA).

[0237] In some embodiments, said first dose is about 1.5 mg / kg of said polynucleotide (e.g., mRNA), said second dose is about 1.5 mg / kg of said polynucleotide (e.g., mRNA), and said third dose is about 1.5 mg / kg of said polynucleotide (e.g., mRNA).

[0238] In some embodiments, said subject is further administered one or more additional doses of said pharmaceutical composition following administration of said third dose (e.g., a fourth dose, a fifth dose, a sixth dose). In some such embodiments, further doses are each administered about 4 weeks following administration of the previous dose. In some such embodiments, further doses are each administered about 5 weeks following administration of the previous dose. In some such embodiments, further doses are each administered about 6 weeks following administration of the previous dose. In some such embodiments, further doses are each administered about 7 weeks following administration of the previous dose. In some such embodiments, further doses are each administered about 8 weeks following administration of the previous dose. In some such embodiments, further doses are each administered about 9 weeks following administration of the previous dose. In some such embodiments, further doses are each administered about 10 weeks following administration of the previous dose. In some such embodiments, further doses are each administered about 11 weeks following administration of the previous dose. In some such embodiments, further doses are each administered about 12 weeks following administration of the previous dose. In some such embodiments, further doses are each administered up to about 52 weeks following administration of the previous dose.

[0239] In some embodiments, said subject is administered a steroid between about 4-24 hours prior to administration of said pharmaceutical composition.

[0240] In some embodiments, said subject is administered a steroid between about 6-18 hours prior to administration of said pharmaceutical composition. In some embodiments, said subject is administered a steroid between about 10-14 hours prior to administration of said pharmaceutical composition. In some embodiments, said subject is administered a steroid about 8-12 hours prior to administration of said pharmaceutical composition. In some embodiments, said subject is WBD (US) 4897-9494-7440vl 18 Aty Docket No. P89339 2080WO (01275) administered a steroid about 12 hours prior to administration of said pharmaceutical composition. In some embodiments, said steroid is administered orally. In some embodiments, said steroid is dexamethasone. In some embodiments, said dexamethasone is administered at a dose of about 8 mg.

[0241] In some embodiments, said subject is administered a steroid between about 1-3 hours prior to administration of said pharmaceutical composition. In some embodiments, said subject is administered a steroid between about 1-2 hours prior to administration of said pharmaceutical composition. In some embodiments, said steroid is administered intravenously. In some embodiments, said steroid is dexamethasone. In some embodiments, said dexamethasone is administered at a dose of about 10 mg.

[0242] In some embodiments, said subject is administered a histamine receptor 1 (Hl) blocker between about 1-3 hours prior to administration of said pharmaceutical composition. In some embodiments, said subject is administered an Hl blocker between about 1-2 hours prior to administration of said pharmaceutical composition. In some embodiments, said Hl blocker is administered intravenously. In some embodiments, said Hl blocker is diphenhydramine. In some embodiments, said diphenhydramine is administered at a dose of about 50 mg. In some embodiments, said Hl blocker is administered orally. In some embodiments, said Hl blocker is cetirizine. In some embodiments, said cetirizine is administered at a dose of about 10 mg.

[0243] In some embodiments, said subject is administered a histamine receptor 2 (H2) blocker between about 1-3 hours prior to administration of said pharmaceutical composition. In some embodiments, said subject is administered an H2 blocker between about 1-2 hours prior to administration of said pharmaceutical composition. In some embodiments, said H2 blocker is administered orally or intravenously. In some embodiments, said H2 blocker is famotidine. In some embodiments, said famotidine is administered at a dose of about 20 mg.

[0244] In some embodiments, said subject is administered a steroid between about 2-6 hours following administration of said pharmaceutical composition. In some embodiments, said subject is administered a steroid about 4 hours following administration of said pharmaceutical composition. In some embodiments, said steroid is administered orally. In some embodiments, said steroid is dexamethasone. In some embodiments, said dexamethasone is administered at a dose of about 4 mg.

[0245] In some embodiments, said subject is administered: a) a steroid (e.g., dexamethasone orally at a dose of about 10 mg) about 12 hours prior to administration of said pharmaceutical composition; b) a steroid (e.g., dexamethasone intravenously at a dose of about 10 mg) about 1-2 hours prior to administration of said pharmaceutical composition; c) an Hl blocker (e.g., diphenhydramine intravenously at a dose of about 50 mg; or cetirizine orally at a dose of about 10

[0246] WBD (US) 4897-9494-7440vl 19 Aty Docket No. P89339 2080WO (01275) mg) about 1-2 hours prior to administration of said pharmaceutical composition; d) a H2 blocker (e.g., famotidine intravenously or orally at a dose of about 20 mg) about 1-2 hours prior to administration of said pharmaceutical composition; and e) a steroid (e.g., dexamethasone orally at a dose of about 4 mg) about 4 hours following administration of said pharmaceutical composition.

[0247] In some embodiments, said subject has been administered a nucleos(t)ide analogue therapy immediately prior to administration of said pharmaceutical composition and achieved an adequate virologic response.

[0248] In some embodiments, said subject has been administered a nucleos(t)ide analogue therapy for at least 6 months immediately prior to administration of said pharmaceutical composition and achieved an adequate virologic response.

[0249] In some embodiments, said subject continues to be administered said nucleos(t)ide analogue therapy following administration of said pharmaceutical composition.

[0250] In some embodiments, said subject continues to be administered said nucleos(t)ide analogue therapy for at least 8 weeks following the last administration of said pharmaceutical composition.

[0251] In some embodiments, said nucleos(t)ide analogue therapy is discontinued if: a) serum HBsAg concentrations are less than 0.05 lU / mL, serum HBV DNA concentrations are less than 10 HJ / mL, and serum alanine transaminase (ALT) concentrations are less than 1.5 x upper limit of normal (ULN); or b) serum HBsAg concentrations are greater than 0.05 HJ / mL and less than 50 lU / mL, serum HBV DNA concentrations are less than 10 HJ / mL, and ALT concentrations are less than 1.5 x upper limit of normal (ULN), serum HBV RNA concentrations are undetectable, and HBcrAg concentrations are undetectable, wherein the conditions of a) or b) must be met on two occasions at least 4 weeks apart, and no earlier than 8 weeks since the last dose of said pharmaceutical composition.

[0252] In some embodiments, said subject is monitored for about 48 weeks following discontinuation of nucleos(t)ide analogue therapy for achievement of a functional cure or a partial cure.

[0253] In some embodiments, said functional cure comprises sustained serum HBsAg concentrations below 0.05 lU / mL with or without seroconversion of antibody to HBsAg for at least 24 weeks following the last dose of said pharmaceutical composition.

[0254] In some embodiments, said partial cure comprises sustained reductions of serum HBsAg concentrations to less than 50 lU / mL but greater than 0.05 lU / mL, serum HBV DNA concentrations less than 10 lU / mL, and serum ALT concentrations less than 1.5 x ULN for at least 24 weeks following the last dose of said pharmaceutical composition.

[0255] In some embodiments, said subject has chronic hepatitis B with compensated liver disease.

[0256] In some embodiments, said subject has received a nucleos(t)ide analogue therapy immediately prior

[0257] WBD (US) 4897-9494-7440vl 20 Aty Docket No. P89339 2080WO (01275) to administration of said pharmaceutical composition and achieved an adequate virologic response. In some embodiments, said subject has received a nucleos(t)ide analogue therapy for at least 6 months immediately prior to administration of said pharmaceutical composition.

[0258] In some embodiments, the serum HBsAg concentration is reduced in said subject following administration of said pharmaceutical composition compared to the serum HBsAg concentration prior to said administration.

[0259] In some embodiments, the serum HBsAg concentration is reduced by about 50%-100% in said subject following administration of said pharmaceutical composition compared to the serum HBsAg concentration prior to said administration.

[0260] In some embodiments, the serum HBsAg concentration is reduced by about 90%-100% in said subject following administration of said pharmaceutical composition compared to the serum HBsAg concentration prior to said administration.

[0261] In some embodiments, the serum HBsAg concentration is reduced by about 95%-100% in said subject following administration of said pharmaceutical composition compared to the serum HBsAg concentration prior to said administration.

[0262] In some embodiments, the serum HBsAg concentration is reduced by about 99%-100% in said subject following administration of said pharmaceutical composition compared to the serum HBsAg concentration prior to said administration.

[0263] In some embodiments, the serum HBsAg concentration is reduced by 100% in said subject following administration of said pharmaceutical composition compared to the serum HBsAg concentration prior to said administration.

[0264] In some embodiments, the serum HBsAg concentration in said subject is less than 50 lU / mL following administration of said pharmaceutical composition.

[0265] In some embodiments, the serum HBsAg concentration in said subject is less than 0.05 HJ / mL following administration of said pharmaceutical composition.

[0266] In some embodiments, the serum HBsAg concentration in said subject is undetectable following administration of said pharmaceutical composition.

[0267] In some embodiments, the plasma HBV DNA concentration is reduced in said subject following administration of said pharmaceutical composition compared to the plasma HBV DNA concentration prior to said administration.

[0268] In some embodiments, the plasma HBV DNA concentration is reduced by about 50%-100% in said subject following administration of said pharmaceutical composition compared to the plasma HBV DNA concentration prior to said administration.

[0269] WBD (US) 4897-9494-7440vl 21 Atty Docket No. P89339 2080WO (01275) In some embodiments, the plasma HBV DNA concentration is reduced by about 90%-100% in said subject following administration of said pharmaceutical composition compared to the plasma HBV DNA concentration prior to said administration.

[0270] In some embodiments, the plasma HBV DNA concentration is reduced by about 95%-100% in said subject following administration of said pharmaceutical composition compared to the plasma HBV DNA concentration prior to said administration.

[0271] In some embodiments, the plasma HBV DNA concentration is reduced by about 99%-100% in said subject following administration of said pharmaceutical composition compared to the plasma HBV DNA concentration prior to said administration.

[0272] In some embodiments, the plasma HBV DNA concentration is reduced by 100% in said subject following administration of said pharmaceutical composition compared to the plasma HBV DNA concentration prior to said administration.

[0273] In some embodiments, the plasma HBV DNA concentration is less than 1 lU / m following administration of said pharmaceutical composition.

[0274] In some embodiments, the plasma HBV DNA concentration is undetectable following administration of said pharmaceutical composition.

[0275] In some embodiments, the serum HBV RNA concentration is reduced in said subject following administration of said pharmaceutical composition compared to the serum HBV RNA concentration prior to said administration.

[0276] In some embodiments, the serum HBV RNA concentration is reduced by about 50%-100% in said subject following administration of said pharmaceutical composition compared to the serum HBV RNA concentration prior to said administration.

[0277] In some embodiments, the serum HBV RNA concentration is reduced by about 90%-100% in said subject following administration of said pharmaceutical composition compared to the serum HBV RNA concentration prior to said administration.

[0278] In some embodiments, the serum HBV RNA concentration is reduced by about 95%-100% in said subject following administration of said pharmaceutical composition compared to the serum HBV RNA concentration prior to said administration.

[0279] In some embodiments, the serum HBV RNA concentration is reduced by about 99%-100% in said subject following administration of said pharmaceutical composition compared to the serum HBV RNA concentration prior to said administration.

[0280] In some embodiments, the serum HBV RNA concentration is reduced by 100% in said subject following administration of said pharmaceutical composition compared to the serum HBV RNA concentration prior to said administration.

[0281] WBD (US) 4897-9494-7440vl 22 Atty Docket No. P89339 2080WO (01275) In some embodiments, the serum HBV RNA concentration is undetectable following administration of said pharmaceutical composition.

[0282] In some embodiments, the serum HBcrAg concentration is reduced in said subject following administration of said pharmaceutical composition compared to the serum HBcrAg concentration prior to said administration.

[0283] In some embodiments, the serum HBcrAg concentration is reduced by about 50%-100% in said subject following administration of said pharmaceutical composition compared to the serum HBcrAg concentration prior to said administration.

[0284] In some embodiments, the serum HBcrAg concentration is reduced by about 90%-100% in said subject following administration of said pharmaceutical composition compared to the serum HBcrAg concentration prior to said administration.

[0285] In some embodiments, the serum HBcrAg concentration is reduced by about 95%-100% in said subject following administration of said pharmaceutical composition compared to the serum HBcrAg concentration prior to said administration.

[0286] In some embodiments, the serum HBcrAg concentration is reduced by about 99%-100% in said subject following administration of said pharmaceutical composition compared to the serum HBcrAg concentration prior to said administration.

[0287] In some embodiments, the serum HBcrAg concentration is reduced by 100% in said subject following administration of said pharmaceutical composition compared to the serum HBcrAg concentration prior to said administration.

[0288] In some embodiments, the serum HBcrAg concentration is undetectable following administration of said pharmaceutical composition.

[0289] In some embodiments, the HBV cccDNA concentration in said target cells is reduced in said subject following administration of said pharmaceutical composition compared to an appropriate control.

[0290] In some embodiments, the HBV cccDNA concentration in target cells in said subject is reduced by about 50%-100% following administration of said pharmaceutical composition compared to an appropriate control.

[0291] In some embodiments, the HBV cccDNA concentration in target cells in said subject is reduced by about 90%-100% following administration of said pharmaceutical composition compared to an appropriate control.

[0292] In some embodiments, the HBV cccDNA concentration in target cells in said subject is reduced by about 95%-100% following administration of said pharmaceutical composition compared to an appropriate control.

[0293] WBD (US) 4897-9494-7440vl 23 Atty Docket No. P89339 2080WO (01275) In some embodiments, the HBV cccDNA concentration in target cells in said subject is reduced by about 99%-100% following administration of said pharmaceutical composition compared to an appropriate control.

[0294] In some embodiments, the HBV cccDNA concentration in target cells in said subject is reduced by 100% following administration of said pharmaceutical composition compared to an appropriate control.

[0295] In some embodiments, the HBV cccDNA concentration in target cells in said subject is undetectable following administration of said pharmaceutical composition.

[0296] In some embodiments, between about 50%-100% of HBV cccDNA present in said target cells comprises an indel at said recognition sequence after administration of said pharmaceutical composition.

[0297] In some embodiments, between about 80%-100% of HBV cccDNA present in said target cells comprises an indel at said recognition sequence after administration of said pharmaceutical composition.

[0298] In some embodiments, between about 90%-100% of HBV cccDNA present in said target cells comprises an indel at said recognition sequence after administration of said pharmaceutical composition.

[0299] In some embodiments, between about 95%-100% of HBV cccDNA present in said target cells comprises an indel at said recognition sequence after administration of said pharmaceutical composition.

[0300] In some embodiments, said indel inactivates said pol gene.

[0301] In some embodiments, said indel inactivates an HbsAg gene.

[0302] In some embodiments, between about 50%-100% of integrated HBV genomes or genome fragments present in the genome of said target cells comprises an indel at said recognition sequence after administration of said pharmaceutical composition.

[0303] In some embodiments, between about 80%-100% of integrated HBV genomes present in the genome of said target cells comprises an indel at said recognition sequence after administration of said pharmaceutical composition.

[0304] In some embodiments, between about 90%-100% of integrated HBV genomes present in the genome of said target cells comprises an indel at said recognition sequence after administration of said pharmaceutical composition.

[0305] In some embodiments, between about 95%-100% of integrated HBV genomes present in the genome of said target cells comprises an indel at said recognition sequence after administration of said pharmaceutical composition.

[0306] In some embodiments, said indel inactivates said pol gene.

[0307] WBD (US) 4897-9494-7440vl 24 Aty Docket No. P89339 2080WO (01275) In some embodiments, said indel inactivates an HbsAg gene.

[0308] BRIEF DESCRIPTION OF THE FIGURES

[0309] Figure 1. HBV 11-12 recognition sequence (SEQ ID NO: 3) and its reverse complement sequence (SEQ ID NO: 4) in the HBV genome.

[0310] Figure 2. Schematic depiction of the mRNA sequence encoding the HBV 11-12L.1090 E80 meganuclease.

[0311] Figure 3. Graphs of plasma concentration over time of 2- [2-(o -methoxy (polyethyleneglycol2000) ethoxy]-N,N-ditetradecylacetamide (“Lipid 2”) following administration of PBGENE-HBV to non-human primates (NHP) on Day 1 and Day 29 in the study described in the present disclosure, “h” stands for hour. “SD” stands for standard deviation. The Y axis is a logarithmic scale. Data depicts the mean ± SD concentration of Lipid 2 from both males and females combined.

[0312] Figure 4. Graphs of plasma concentration over time of bis(2 -butyloctyl) 10-(N-(3- (pyrrolidin-l-yl)propyl)nonanamido)nonadecanedioate (“Lipid 1”) following administration of PBGENE-HBV to NHP on Day 1 and Day 29 in the study described in the present disclosure, “h” stands for hour. “SD” stands for standard deviation. The Y axis is a logarithmic scale. Data depicts the mean ± SD concentration of Lipid 1 from both males and females combined.

[0313] Figure 5. Graphs of plasma concentration over time of nuclease mRNA following administration of PBGENE-HBV to NHP on Day 1 and Day 29 in the study described in the present disclosure, “h” stands for hour. “SD” stands for standard deviation. The Y axis is a logarithmic scale. Data depicts the mean ± SD concentration of nuclease mRNA from both males and females combined.

[0314] Figure 6. Graphs of mean tissue concentrations (in ng / mg tissue) of Lipid 1 and Lipid 2 in the specified tissues following the first and second administration of PBGENE-HBV to NHP in the study described in the present disclosure. “SOI” stands for start of infusion. Data depicts the mean tissue concentration from both males and females. Day 43 (male) and Day 44 (female) data (336 and 360 hours after Dose 2, respectively) were averaged and plotted as a single point at 348 hours post SOI. The liver concentration displayed is the mean concentration of caudate, medial, and lateral liver lobes. Tissues not displayed for Lipid 2 are those where concentrations were never above 5000 ng / g (i.e., brain, heart, lymph nodes, spinal cord, and testes). Tissues not displayed for Lipid 1 are those where concentrations were never above 25,000 ng / g (i.e., bone marrow, brain, heart, kidney, lymph nodes, spinal cord, and testes).

[0315] Figure 7. Graphs of mean tissue concentrations (in ng / mg tissue) of nuclease mRNA and nuclease protein in the liver and spleen following the first and second administration of PBGENE- WBD (US) 4897-9494-7440vl 25 Aty Docket No. P89339 2080WO (01275) HBV to NHP in the study described in the present disclosure. “SOI” stands for start of infusion. Data depicts the mean tissue concentration from both males and females. Day 43 (male) and Day 44 (female) data (336 and 360 hours after Dose 2) were averaged and plotted as a single point at 348 hours post SOI. The liver concentration displayed is the mean concentration of caudate, medial, and lateral liver lobes. Tissues not displayed for nuclease mRNA are those where tissue concentrations were never above 5 x io8copies / mg (i.e., adrenal, bone marrow, brain, heart, kidney, lung, lymph nodes, spinal cord, testes, and ovary). Tissues not displayed for nuclease protein are those where tissue concentrations were BLQ at all timepoints (i.e., bone marrow, brain, heart, kidney, lung, lymph nodes, spinal cord, and testes) and the adrenal gland, where only 1 sample from 1 female showed nuclease protein at 0.813 ng nuclease protein / mg tissue.

[0316] Figure 8. Representative images by chromogenic RNAscope of E80 nuclease mRNA biodistribution from cynomolgus monkeys given 1.5 mg / kg PBGENE-HBV on Day 1 and Day 29. Representative sections of untreated liver and E80 nuclease-treated liver are shown in the left and right panels, respectively. A representative section of E80 nuclease-treated liver, isolated 4 hours post first dose, and magnified 40X is shown in the lower panel.

[0317] Figures 9A and 9B. Representative images by RNAscope of E80 nuclease FISH + immunofluorescence DDX4 staining in ovary (Figure 8 A) and testes (Figure 8B) from cynomolgus monkeys given 1.5 mg / kg PBGENE-HBV on Day 1 and Day 29. “FISH” stands for fluorescence in situ hybridization. “HBV11-12L” refers to a nuclease. White arrows mark nuclease mRNA (green dots) detected by RNAscope FISH, with germ cells expressing DDX4 protein shown as red staining.

[0318] Figure 10. The study design of the phase 1, open-label, dose escalation and dose expansion study to evaluate the safety, tolerability, pharmacokinetics (PK), and antiviral activity of multiple doses of PBGENE-HBV in adult participants with hepatitis B e antigen (HBeAg)-negative CHB who are receiving a nucleos(t)ide analog therapy (NA therapy) described herein. “DMC” stands for data monitoring committee. “N” refers to number of participants.

[0319] Figure 11. A schematic depiction of participant management in Parts 1 and 2 of the study described herein.

[0320] Figure 12. A table depicting visits required for each dosing interval (through Day 42) in the study described herein.

[0321] Figure 13. A table depicting the schedule of events for screening and dosing periods 1, 2, and 3 through Day 42 (Parts 1 and 2) in the study described herein.

[0322] Figure 14. Atable depicting the schedule of follow-up visits weeks 10 through 48 (Parts 1 and 2) in the study described herein.

[0323] WBD (US) 4897-9494-7440vl 26 Atty Docket No. P89339 2080WO (01275) Figure 15. A table depicting post-NA discontinuation follow-up (Parts 1 and 2) in the study described herein.

[0324] Figure 16. A schematic depiction of individual participant dosing criteria for the second dose in the study described herein.

[0325] Figure 17. A schematic depiction of individual participant dosing criteria for the third dose in the study described herein.

[0326] Figure 18. Depiction of an algorithm of the step 1 dose escalation guidelines in Part 1 of the study described herein.

[0327] Figure 19. Depiction of an algorithm of the step 2 dose escalation guidelines in Part 1 of the study described herein.

[0328] Figures 20A-B. Summary of baseline participant characteristics of subjects. Figure 20A provides baseline participant characteristics of subjects in cohort 1 (0.2 mg / kg). Figure 20B provides baseline participant characteristics of subjects in cohort 2 (0.4 mg / kg).

[0329] Figures 21A-B. Summary of treatment-related and possibly treatment-related adverse events (AEs). Figure 21 A provides a summary of treatment-related and possibly treatment-related AEs for subjects in cohort 1 (0.2 mg / kg). Figure 21 A provides a summary of treatment-related and possibly treatment-related AEs for subjects in cohort 2 (0.4 mg / kg).

[0330] Figures 22A-G. Clinical chemistry, hematology, and coagulation values from subjects in cohort 1 (0.2 mg / kg). Figure 22A provides alanine transaminase (ALT) levels. Figure 22B provides aspartate transaminase (AST) levels. Figure 22C provides creatinine levels. Figure 22D provides platelet counts. Figure 22E provides alkaline phosphatase (ALP) levels. Figure 22F provides bilirubin levels. Figure 22G provides prothrombin time.

[0331] Figures 23A-G. Clinical chemistry, hematology, and coagulation values from subjects in cohort 2 (0.4 mg / kg). Figure 23 A provides alanine transaminase (ALT) levels. Figure 23B provides aspartate transaminase (AST) levels. Figure 23C provides creatinine levels. Figure 23D provides platelet counts. Figure 23E provides alkaline phosphatase (ALP) levels. Figure 23F provides bilirubin levels. Figure 23G provides prothrombin time.

[0332] Figure 24. Pharmacokinetic data of PBGENE-HBV in subjects from cohort 1 (0.2 mg / kg).

[0333] Figure 25. Summary of Hepatitis B Surface antigen (HBsAg) levels over time in one subject following three doses of PBGENE-HBV at 0.2 mg / kg.

[0334] BRIEF DESCRIPTION OF THE SEQUENCES

[0335] SEQ ID NO: 1 sets forth the amino acid sequence of the wild-type I-Crel meganuclease from Chlamydomonas reinhardtii .

[0336] SEQ ID NO: 2 sets forth the amino acid sequence of the LAGLID ADG motif.

[0337] WBD (US) 4897-9494-7440vl 27 Atty Docket No. P89339 2080WO (01275) SEQ ID NO: 3 sets forth the nucleic acid sequence of the sense strand of the HBV 11-12 recognition sequence.

[0338] SEQ ID NO: 4 sets forth the nucleic acid sequence of the reverse complement (antisense strand) of the HBV 11-12 recognition sequence.

[0339] SEQ ID NO: 5 sets forth the amino acid sequence of the HBV 11-12L.1090 E80 meganuclease having the Ml residue.

[0340] SEQ ID NO: 6 sets forth the amino acid sequence of the HBV 11-12L.1090 E80 meganuclease without the Ml residue.

[0341] SEQ ID NO: 7 sets forth the nucleic acid sequence of the HBV 11-12L.1090 E80 meganuclease mRNA having the Ml residue.

[0342] SEQ ID NO: 8 sets forth the nucleic acid sequence of the HBV 11-12L.1090 E80 meganuclease mRNA without the Ml residue.

[0343] SEQ ID NO: 9 sets forth the nucleic acid sequence of the HBV 11-12L.1090 E80 meganuclease DNA having the Ml residue.

[0344] SEQ ID NO: 10 sets forth the nucleic acid sequence of the HBV 11-12L.1090 E80 meganuclease DNA without the Ml residue.

[0345] SEQ ID NO: 11 sets forth the amino acid sequence of an SV40 NLS.

[0346] SEQ ID NO: 12 sets forth the amino acid sequence of an SV40 NLS.

[0347] SEQ ID NO: 13 sets forth an mRNA sequence of the 5’ ALB untranslated region (UTR).

[0348] SEQ ID NO: 14 sets forth a DNA sequence of the 5’ ALB UTR.

[0349] SEQ ID NO: 15 sets forth an mRNA sequence of the 3’ SNRPB UTR.

[0350] SEQ ID NO: 16 sets forth a DNA sequence of the 3’ SNRPB UTR.

[0351] SEQ ID NO: 17 sets forth an mRNA sequence of a Kozak sequence.

[0352] SEQ ID NO: 18 sets forth a DNA sequence of a Kozak sequence.

[0353] SEQ ID NO: 19 sets forth a nucleic acid sequence of a poly A termination sequence.

[0354] SEQ ID NO: 20 sets forth a nucleic acid sequence encoding an mRNA construct encoding the HBV 11-12L.1090 E80 meganuclease clinical candidate having the Ml residue. Each U in SEQ ID NO: 20 can be uridine or a modified uridine nucleotide (e.g., pseudouridine, N1 -methylpseudouridine, 5-methoxyuridine, or 2-thiouridine). Each C in SEQ ID NO: 20 can be cytosine or a modified cytosine nucleotide (e.g., 5-methylcytidine, 2'-O-methylcytidine, or N4-acetyl-cytosine). Each A in SEQ ID NO: 20 can be adenine or a modified adenine nucleotide (e.g., N6- methyladenosine or Nl-methyl-adenine).

[0355] SEQ ID NO: 21 sets forth a nucleic acid sequence encoding an mRNA construct encoding the HBV 11-12L.1090 E80 meganuclease clinical candidate without the Ml residue. Each U in SEQ ID NO: 21 can be uridine or a modified uridine nucleotide (e.g., pseudouridine, Nl-methyl- WBD (US) 4897-9494-7440vl 28 Aty Docket No. P89339 2080WO (01275) pseudouridine, 5-methoxyuridine, or 2-thiouridine). Each C in SEQ ID NO: 21 can be cytosine or a modified cytosine nucleotide (e.g., 5-methylcytidine, 2'-O-methylcytidine, or N4-acetyl-cytosine). Each A in SEQ ID NO: 21 can be adenine or a modified adenine nucleotide (e.g., N6- methyladenosine or Nl-methyl-adenine).

[0356] SEQ ID NO: 22 sets forth a nucleic acid sequence encoding a DNA construct encoding the HBV 11-12L.1090 E80 meganuclease clinical candidate having the Ml residue.

[0357] SEQ ID NO: 23 sets forth a nucleic acid sequence encoding a DNA construct encoding the HBV 11-12L.1090 E80 meganuclease clinical candidate without the Ml residue.

[0358] DETAILED DESCRIPTION OF THE INVENTION

[0359] 1.1 References and Definitions

[0360] The patent and scientific literature referred to herein establishes knowledge that is available to those of skill in the art. The issued US patents, allowed applications, published foreign applications, and references, including GenBank database sequences, which are cited herein are hereby incorporated by reference to the same extent as if each was specifically and individually indicated to be incorporated by reference.

[0361] The present disclosure can be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art. For example, features illustrated with respect to one embodiment can be incorporated into other embodiments, and features illustrated with respect to a particular embodiment can be deleted from that embodiment. In addition, numerous variations and additions to the embodiments suggested herein will be apparent to those skilled in the art in light of the instant disclosure, which do not depart from the instant disclosure.

[0362] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The terminology used in the description of the disclosure herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure.

[0363] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference herein in their entirety.

[0364] As used herein, “a,” “an,” or “the” can mean one or more than one. For example, “a” cell can mean a single cell or a multiplicity of cells.

[0365] As used herein, unless specifically indicated otherwise, the word “or” is used in the inclusive sense of “and / or” and not the exclusive sense of “either / or.”

[0366] WBD (US) 4897-9494-7440vl 29 Atty Docket No. P89339 2080WO (01275) As used herein, the terms “nuclease” and “endonuclease” are used interchangeably to refer to naturally-occurring or engineered enzymes, which cleave a phosphodiester bond within a polynucleotide chain.

[0367] As used herein, the terms “cleave” or “cleavage” refer to the hydrolysis of phosphodiester bonds within the backbone of a recognition sequence within a target sequence that results in a double-stranded break within the target sequence, referred to herein as a “cleavage site”.

[0368] As used herein, the term “meganuclease” refers to an endonuclease that binds doublestranded DNA at a recognition sequence that is greater than 12 base pairs. In some embodiments, the recognition sequence for a meganuclease of the present disclosure is 22 base pairs. A meganuclease can be an endonuclease that is derived from I-Crel (SEQ ID NO: 1), and can refer to an engineered variant of I-Crel that has been modified relative to natural I-Crel with respect to, for example, DNA-binding specificity, DNA cleavage activity, DNA-binding affinity, or dimerization properties. Methods for producing such modified variants of I-Crel are known in the art (e.g., WO 2007 / 047859, incorporated by reference in its entirety). A meganuclease as used herein binds to double-stranded DNA as a heterodimer. A meganuclease may also be a “single-chain meganuclease” in which a pair of DNA-binding domains is joined into a single polypeptide using a peptide linker. The term “homing endonuclease” is synonymous with the term “meganuclease.” Meganucleases of the present disclosure are substantially non-toxic when expressed in the targeted cells as described herein such that cells can be transfected and maintained at 37°C without observing deleterious effects on cell viability or significant reductions in meganuclease cleavage activity when measured using the methods described herein.

[0369] As used herein, the term “single-chain meganuclease” refers to a polypeptide comprising a pair of nuclease subunits joined by a linker. A single-chain meganuclease has the organization: N- terminal subunit - Linker - C-terminal subunit. The two meganuclease subunits will generally be non-identical in amino acid sequence and will bind non-identical DNA sequences. Thus, singlechain meganucleases typically cleave pseudo-palindromic or non-palindromic recognition sequences. A single-chain meganuclease may be referred to as a “single-chain heterodimer” or “single-chain heterodimeric meganuclease” although it is not, in fact, dimeric. For clarity, unless otherwise specified, the term “meganuclease” can refer to a dimeric or single-chain meganuclease.

[0370] As used herein, the term “linker” refers to an exogenous peptide sequence used to join two meganuclease subunits into a single polypeptide. A linker may have a propensity to form a specific three-dimensional structure under physiological conditions, such as turns and / or coils.

[0371] As used herein, the terms “recombinant” or “engineered,” with respect to a protein, means having an altered amino acid sequence as a result of the application of genetic engineering techniques to nucleic acids that encode the protein and cells or organisms that express the protein. WBD (US) 4897-9494-7440vl 30 Aty Docket No. P89339 2080WO (01275) With respect to a nucleic acid, the term “recombinant” or “engineered” means having an altered nucleic acid sequence as a result of the application of genetic engineering techniques. Genetic engineering techniques include, but are not limited to, PCR and DNA cloning technologies; transfection, transformation, and other gene transfer technologies; homologous recombination; site- directed mutagenesis; and gene fusion. In accordance with this definition, a protein having an amino acid sequence identical to a naturally-occurring protein, but produced by cloning and expression in a heterologous host, is not considered recombinant or engineered.

[0372] As used herein, the term “wild-type” refers to the most common naturally occurring allele (i.e., polynucleotide sequence) in the allele population of the same type of gene, wherein a polypeptide encoded by the wild-type allele has its original functions. The term “wild-type” also refers to a polypeptide encoded by a wild-type allele. Wild-type alleles (i.e., polynucleotides) and polypeptides are distinguishable from mutant or variant alleles and polypeptides, which comprise one or more mutations and / or substitutions relative to the wild-type sequence(s). Whereas a wildtype allele or polypeptide can confer a normal phenotype in an organism, a mutant or variant allele or polypeptide can, in some instances, confer an altered phenotype. Wild-type nucleases are distinguishable from recombinant or non-naturally-occurring nucleases. The term “wild-type” can also refer to a cell, an organism, and / or a subject which possesses a wild-type allele of a particular gene, or a cell, an organism, and / or a subject used for comparative purposes.

[0373] As used herein, the term “genetically-modified” refers to a cell or organism in which, or in an ancestor of which, a genomic DNA sequence has been deliberately modified by recombinant technology. As used herein, the term “genetically-modified” encompasses the term “transgenic.”

[0374] As used herein, the term with respect to recombinant proteins, the term “modification” means any insertion, deletion, or substitution of an amino acid residue in the recombinant sequence relative to a reference sequence (e.g., a wild-type or a native sequence).

[0375] As used herein, the terms “recognition sequence” or “recognition site” refers to a DNA sequence that is bound and cleaved by a nuclease. In the case of a meganuclease, a recognition sequence comprises a pair of inverted, 9 basepair “half sites” which are separated by four basepairs. In the case of a single-chain meganuclease, the N-terminal domain of the protein contacts a first half-site and the C-terminal domain of the protein contacts a second half-site. Cleavage by a meganuclease produces four basepair 3’ “overhangs.” “Overhangs,” or “sticky ends” are short, single-stranded DNA segments that can be produced by endonuclease cleavage of a doublestranded DNA sequence. In the case of meganucleases and single-chain meganucleases derived from I-Crel, the overhang comprises bases 10-13 of the 22 basepair recognition sequence.

[0376] As used herein, the terms “target site” or “target sequence” refers to a region of the chromosomal DNA of a cell comprising a recognition sequence for a nuclease.

[0377] WBD (US) 4897-9494-7440vl 31 Aty Docket No. P89339 2080WO (01275) As used herein, the terms “DNA-binding affinity” or “binding affinity” means the tendency of a meganuclease to non-covalently associate with a reference DNA molecule (e.g., a recognition sequence or an arbitrary sequence). Binding affinity is measured by a dissociation constant, Kd. As used herein, a nuclease has “altered” binding affinity if the Kd of the nuclease for a reference recognition sequence is increased or decreased by a statistically significant percent change relative to a reference nuclease.

[0378] As used herein, the term “specificity” refers to the ability of a nuclease to recognize and cleave double-stranded DNA molecules only at a particular sequence of base pairs referred to as the recognition sequence, or only at a particular set of recognition sequences. The set of recognition sequences will share certain conserved positions or sequence motifs, but may be degenerate at one or more positions. A highly-specific nuclease is capable of cleaving only one or a very few recognition sequences. Specificity can be determined by any method known in the art, such as unbiased identification of DSBs enabled by sequencing (GUIDE-seq), oligonucleotide (oligo) capture assay, whole genome sequencing, and long-range next generation sequencing of the recognition sequence. In some embodiments, specificity is measured using GUIDE-seq. As used herein, “specificity” is synonymous with a low incidence of cleavage of sequences different from the target sequences (non-target sequences), i.e., off-target cutting. A low incidence of off-target cutting may comprise an incidence of cleavage of non-target sequences of less than 25%, less than 20%, less than 18%, less than 15%, less than 12.5%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2.5%, less than 2%, less than 1.5%, less than 1%, less than 0.75%, less than 0.5%, or less than 0.25%.

[0379] As used herein, a meganuclease has “altered” specificity if it binds to and cleaves a recognition sequence which is not bound to and cleaved by a reference meganuclease (e.g., a wildtype) under physiological conditions, or if the rate of cleavage of a recognition sequence is increased or decreased by a biologically significant amount (e.g., at least 2x, or 2x-10x) relative to a reference meganuclease.

[0380] In some embodiments, the presently disclosed engineered meganucleases have improved (i.e., increased) specificity for the target recognition sequence that comprises SEQ ID NO: 3 (i.e., HBV 11-12) as compared to previously described megenucleases, e.g., the HBV 11-12L.1090QQ Linkerl meganuclease (described in WO 2021 / 113765, which is incorporated by reference in its entirety, and set forth as SEQ ID NO: 12 therein). Thus, in certain embodiments, the presently disclosed engineered meganucleases exhibit reduced off-target cleavage as compared to the HBV 11-12L.1090QQ Linkerl meganuclease. Off-target cleavage by a meganuclease can be measured using any method known in the art, including for example, oligo capture analysis as described here, a T7 endonuclease (T7E) assay as described herein, digital PCR as described herein, targeted WBD (US) 4897-9494-7440vl 32 Aty Docket No. P89339 2080WO (01275) sequencing of particular off-target sites, exome sequencing, whole genome sequencing, direct in situ breaks labeling enrichment on streptavidin and next-generation sequencing (BLESS), genomewide, GUIDE-seq, and linear amplification-mediated high-throughput genome-wide translocation sequencing (LAM-HTGTS) (see, e.g., Zischewski et al. (2017), Biotechnology Advances 35(1) :95- 104, which is incorporated by reference in its entirety).

[0381] As used herein, the term “efficiency of cleavage” refers to the incidence by which a meganuclease cleaves a recognition sequence in a double-stranded DNA molecule relative to the incidence of all cleavage events by the meganuclease on the DNA molecule. “Efficiency of cleavage” is synonymous with DNA editing efficiency or on-target editing. Efficiency of cleavage and / or indel formation by a meganuclease can be measured using any method known in the art, including T7E assay, digital PCR (ddPCR), mismatch detection assays, mismatch cleavage assay, high-resolution melting analysis (EIRMA), heteroduplex mobility assay, sequencing, and fluorescent PCR capillary gel electrophoresis (see, e.g., Zischewski et al. (2017) Biotechnology Advances 35(1):95- 104, which is incorporated by reference in its entirety). In some embodiments, efficiency of cleavage is measured by ddPCR. In some embodiments, the disclosed meganucleases generate efficiencies of cleavage of at least about 35%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 99% at the recognition sequence.

[0382] An “indel”, as used herein, refers to the insertion or deletion of a nucleobase within a nucleic acid, such as DNA. In some embodiments, it is desirable to generate one or more insertions or deletions (i.e., indels) in the nucleic acid, e.g., in a foreign nucleic acid such as viral DNA. Accordingly, as used herein, “efficiency of indel formation” refers to the incidence by which a meganuclease generates one or more indels through cleavage of a recognition sequence relative to the incidence of all cleavage events by the meganuclease on the DNA molecule. In some embodiments, efficiency of indel formation is measured by ddPCR. In some embodiments, the disclosed meganucleases generate efficiencies of indel formation of at least about 35%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 99% at the recognition sequence. The disclosed meganucleases may generate efficiencies of cleavage and / or efficiencies of indel formation of at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80% at the recognition sequence.

[0383] As used herein, the term “homologous recombination” or “HR” refers to the natural, cellular process in which a double-stranded DNA-break is repaired using a homologous DNA sequence as the repair template (see, e.g. Cahill et al. (2006), Front. Biosci. 11 : 1958-1976). The homologous DNA sequence may be an endogenous chromosomal sequence or an exogenous nucleic acid that was delivered to the cell.

[0384] WBD (US) 4897-9494-7440vl 33 Atty Docket No. P89339 2080WO (01275) As used herein, the term “non-homologous end-joining” or “NHEJ” refers to the natural, cellular process in which a double-stranded DNA-break is repaired by the direct joining of two non- homologous DNA segments (see, e.g. Cahill et al. (2006), Front. Biosci. 11 : 1958-1976). DNA repair by non-homologous end-joining is error-prone and frequently results in the untemplated addition or deletion of DNA sequences at the site of repair. In some instances, cleavage at a target recognition sequence results in NHEJ at a target recognition site. Nuclease-induced cleavage of a target site in the coding sequence of a gene followed by DNA repair by NHEJ can introduce mutations into the coding sequence, such as frameshift mutations, that disrupt gene function. Thus, engineered meganucleases can be used to effectively knock-out a gene in a population of cells.

[0385] As used herein, the term “homology arms” or “sequences homologous to sequences flanking a meganuclease cleavage site” refer to sequences flanking the 5’ and 3’ ends of a nucleic acid molecule which promote insertion of the nucleic acid molecule into a cleavage site generated by a meganuclease. In general, homology arms can have a length of at least 50 base pairs, preferably at least 100 base pairs, and up to 2000 base pairs or more, and can have at least 90%, preferably at least 95%, or more, sequence homology to their corresponding sequences in the genome. In some embodiments, the homology arms are about 500 base pairs.

[0386] As used herein with respect to both amino acid sequences and nucleic acid sequences, the terms “percent identity,” “sequence identity,” “percentage similarity,” “sequence similarity” and the like refer to a measure of the degree of similarity of two sequences based upon an alignment of the sequences that maximizes similarity between aligned amino acid residues or nucleotides, and which is a function of the number of identical or similar residues or nucleotides, the number of total residues or nucleotides, and the presence and length of gaps in the sequence alignment. A variety of algorithms and computer programs are available for determining sequence similarity using standard parameters. As used herein, sequence similarity is measured using the BLASTp program for amino acid sequences and the BLASTn program for nucleic acid sequences, both of which are available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ), and are described in, for example, Altschul et al. (1990), J. Mol. Biol. 215:403-410; Gish and States (1993), Nature Genet. 3:266-272; Madden et al. (1996), Meth. Enzymol.266: 131-141; Altschul et al. (1997), Nucleic Acids Res. 25:33 89-3402); Zhang et al. (2000), J. Comput. Biol. 7(l-2):203-14. As used herein, percent similarity of two amino acid sequences is the score based upon the following parameters for the BLASTp algorithm: word size=3; gap opening penalty=-ll; gap extension penalty=-l; and scoring matrix=BLOSUM62. As used herein, percent similarity of two nucleic acid sequences is the score based upon the following parameters for the BLASTn algorithm: word size=ll; gap opening penalty=-5; gap extension penalty=-2; match reward=l; and mismatch penalty=-3.

[0387] WBD (US) 4897-9494-7440vl 34 Aty Docket No. P89339 2080WO (01275) As used herein, the term “corresponding to” with respect to modifications of two proteins or amino acid sequences, is used to indicate that a specified modification in the first protein is a substitution of the same amino acid residue as in the modification in the second protein, and that the amino acid position of the modification in the first protein corresponds to or aligns with the amino acid position of the modification in the second protein when the two proteins are subjected to standard sequence alignments (e.g., using the BLASTp program) and aligned for maximum sequence identity across the entire subunit or protein. Thus, the modification of residue “X” to amino acid “A” in the first protein will correspond to the modification of residue “Y” to amino acid “A” in the second protein if residues X and Y correspond to each other in a sequence alignment, and despite the fact that X and Y may be at different positions relative to the N-terminus or C- terminus.

[0388] As used herein, the term “recognition half-site,” “recognition sequence half-site,” or simply “half-site” means a nucleic acid sequence in a double-stranded DNA molecule that is recognized and bound by a monomer of a homodimeric or heterodimeric meganuclease or by one subunit of a single-chain meganuclease or by one subunit of a single-chain meganuclease.

[0389] As used herein, the term “hypervariable region” refers to a localized sequence within a meganuclease monomer or subunit that comprises amino acids with relatively high variability. A hypervariable region can comprise about 50-60 contiguous residues, about 53-57 contiguous residues, or preferably about 56 residues. A hypervariable region can comprise one or more residues that contact DNA bases in a recognition sequence and can be modified to alter base preference of the monomer or subunit. A hypervariable region can also comprise one or more residues that bind to the DNA backbone when the meganuclease associates with a double-stranded DNA recognition sequence. Such residues can be modified to alter the binding affinity of the meganuclease for the DNA backbone and the target recognition sequence. In different embodiments, a hypervariable region may comprise between 1-20 residues that exhibit variability and can be modified to influence base preference and / or DNA-binding affinity. In particular embodiments, a hypervariable region comprises between about 15-20 residues that exhibit variability and can be modified to influence base preference and / or DNA-binding affinity.

[0390] The terms “recombinant DNA construct,” “recombinant construct,” “expression cassette,” “cassette,” “expression construct,” “chimeric construct,” “construct,” and “recombinant DNA fragment” are used interchangeably herein and are single or double-stranded polynucleotides. A recombinant construct comprises an artificial combination of nucleic acid fragments, including, without limitation, regulatory and coding sequences that are not found together in nature. For example, a recombinant DNA construct may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from WBD (US) 4897-9494-7440vl 35 Aty Docket No. P89339 2080WO (01275) the same source and arranged in a manner different than that found in nature. Such a construct may be used by itself or may be used in conjunction with a vector.

[0391] As used herein, a “vector” or “recombinant DNA vector” may be a construct that includes a replication system and sequences that are capable of transcription and translation of a polypeptide- encoding sequence in a given host cell. If a vector is used, then the choice of vector is dependent upon the method that will be used to transform host cells as is well known to those skilled in the art. Vectors can include, without limitation, plasmid vectors and recombinant AAV vectors, or any other vector known in the art suitable for delivering a gene to a target cell. The skilled artisan is well aware of the genetic elements that must be present on the vector in order to successfully transform, select and propagate host cells comprising any of the isolated nucleotides or nucleic acid sequences described herein. In some embodiments, a “vector” also refers to a viral vector. Viral vectors can include, without limitation, retroviral vectors, lentiviral vectors, adenoviral vectors, and adeno-associated viral vectors (AAV).

[0392] As used herein, the term “operably linked” is intended to mean a functional linkage between two or more elements. For example, an operable linkage between a nucleic acid sequence encoding a nuclease as described herein and a regulatory sequence (e.g., a promoter) is a functional link that allows for expression of the nucleic acid sequence encoding the nuclease. Operably linked elements may be contiguous or non-contiguous. When used to refer to the joining of two protein coding regions, by operably linked is intended that the coding regions are in the same reading frame.

[0393] As used herein, “uridine depletion” refers to the removal or replacement of uridine residues from an RNA molecule (e.g., mRNA) or thymidine residues from a DNA molecule that encodes for an RNA molecule. The uridines can be replaced with any other residue, such as adenosine or pseudouridine, within any region of the RNA molecule, but in some embodiments, the uridines are removed and replaced within the non-coding region of an RNA molecule.

[0394] As used herein, “codon-optimized” refers to a coding sequence that has been modified to improve gene expression and increase the translational efficiency by accommodating the codon bias of the host organism or tissue, wherein the limitations associated with species-specific and tissue-specific differences in codon usage and transfer RNA (tRNA) abundance are lessened. The coding sequence can be codon-optimized for expression in mammalian cells and / or specific tissues. As it has been shown that the codon usage bias of genes specifically expressed in the human liver differ from the codon usage bias of the human genome coding DNA sequences (Dittmar et al. (2006) PLoS Genet. 2:e221), in some embodiments, sequences encoding an engineered meganuclease described herein are optimized for expression in liver.

[0395] As used herein, “inactivating” or “inactivates” in reference to a gene refers to either the introduction of a mutation into the gene such that the resultant inactivated gene does not encode an WBD (US) 4897-9494-7440vl 36 Aty Docket No. P89339 2080WO (01275) active and / or full-length protein or the gene is eliminated (i.e., degraded). An HBV genome or fragment thereof can be eliminated, thus inactivating any HBV genes present in the HBV genome or fragment thereof, through the cleavage of the genome or genome fragment that results in the subsequent degradation of the genome or fragment thereof.

[0396] As used herein, a “control” or “control cell” refers to a cell that provides a reference point for measuring changes in genotype or phenotype of a genetically-modified cell. A control cell may comprise, for example: (a) a wild-type cell, i.e., of the same genotype as the starting material for the genetic alteration which resulted in the genetically-modified cell; (b) a cell of the same genotype as the genetically-modified cell but which has been transformed with a null construct (i.e., with a construct which has no known effect on the trait of interest); or, (c) a cell genetically identical to the genetically-modified cell but which is not exposed to conditions or stimuli or further genetic modifications that would induce expression of altered genotype or phenotype. A control subject may comprise, for example: a wild-type subject, i.e., a subject not having an HBV infection, which is not exposed to conditions or stimuli or further genetic modifications (e.g., administration of an engineered meganuclease described herein). Alternatively, a control subject may comprise, for example: a subject having an HBV infection, which is not exposed to conditions or stimuli or further genetic modifications (e.g., administration of an engineered meganuclease described herein) that can alter the HBV infection status of the subject.

[0397] As used herein, the terms “treatment” or “treating a subject” refers to the administration of an engineered meganuclease of the disclosure, or a nucleic acid encoding an engineered meganuclease of the disclosure to a subject infected with HBV for the purpose of slowing or stopping the rate of HBV proliferation of the virus by cleaving the genome of at least one HBV particle. Such treatment reduces or prevents transfection and replication of HBV in the subject, and provides either partial or complete relief of one or more symptoms of HBV infection or a disease associated with HBV infection in the subject. Means to assess alleviation of symptoms of HBV infection or a disease associated with HBV infection may include measurement of liver functions by determining levels of the enzyme alanine aminotransferase (ALT) or by measuring sero conversion, namely disappearance and / or reduction of the circulating HBeAg and / or HBsAg levels. Further, alleviation or reduction of symptoms of HBV infection or a disease associated with HBV infection can be determined by examining liver biopsies and measuring the level of tissue fibrosis by methods well known in the art. The number of circulating viral particles can be determined for example by measuring HBV DNA levels using PCR or by detecting HBsAg levels in the blood. The terms “treatment” or “treating a subject” can further refer to the administration of a cell (e.g., hepatocyte cell) comprising a nucleic acid encoding an engineered meganuclease, wherein the cell is delivered to a target tissue (e.g., liver) and produces the engineered meganuclease in an amount WBD (US) 4897-9494-7440vl 37 Aty Docket No. P89339 2080WO (01275) sufficient to treat an HBV infection or a disease associated with an HBV infection in the subject, thereby resulting in either partial or complete relief of one or more symptoms of the HBV infection or the disease associated with the HBV infection. In some aspects, an engineered meganuclease of the disclosure or a nucleic acid encoding the same is administered during treatment in the form of a pharmaceutical composition of the disclosure.

[0398] The term “a disease associated with Hepatitis B virus infection” refers to any condition related to or resulting from infection with a Hepatitis B virus, such as chronic liver diseases / disorders, inflammations, fibrotic conditions, and proliferative disorders, such as liver cancers. Chronic persistent HBV infection can cause, for example, fatigue, liver damage, cirrhosis of the liver, and hepatocellular carcinoma, a primary liver cancer.

[0399] The terms “proliferating” and “proliferation” as used herein refer to HBV viruses or HBV covalently closed circular DNA (cccDNA) actively dividing and / or infecting human cells. Thus, reduction in proliferation refers to any decrease in the proliferation of HBV including reduction of at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% when compared to an appropriate control not having been administered the engineered meganuclease, or nucleic acid encoding the engineered meganuclease, described herein. Throughout this application, the term “proliferative disorder” refers to any disease / disorder marked by unwanted or aberrant proliferation of cells or tissue. As used herein, the term “proliferative disorder” also refers to conditions in which the unregulated and / or abnormal growth of cells can lead to the development of an unwanted condition or disease, which can be cancerous or non-cancerous.

[0400] As used herein, the term “effective amount” or “therapeutically effective amount” refers to an amount sufficient to effect beneficial or desirable biological and / or clinical results. The therapeutically effective amount will vary depending on the formulation or composition used, the disease and its severity and the age, weight, physical condition, and responsiveness of the subject to be treated. In specific embodiments, an effective amount of the engineered meganuclease or pharmaceutical compositions described herein reduces the level or proliferation of HBV or reduces at least one symptom of a disease associated with HBV infection in a subject with an HBV infection.

[0401] The term “gc / kg” or “gene copies / kilogram” refers to the number of copies of a nucleic acid encoding an engineered meganuclease described herein per weight in kilograms of a subject that is administered the nucleic acid encoding the engineered meganuclease.

[0402] The term “lipid nanoparticle” refers to a lipid composition having a typically spherical structure with an average diameter between 10 and 1000 nanometers. In some formulations, lipid nanoparticles can comprise at least one cationic lipid, at least one non-cationic lipid, and at least WBD (US) 4897-9494-7440vl 38 Aty Docket No. P89339 2080WO (01275) one conjugated lipid. Lipid nanoparticles known in the art that are suitable for encapsulating nucleic acids, such as mRNA, are contemplated for use.

[0403] As used herein, the recitation of a numerical range for a variable is intended to convey that the present disclosure may be practiced with the variable equal to any of the values within that range. Thus, for a variable which is inherently discrete, the variable can be equal to any integer value within the numerical range, including the end-points of the range. Similarly, for a variable which is inherently continuous, the variable can be equal to any real value within the numerical range, including the end-points of the range. As an example, and without limitation, a variable which is described as having values between 0 and 2 can take the values 0, 1 or 2 if the variable is inherently discrete, and can take the values 0.0, 0.1, 0.01, 0.001, or any other real values =0 and =2 if the variable is inherently continuous.

[0404] 2, 1 Principle of the Invention

[0405] The present disclosure encompasses lipid nanoparticles (LNPs) comprising a polynucleotide encoding an engineered HBV meganuclease which has improved properties, such as improved (i.e., increased) specificity resulting in reduced off-target cutting, and enhanced (i.e., increased) efficiency of cleavage and indel formation at the HBV 11-12 recognition sequence, particularly in cells comprising an HBV genome or HBV genome fragment, which can be present within HBV cccDNA and / or can be integrated the genome of the cell. The present disclosure also encompasses a pharmaceutical composition comprising the LNPs, methods of inactivating a pol gene of an HBV genome or an HBV genome fragment in a cell in vitro or in vivo, and methods of treating a disease associated with hepatitis B virus infection (e.g., such as CHB with compensated liver disease) using the LNPs or the pharmaceutical compositions.

[0406] The meganucleases of the present disclosure include HBV 11-12 meganucleases that recognize the HBV 11-12 recognition sequence (SEQ ID NO: 3) in the polymerase (P) gene of the Hepatitis B virus genome that encodes the viral DNA polymerase. The HBV 11-12 recognition sequence is conserved across at least HBV genotypes A-G, which advantageously allows for the presently disclosed engineered meganucleases to target HBV infections around the globe. The amino acid sequence of the HBV 11-12L.1090 E80 meganuclease is set forth as SEQ ID NO: 5, and the HBV 11-12L.1090 E80 sequence missing the Ml residue is set forth as SEQ ID NO: 6. The nomenclature “E80” refers to the presence of an amino acid residue Q at position 80 of SEQ ID NO: 5 and the presence of an amino acid residue E at position 260 of SEQ ID NO: 5. Linkerl923, which corresponds to residues 154-184 of SEQ ID NO: 5, joins the first and second subunits of these engineered meganucleases. The first and second subunits of the HBV 11-12L.1090 E80 meganuclease comprise certain amino acid modifications that, without being held to any particular

[0407] WBD (US) 4897-9494-7440vl 39 Aty Docket No. P89339 2080WO (01275) theory or mechanism of action, are believed to interact with Linkerl923 within the I-Crel scaffold, leading to stabilization of the engineered meganuclease.

[0408] The mRNA sequence encoding the HBV 11-12L.1090 E80 meganuclease clinical candidate has been developed. The mRNA sequence of the clinical candidate includes a Cap-1 structure at the 5’ terminus (m7G(5’)ppp(5’)(2’OMeA)pG), a 5’ UTR from the albumin gene (ALB) (SEQ ID NO: 13), an open reading frame (ORF) containing the coding sequence of the meganuclease (SEQ ID NO: 7 or 8), sequences encoding simian virus 40 nuclear localization signals (SV40 NLS) at 5’ and 3’ extremes of the ORF (SEQ ID NOs: 11 and 12 at the 5’ and 3’ ends, respectively), a 3’ UTR from the small nuclear ribonucleoprotein polypeptides B and Bl genes (SNRPB) (SEQ ID NO: 15), and finally a poly-A tail (SEQ ID NO: 19), as shown in Figure 2. The HBV 11-12L.1090 E80 nuclease comprises a polypeptide linker designed to covalently connect two engineered, I-Crel-derived subunits and develop electrostatic contacts within the backbone of the nuclease. The codon sequence of the meganuclease has been optimized to improve mRNA translation and stability (referred to as a “MAX construct”). Uridine (“U”) bases in the mRNA sequence can be uridine or substituted with a modified uridine nucleotide such as, for example, pseudouridine, Nl-methyl-pseudouridine, 5- methoxyuridine, or 2-thiouridine. Cytosine (“C”) bases can be cytosine or substituted with a modified cytosine nucleotide such as, for example, 5-methylcytidine, 2'-O-methylcytidine, or N4- acetyl-cytosine. Adenine (“A”) bases in the mRNA sequence can be adenine or substituted with a modified adenine nucleotide such as, for example, N6-m ethyladenosine or Nl-methyl-adenine. The complete sequence of the mRNA and DNA sequences of the clinical candidate HBV 11-12L.1090 E80 nuclease are set forth in SEQ ID NOs: 20 and 22, respectively. The complete sequence of the mRNA and DNA sequences of the clinical candidate HBV 11-12L.1090 E80 nuclease lacking the Ml residue are set forth in SEQ ID NOs: 21 and 23, respectively.

[0409] The LNPs of the present disclosure comprise Bis(2-butyloctyl) 10-(N-(3-(pyrrolidin-l- yl)propyl)nonanamido)nonadecanedioate (“Lipid 1”); 2-[2-(o -methoxy (polyethyleneglycol2000) ethoxy]-N,N-ditetradecylacetamide (“Lipid 2”); l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); and cholesterol, and contains a polynucleotide encoding the engineered HBV meganuclease therein (e.g., an mRNA), for delivery to a target cell, such as a liver cell, such as a hepatocyte.

[0410] In a Good Laboratory Practice (GLP) toxicology study conducted in NHP, the LNPs and pharmaceutical compositions provided herein were considered well tolerated following repeated administration at doses up to 1.5 mg / kg / dose as no mortality was seen and no adverse effects were noted in in any parameter examined through the interim necropsy interval, including postmortem evaluations up to Day 86. A dose escalation (Part 1) and expansion (Part 2) study to evaluate the

[0411] WBD (US) 4897-9494-7440vl 40 Atty Docket No. P89339 2080WO (01275) activity of the LNPs and pharmaceutical compositions provided herein in human subjects with CHB is provided.

[0412] 2,2 Meganucleases that Recognize and Cleave the HBV 11-12 Recognition Sequence Within the Hepatitis B Viral Genome

[0413] Recognition Sequences

[0414] It is known in the art that it is possible to use a site-specific nuclease to make a DNA break in the genome of a virus, and that such a DNA break can result in permanent modification of the genome via mutagenic NHEJ repair or via homologous recombination with a transgenic DNA sequence such that the HBV virion can no longer divide / replicate or infect human cells.

[0415] Engineered meganucleases of the disclosure have been designed to bind and cleave an HBV 11-12 recognition sequence (SEQ ID NO: 3). The HBV 11-12 recognition sequence is positioned within the polymerase gene ORF multiple HBV genotypes, including at least genotypes A, B, C, D, E, F, and G.

[0416] As shown in Figure 1, the HBV 11-12 recognition sequence, targeted by engineered meganucleases of the present disclosure, comprises two recognition half-sites. Each recognition half-site comprises 9 base pairs, separated by a 4 base pair central sequence. The HBV 11-12 recognition sequence (SEQ ID NO: 3) comprises two recognition half-sites referred to as HBV11 and HBV12. SEQ ID NO: 4 is a reverse complement of SEQ ID NO: 3.

[0417] Exemplary Engineered Meganucleases

[0418] It is known in the art that it is possible to use a site-specific nuclease to make a DNA break in the genome of a virus, and that such a DNA break can result in permanent modification of the genome via NHEJ such that the HBV virion can no longer divide / replicate or infect human cells. Generating a DNA break in a viral genome can also lead to degradation of the viral genome, rendering it unable to divide, replicate, or be infective.

[0419] Thus, in some embodiments, the present disclosure provides engineered nucleases, particularly engineered meganucleases. In particular embodiments, the meganucleases are singlechain meganucleases. A single-chain meganuclease comprises an N-terminal subunit and a C- terminal subunit joined by a linker peptide. Each of the two domains recognizes half of the recognition sequence (i.e., a recognition half-site) and the site of DNA cleavage is at the middle of the recognition sequence near the interface of the two subunits. DNA strand breaks are offset by four base pairs such that DNA cleavage by a meganuclease generates a pair of four base pair, 3’ single-strand overhangs.

[0420] WBD (US) 4897-9494-7440vl 41 Atty Docket No. P89339 2080WO (01275) In some embodiments, the presently disclosed engineered meganucleases exhibit at least one optimized characteristic in comparison to the previously described meganuclease HBV 11- 12L.1090QQ Linkerl. Such optimized characteristics include improved (i.e. increased) specificity resulting in reduced off-target cutting, and enhanced (i.e., increased) efficiency of cleavage and indel (i.e., insertion or deletion) formation at the HBV 11-12 recognition sequence, particularly in cells comprising an integrated copy of the HBV genome. Thus, in particular embodiments, the presently disclosed engineered meganucleases, when delivered to a population of HBV-infected target cells, is able to generate a greater percentage of virions or cells with a cleavage and / or an indel in the HBV genome (either incorporated or unincorporated). In some of these embodiments, the population of HBV or target cells comprises at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% of HBV or target cells comprising a cleavage and / or an indel in the HBV genome (either incorporated or unincorporated). Cleavage and / or indel formation by a meganuclease can be measured using any method known in the art, including T7E assay, digital PCR, mismatch detection assays, mismatch cleavage assay, high-resolution melting analysis (HRMA), heteroduplex mobility assay, sequencing, and fluorescent PCR capillary gel electrophoresis (see, e.g., Zischewski et al. (2017) Biotechnology Advances 35(l):95-104, which is incorporated by reference in its entirety).

[0421] In some embodiments, the target cell is a liver cell. In some embodiments, the liver cell is a hepatocyte. In some embodiments, the target cell is a primary human hepatocyte (PHH). In some embodiments, the target cell is a non-human, mammalian hepatocyte.

[0422] Engineered meganucleases described herein comprise a first subunit, comprising a first hypervariable (HVR1) region, and a second subunit, comprising a second hypervariable (HVR2) region. Further, the first subunit binds to a first recognition half-site in the recognition sequence (i.e., the HBV11 half-site), and the second subunit binds to a second recognition half-site in the recognition sequence (i.e., the HBV12 half-site). In embodiments where the engineered meganuclease is a single-chain meganuclease, the first and second subunits can be oriented such that the first subunit, which comprises the HVR1 region and binds the first half-site, is positioned as the N-terminal subunit, and the second subunit, which comprises the HVR2 region and binds the second half-site, is positioned as the C-terminal subunit. In alternative embodiments, the first and second subunits can be oriented such that the first subunit, which comprises the HVR1 region and binds the first half-site, is positioned as the C-terminal subunit, and the second subunit, which comprises the HVR2 region and binds the second half-site, is positioned as the N-terminal subunit.

[0423] WBD (US) 4897-9494-7440vl 42 Atty Docket No. P89339 2080WO (01275) Cleavage at the HBV 11-12 recognition sequence can allow for non-homologous end joining (NHEJ) at the cleavage site and can disrupt expression of one or more viral proteins (e.g., viral DNA polymerase) due to NHEJ at the cleavage site that results in insertions, deletions, or frameshift mutations. Alternatively, cleavage of the HBV genome at the HBV 11-12 recognition sequence may promote degradation of the HBV genome and / or HBV cccDNA. Disruption of the expression of the viral protein(s) can reduce or eliminate the infection and / or proliferation of HBV. Additionally, cleavage at the HBV 11-12 recognition sequence can further allow for homologous recombination of exogenous nucleic acid sequences directly into the HBV genome to disrupt the expression of one or more viral proteins. For example, a “suicide gene” can be introduced into an HBV genome via homologous recombination.

[0424] Amino acid sequences of exemplary engineered meganucleases that recognize and cleave the HBV 11-12 recognition sequence are provided in SEQ ID NOs: 5 and 6 and are further described below.

[0425] HBV 11-12L.1090 E80 (SEQ ID NO: 5 or 6)

[0426] In some embodiments, the HVR1 region comprises an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, sequence identity to residues 204-259 of SEQ ID NO: 5.

[0427] In the HBV 11-12L.1090 meganucleases described herein that comprise the Linkerl923 sequence, the N-terminal subunit can include an A at position 96, an A at position 99, and a D at position 100, and the C-terminal subunit includes a Y at a position corresponding to position 57 of SEQ ID NO: 1 (i.e., position 237 of SEQ ID NO: 5) and a T at a position corresponding to position 61 of SEQ ID NO: 1 (i.e., position 241 of SEQ ID NO: 5).

[0428] In some embodiments, the HVR1 region comprises one or more residues corresponding to residues 204, 206, 208, 210, 212, 213, 218, 220, 222, 224, 226, 248, 250, 255, and 257 of SEQ ID NO: 5.

[0429] In some embodiments, the HVR1 region comprises residues corresponding to residues 204, 206, 208, 210, 212, 213, 218, 220, 222, 224, 226, 248, 250, 255, and 257 of SEQ ID NO: 5.

[0430] In some embodiments, the HVR1 region comprises a residue corresponding to residue 237 of SEQ ID NO: 5.

[0431] In some embodiments, the HVR1 region comprises a residue corresponding to residue 241 of SEQ ID NO: 5.

[0432] In some embodiments, the HVR1 region comprises a residue corresponding to residue 251 of SEQ ID NO: 5.

[0433] WBD (US) 4897-9494-7440vl 43 Atty Docket No. P89339 2080WO (01275) In some embodiments, the HVR1 region comprises a residue corresponding to residue 252 of SEQ ID NO: 5.

[0434] In some embodiments, the HVR1 region comprises a residue corresponding to residue 253 of SEQ ID NO: 5.

[0435] In some embodiments, the HVR1 region comprises Y, R, K, or D at a residue corresponding to residue 246 of SEQ ID NO: 5.

[0436] In some embodiments, the HVR1 region comprises residues 204-259 of SEQ ID NO: 5.

[0437] In some embodiments, the first subunit comprises an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, sequence identity to residues 187-333 of SEQ ID NO: 5.

[0438] In some embodiments, the first subunit comprises an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, sequence identity to residues 185-343 of SEQ ID NO: 5.

[0439] In some embodiments, the first subunit comprises a residue corresponding to residue 260 of SEQ ID NO: 5.

[0440] In some embodiments, the first subunit comprises G, S, or A at a residue corresponding to residue 199 of SEQ ID NO: 5.

[0441] In some embodiments, the first subunit comprises E, Q, or K at a residue corresponding to residue 260 of SEQ ID NO: 5.

[0442] In some embodiments, the first subunit comprises residues 187-333 of any one of SEQ ID NO: 5.

[0443] In some embodiments, the first subunit comprises residues 185-343 of any one of SEQ ID NO: 5.

[0444] In some embodiments, the HVR2 region comprises an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, sequence identity to residues 24-79 of SEQ ID NO: 5.

[0445] In some embodiments, the HVR2 region comprises one or more residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 5.

[0446] In some embodiments, the HVR2 region comprises residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 5.

[0447] In some embodiments, the HVR2 region comprises a residue corresponding to residue 51 of

[0448] SEQ ID NO: 5.

[0449] WBD (US) 4897-9494-7440vl 44 Atty Docket No. P89339 2080WO (01275) In some embodiments, the HVR2 region comprises Y, R, K, or D at a residue corresponding to residue 66 of SEQ ID NO: 5.

[0450] In some embodiments, the HVR2 region comprises residues 24-79 of SEQ ID NO: 5.

[0451] In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, sequence identity to residues 7-153 of SEQ ID NO: 5.

[0452] In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, sequence identity to residues 6-153 of SEQ ID NO: 5.

[0453] In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, sequence identity to residues 5-153 of SEQ ID NO: 5.

[0454] In some embodiments, the second subunit is an N-terminal subunit and comprises an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, sequence identity to residues 4-153 of SEQ ID NO: 5.

[0455] In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, sequence identity to residues 3-153 of SEQ ID NO: 5.

[0456] In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, sequence identity to residues 2-153 of SEQ ID NO: 5. In some embodiments, the second subunit comprises a residue other than M at a position corresponding to position 1 of SEQ ID NO: 5.

[0457] In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, sequence identity to residues 1-153 of SEQ ID NO: 5.

[0458] WBD (US) 4897-9494-7440vl 45 Atty Docket No. P89339 2080WO (01275) In some embodiments, the second subunit comprises a residue corresponding to residue 19 of SEQ ID NO: 5.

[0459] In some embodiments, the second subunit comprises a residue corresponding to residue 80 of SEQ ID NO: 5.

[0460] In some embodiments, the second subunit comprises a residue corresponding to residue 96 of SEQ ID NO: 5.

[0461] In some embodiments, the second subunit comprises a residue corresponding to residue 99 of SEQ ID NO: 5.

[0462] In some embodiments, the second subunit comprises a residue corresponding to residue 100 of SEQ ID NO: 5.

[0463] In some embodiments, the second subunit comprises G, S, or A at a residue corresponding to residue 19 of SEQ ID NO: 5.

[0464] In some embodiments, the second subunit comprises E, Q, or K at a residue corresponding to residue 80 of SEQ ID NO: 5.

[0465] In some embodiments, the second subunit comprises residues 7-153 of SEQ ID NO: 5.

[0466] In some embodiments, the second subunit comprises residues 6-153 of SEQ ID NO: 5.

[0467] In some embodiments, the second subunit comprises residues 5-153 of SEQ ID NO: 5.

[0468] In some embodiments, the second subunit comprises residues 4-153 of SEQ ID NO: 5.

[0469] In some embodiments, the second subunit comprises residues 3-153 of SEQ ID NO: 5.

[0470] In some embodiments, the second subunit comprises residues 2-153 of SEQ ID NO: 5. In some embodiments, the second subunit comprises a residue other than M at a position corresponding to position 1 of SEQ ID NO: 5 or SEQ ID NO: 5.

[0471] In some embodiments, the second subunit comprises residues 1-153 of SEQ ID NO: 5.

[0472] In some embodiments, the engineered meganuclease is a single-chain meganuclease comprising a linker and wherein the linker covalently joins the first subunit and the second subunit.

[0473] In some embodiments, the N-terminus of the linker is fused to the residue (i.e., a D residue) corresponding to residue 153 of SEQ ID NO: 5, and the C-terminus of the linker is fused to the residue (i.e., a Y residue) corresponding to residue 185 of SEQ ID NO: 5.

[0474] In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, sequence identity to residues 4-343 of SEQ ID NO: 5.

[0475] In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least

[0476] WBD (US) 4897-9494-7440vl 46 Atty Docket No. P89339 2080WO (01275) 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, sequence identity to residues 3-343 of SEQ ID NO: 5.

[0477] In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, sequence identity to residues 2-343 of SEQ ID NO: 5. In some embodiments, the second subunit comprises a residue other than M at a position corresponding to position 1 of SEQ ID NO: 5.

[0478] In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, sequence identity to SEQ ID NO: 5.

[0479] In some embodiments, the engineered meganuclease comprises an amino acid sequence of residues 4-343 of SEQ ID NO: 5.

[0480] In some embodiments, the engineered meganuclease comprises an amino acid sequence of residues 3-343 of SEQ ID NO: 5.

[0481] In some embodiments, the engineered meganuclease comprises an amino acid sequence of residues 2-343 of SEQ ID NO: 5 (i.e., SEQ ID NO: 6). In some embodiments, the second subunit comprises a residue other than M at a position corresponding to position 1 of SEQ ID NO: 5.

[0482] In some embodiments, the engineered meganuclease comprises an amino acid sequence of SEQ ID NO: 5.

[0483] In some embodiments, the engineered meganuclease is encoded by a nucleic sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, sequence identity to a nucleic acid sequence of any one of SEQ ID NOs: 7-10.

[0484] In some embodiments, the engineered meganuclease comprises an NLS.

[0485] In some embodiments, the NLS is at the N-terminus of the engineered meganuclease.

[0486] In some embodiments, the NLS is at the C-terminus of the engineered meganuclease.

[0487] In some embodiments, the engineered meganuclease comprises a first NLS at the N- terminus and a second NLS at the C-terminus.

[0488] In certain embodiments of the engineered meganucleases described herein, the first subunit (i.e., comprising HVR1) can be positioned as the C-terminal subunit, and the second subunit (i.e., comprising HVR2) can be positioned as the N-terminal subunit. In some embodiments of such a configuration, such as those exemplified in SEQ ID NO: 5 and SEQ ID NO: 6, the first subunit WBD (US) 4897-9494-7440vl 47 Aty Docket No. P89339 2080WO (01275) (i.e., the C-terminal subunit) can lack residues at its N-terminus that correspond to residues 1-4 of wild-type I-Crel because the binding site of the polypeptide linker is at the Y residue corresponding to position 5 of wild-type I-Crel. The first subunit can further comprise residues at its C-terminus that correspond to residues 154-163 of wild-type I-Crel. Also, in some embodiments of such a configuration, the second subunit (i.e., the N-terminal subunit) can lack residues at its C-terminus that correspond to residues 154-163 of wild-type I-Crel because the binding site of the polypeptide linker is at the D residue corresponding to position 153 of wild-type I-Crel. The second subunit can further comprise one or more residues at its N-terminus that correspond to one or more of residues 1-6 of wild-type I-Crel (e.g., residues 1-6, 2-6, 3-6, 4-6, or 5-6).

[0489] In other embodiments of the engineered meganucleases described herein, the first subunit (i.e., comprising HVR1) can be positioned as the N-terminal subunit, and the second subunit (i.e., comprising HVR2) can be positioned as the C-terminal subunit. In some embodiments of such a configuration, the first subunit (i.e., the N-terminal subunit) can lack residues at its C-terminus that correspond to residues 154-163 of wild-type I-Crel because the binding site of the polypeptide linker is at the D residue corresponding to position 153 of wild-type I-Crel. The first subunit can further comprise one or more residues at its N-terminus that correspond to one or more of residues 1-6 of wild-type I-Crel (e.g., residues 1-6, 2-6, 3-6, 4-6, or 5-6). Also, in some embodiments of such a configuration, the second subunit (i.e., the C-terminal subunit) can lack residues at its N- terminus that correspond to residues 1-4 of wild-type I-Crel because the binding site of the polypeptide linker is at the Y residue corresponding to position 5 of wild-type I-Crel. The second subunit can further comprise residues at its C-terminus that correspond to residues 154-163 of wildtype I-Crel.

[0490] In specific embodiments, the engineered meganuclease provided herein is encoded by an mRNA sequence that includes a Cap-1 structure at the 5’ terminus (m7G(5’)ppp(5’)(2’OMeA)pG), a 5’ UTR from the albumin gene (ALB) (e.g., SEQ ID NO: 13), an ORF containing the coding sequence of the meganuclease (e.g., SEQ ID NO: 7 or 8), sequences encoding simian virus 40 nuclear localization signals (SV40 NLS) at the 5’ and 3’ extremes of the ORF (SEQ ID NOs: 11 and 12 at the 5’ and 3’ ends, respectively), a 3’ UTR from the small nuclear ribonucleoprotein polypeptides B and Bl genes (SNRPB) (SEQ ID NO: 15), and finally a poly- A tail (SEQ ID NO: 19), as shown in Figure 2. In specific embodiments, the engineered meganuclease provided herein is encoded by an mRNA sequence set forth in SEQ ID NO: 20 or 21, or a DNA sequence set forth in SEQ ID NO: 22 or 23.

[0491] 2,3 Engineered Meganuclease Variants

[0492] WBD (US) 4897-9494-7440vl 48 Atty Docket No. P89339 2080WO (01275) Embodiments of the disclosure encompass the engineered meganucleases described herein, and variants thereof. Further embodiments of the disclosure encompass polynucleotides comprising a nucleic acid sequence encoding the meganucleases described herein, and variants of such polynucleotides.

[0493] As used herein, “variants” is intended to mean substantially similar sequences. A “variant” polypeptide is intended to mean a polypeptide derived from the “native” polypeptide by deletion or addition of one or more amino acids at one or more internal sites in the native protein and / or substitution of one or more amino acids at one or more sites in the native polypeptide. As used herein, a “native” polynucleotide or polypeptide comprises a parental sequence from which variants are derived. Variant polypeptides encompassed by the embodiments are biologically active. That is, they continue to possess the desired biological activity of the native protein; i.e., the ability to recognize and cleave the HBV 11-12 recognition sequence (SEQ ID NO: 3) within the genome of a Hepatitis B virus, and in some embodiments, exhibit at least one improved property over previously described engineered HBV meganucleases (e.g., the HBV 11-12L.1090QQ Linkerl meganuclease), such as improved (i.e., increased) specificity and enhanced (i.e., increased) efficiency of cleavage and indel formation. Such variants may result, for example, from human manipulation. Biologically active variants (e.g., SEQ ID NOs: 5 and 6) of a native polypeptide of the embodiments, or biologically active variants of the recognition half-site binding subunits described herein, will have at least about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%, sequence identity to the amino acid sequence of the native polypeptide, native subunit, native HVR1 region, and / or native HVR2 region, as determined by sequence alignment programs and parameters described elsewhere herein. A biologically active variant of a polypeptide or subunit of the embodiments may differ from that polypeptide or subunit by as few as about 1-40 amino acid residues, as few as about 1-20, as few as about 1-10, as few as about 5, as few as 4, 3, 2, or even 1 amino acid residue.

[0494] The polypeptides of the embodiments may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants can be prepared by mutations in the DNA. Methods for mutagenesis and polynucleotide alterations are well known in the art. See, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods in Enzymol. 154:367-382; U.S. Pat. No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York) and the references cited therein. Guidance as to appropriate amino acid substitutions that do not affect biological activity of the WBD (US) 4897-9494-7440vl 49 Aty Docket No. P89339 2080WO (01275) protein of interest may be found in the model of Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.), herein incorporated by reference. Conservative substitutions, such as exchanging one amino acid with another having similar properties, may be optimal.

[0495] In some embodiments, engineered meganucleases described herein can comprise variants of the HVR1 and HVR2 regions described herein. Parental HVR regions can comprise, for example, residues corresponding to residues 24-79 or residues 204-259 of the exemplified engineered meganucleases. Thus, variant HVRs can comprise an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more, sequence identity to an amino acid sequence corresponding to residues 24-79 or residues 204-259 of the engineered meganucleases exemplified herein, such that the variant HVR regions maintain the biological activity of the engineered meganuclease (i.e., binding to and cleaving the recognition sequence). Further, in some embodiments of the disclosure, a variant HVR1 region or variant HVR2 region can comprise residues corresponding to the amino acid residues found at specific positions within the parental HVR. In this context, “corresponding to” means that an amino acid residue in the variant HVR is the same amino acid residue (i.e., a separate identical residue) present in the parental HVR sequence in the same relative position (i.e., in relation to the remaining amino acids in the parent sequence). By way of example, if a parental HVR sequence comprises a serine residue at position 26, a variant HVR that “comprises a residue corresponding to” residue 26 will also comprise a serine at a position that is relative (i.e., corresponding) to parental position 26.

[0496] In particular embodiments, engineered meganucleases described herein comprise an HVR1 that has at least at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to an amino acid sequence corresponding to residues 204-259 of SEQ ID NO: 5 or residues 203-258 of SEQ ID NO: 6.

[0497] In certain embodiments, engineered meganucleases described herein comprise an HVR2 region that has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to an amino acid sequence corresponding to residues 24-79 of SEQ ID NO: 5 or residues 23-78 of SEQ ID NO: 6.

[0498] A substantial number of amino acid modifications to the DNA recognition domain of the wild-type I-Crel meganuclease have previously been identified (e.g., U.S. 8,021,867) which, singly WBD (US) 4897-9494-7440vl 50 Aty Docket No. P89339 2080WO (01275) or in combination, result in engineered meganucleases with specificities altered at individual bases within the DNA recognition sequence half-site, such that the resulting rationally-designed meganucleases have half-site specificities different from the wild-type enzyme. Table 1 provides potential substitutions that can be made in an engineered meganuclease monomer or subunit to enhance specificity based on the base present at each half-site position (-1 through -9) of a recognition half-site.

[0499] Table 1: Potential Substitutions in Engineered Meganuclease Subunit to Enhance Specificity

[0500] WBD (US) 4897-9494-7440vl 51 Aty Docket No. P89339 2080WO (01275)

[0501] An asterisk indicates that the residue contacts the base on the antisense strand.

[0502] Certain modifications can be made in an engineered meganuclease monomer or subunit to modulate DNA-binding affinity and / or activity. For example, an engineered meganuclease monomer or subunit described herein can comprise a G, S, or A at a residue corresponding to position 19 of I-Crel or position 199 of SEQ ID NO: 5 (WO 2009 / 001159), a Y, R, K, or D at a residue corresponding to position 66 of I-Crel or position 246 of SEQ ID NO: 5, and / or an E, Q, or K at a residue corresponding to position 80 of I-Crel or position 260 of SEQ ID NO: 5 (US Pat. No. 8,021,867).

[0503] For polynucleotides, a “variant” comprises a deletion and / or addition of one or more nucleotides at one or more sites within the native polynucleotide. One of skill in the art will recognize that variants of the nucleic acids of the embodiments will be constructed such that the ORF is maintained. For polynucleotides, conservative variants include those sequences that, because of the degeneracy of the genetic code, encode the amino acid sequence of one of the polypeptides of the embodiments. Variant polynucleotides include synthetically derived polynucleotides, such as those generated, for example, by using site-directed mutagenesis but which still encode a recombinant nuclease of the embodiments. Generally, variants of a particular polynucleotide of the embodiments will have at least about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more sequence identity to that particular polynucleotide as determined by sequence alignment programs and parameters described elsewhere herein. Variants of a particular polynucleotide of the embodiments (i.e., the reference polynucleotide) can also be evaluated by comparison of the percent sequence identity between the polypeptide encoded by a variant polynucleotide and the polypeptide encoded by the reference polynucleotide.

[0504] WBD (US) 4897-9494-7440vl 52 Atty Docket No. P89339 2080WO (01275) The deletions, insertions, and substitutions of the protein sequences encompassed herein are not expected to produce radical changes in the characteristics of the polypeptide. However, when it is difficult to predict the exact effect of the substitution, deletion, or insertion in advance of doing so, one skilled in the art will appreciate that the effect will be evaluated by screening the polypeptide its intended activity. For example, variants of an engineered meganuclease would be screened for their ability to preferentially bind and cleave the HBV 11-12 recognition sequence within the genome of a Hepatitis B virus.

[0505] 2,4 Lipid Nanoparticle Compositions

[0506] A lipid nanoparticle (LNP) comprising a polynucleotide is provided. The polynucleotide comprises a nucleic acid sequence encoding an engineered meganuclease that binds and cleaves a recognition sequence comprising SEQ ID NO: 3 in a polymerase (pol) gene of a hepatitis B virus (HBV) genome or HBV genome fragment.

[0507] The term “lipid” refers to a group of organic compounds that include, but are not limited to, esters of fatty acids and are generally characterized by being poorly soluble in water, but soluble in many organic solvents. Lipids are usually divided into at least three classes: (1) “simple lipids,” which include fats and oils as well as waxes; (2) “compound lipids,” which include phospholipids and glycolipids; and (3) “derived lipids” such as steroids.

[0508] A “steroid” is a compound comprising the following carbon skeleton:

[0509] Non-limiting examples of steroids include cholesterol, and the like.

[0510] A “cationic lipid” refers to a lipid capable of being positively charged. Exemplary cationic lipids include one or more amine group(s) which bear the positive charge. Exemplary cationic lipids are ionizable such that they can exist in a positively charged or neutral form depending on pH. The ionization of the cationic lipid affects the surface charge of the lipid nanoparticle under different pH conditions. This charge state can influence plasma protein absorption, blood clearance and tissue distribution (Semple, S.C., et al., Adv. Drug Deliv Rev 32:3-17 (1998)) as well as the ability to form endosomolytic non-bilayer structures (Hafez, I.M., et al., Gene Ther 8: 1188-1196 (2001)) critical to the intracellular delivery of nucleic acids.

[0511] The term “lipid nanoparticle” refers to particles having at least one dimension on the order of nanometers (e.g., 1-1,000 nm) which include one or more of the compounds of formula (I) or

[0512] WBD (US) 4897-9494-7440vl Aty Docket No. P89339 2080WO (01275) other specified cationic lipids. In some embodiments, lipid nanoparticles are included in a formulation that can be used to deliver an active agent or therapeutic agent, such as a nucleic acid (e.g., mRNA) to a target site of interest (e.g., cell, tissue, organ, tumor, and the like). In some embodiments, the lipid nanoparticles of the invention comprise a nucleic acid. Such lipid nanoparticles typically comprise a compound of Formula (I) and one or more excipient selected from neutral lipids, charged lipids, steroids and polymer conjugated lipids. In some embodiments, the active agent or therapeutic agent, such as a nucleic acid, may be encapsulated in the lipid portion of the lipid nanoparticle or an aqueous space enveloped by some or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects induced by the mechanisms of the host organism or cells e.g. an adverse immune response.

[0513] In various embodiments, the lipid nanoparticles have a mean diameter of from about 30 nm to about 150 nm, from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, from about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, from about 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70 nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm, and are substantially non-toxic. In certain embodiments, nucleic acids, when present in the lipid nanoparticles, are resistant in aqueous solution to degradation with a nuclease. Lipid nanoparticles comprising nucleic acids and their method of preparation are disclosed in, e.g., U.S. Patent Publication Nos. 2004 / 0142025, 2007 / 0042031 and PCT Pub. Nos. WO 2013 / 016058 and WO 2013 / 086373, the full disclosures of which are herein incorporated by reference in their entirety for all purposes.

[0514] As used herein, “lipid encapsulated” refers to a lipid nanoparticle that provides an active agent or therapeutic agent, such as a nucleic acid (e.g., mRNA), with full encapsulation, partial encapsulation, or both. In an embodiment, the nucleic acid (e.g., mRNA) is fully encapsulated in the lipid nanoparticle.

[0515] The term “polymer conjugated lipid” refers to a molecule comprising both a lipid portion and a polymer portion. An example of a polymer conjugated lipid is a pegylated lipid. The term “pegylated lipid” refers to a molecule comprising both a lipid portion and a polyethylene glycol portion. Pegylated lipids are known in the art and include l-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG) and the like.

[0516] WBD (US) 4897-9494-7440vl 54 Atty Docket No. P89339 2080WO (01275) The term “neutral lipid” refers to any of a number of lipid species that exist either in an uncharged or neutral zwitterionic form at a selected pH. At physiological pH, such lipids include, but are not limited to, phosphotidylcholines such as l ,2-Distearoyl-.s / / -glycero-3-phosphocholine (DSPC), l ,2-Dipalmitoyl-.s / / -glycero-3 -phosphocholine (DPPC), l ,2-Dimyristoyl-.s / / -glycero-3- phosphocholine (DMPC), I -Pal mi toyl-2-oleoyl-.s / / -glycero-3 -phosphocholine (POPC), 1,2- dioleoyl-sn-glycero-3 -phosphocholine (DOPC), phophatidylethanolamines such as 1 , 2-Di oleoyl -s / / - glycero-3 -phosphoethanolamine (DOPE), sphingomyelins (SM), ceramides, steroids such as sterols and their derivatives. Neutral lipids may be synthetic or naturally derived.

[0517] The term “charged lipid” refers to any of a number of lipid species that exist in either a positively charged or negatively charged form independent of the pH within a useful physiological range e.g. pH ~3 to pH ~9. Charged lipids may be synthetic or naturally derived. Examples of charged lipids include phosphatidylserines, phosphatidic acids, phosphatidylglycerols, phosphatidylinositols, sterol hemi succinates, dialkyl trimethylammonium-propanes, (e.g. DOTAP, DOTMA), dialkyl dimethylaminopropanes, ethyl phosphocholines, dimethylaminoethane carbamoyl sterols (e.g. DC-Chol).

[0518] “Alkyl” refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, having, for example, from one to twenty-four carbon atoms (C1-C24 alkyl), four to twenty carbon atoms (C4-C20 alkyl), six to sixteen carbon atoms (Ce-Ci6 alkyl), six to nine carbon atoms (C6-C9 alkyl), one to fifteen carbon atoms (C1-C15 alkyl), one to twelve carbon atoms (C1-C12 alkyl), one to eight carbon atoms (Ci-Cs alkyl) or one to six carbon atoms (Ci-Ce alkyl) and which is attached to the rest of the molecule by a single bond, e.g., methyl, ethyl, n propyl, 1 methylethyl (iso propyl), n butyl, n pentyl, 1,1 dimethylethyl (t butyl), 3 methylhexyl, 2 methylhexyl, and the like. Unless stated otherwise specifically in the specification, an alkyl group is optionally substituted.

[0519] “Alkylene” or “alkylene chain” refers to a straight or branched divalent hydrocarbon chain linking the rest of the molecule to a radical group, consisting solely of carbon and hydrogen, which is saturated or unsaturated (z.e., contains one or more double and / or triple bonds), and having, for example, from one to twenty-four carbon atoms (C1-C24 alkylene), one to fifteen carbon atoms (Ci- C15 alkylene), one to twelve carbon atoms (C1-C12 alkylene), one to eight carbon atoms (Ci-Cs alkylene), one to six carbon atoms (Ci-Ce alkylene), two to four carbon atoms (C2-C4 alkylene), one to two carbon atoms (C1-C2 alkylene), e.g., methylene, ethylene, propylene, ^-butylene, ethenylene, propenylene, w-butenylene, propynylene, w-butynylene, and the like. The alkylene chain is attached to the rest of the molecule through a single or double bond and to the radical group through a single

[0520] WBD (US) 4897-9494-7440vl 55 Atty Docket No. P89339 2080WO (01275) or double bond. The points of attachment of the alkylene chain to the rest of the molecule and to the radical group can be through one carbon or any two carbons within the chain. Unless stated otherwise specifically in the specification, an alkylene chain may be optionally substituted.

[0521] “Heterocyclyl” or “heterocyclic ring” refers to a stable 3- to 18-membered (e.g., 5, 6 or 7- membered) non-aromatic ring radical having one to twelve ring carbon atoms (e.g., two to twelve) and from one to six ring heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur. Unless stated otherwise specifically in the specification, the heterocyclyl radical may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include fused or bridged ring systems; and the nitrogen, carbon or sulfur atoms in the heterocyclyl radical may be optionally oxidized; the nitrogen atom may be optionally quaternized; and the heterocyclyl radical may be partially or fully saturated. Examples of such heterocyclyl radicals include, but are not limited to, dioxolanyl, thienyl[l,3]dithianyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, and 1,1-dioxo-thiomorpholinyl. Unless stated otherwise specifically in the specification, a heterocyclyl group may be optionally substituted.

[0522] The term “substituted” used herein means any of the above groups (e.g., alkyl, alkylene or heterocyclyl) wherein at least one hydrogen atom (e.g., 1, 2 ,3 or all hydrogen atoms) is replaced by a bond to a non-hydrogen atom such as, but not limited to: a halogen atom such as F, Cl, Br, or I; oxo groups (=0); hydroxyl groups (-0H); C1-C12 alkyl groups; cycloalkyl groups; -(C=0)0R ; - 0(C=0)R ; -C(=0)R ; -OR ;

[0523] S(O)XR ; SSR ; C(=O)SR’; SC(=O)R ; NRR ; NR C(=0)R ; C(=0)NRR ;

[0524] -NR C(=0)NR R ; -0C(=0)NR R ; -NR C(=0)0R ; -NR S(0)xNRR’; -NR S(0)XR ; and -S(O)XNR R , wherein: R is, at each occurrence, independently H, C1-C15 alkyl or cycloalkyl, and x is 0, 1 or 2. In some embodiments the substituent is a C1-C12 alkyl group. In other embodiments, the substituent is a cycloalkyl group. In other embodiments, the substituent is a halo group, such as fluoro. In other embodiments, the substituent is a oxo group. In other embodiments, the substituent is a hydroxyl group. In other embodiments, the substituent is an alkoxy group. In other embodiments, the substituent is a carboxyl group. In other embodiments, the substituent is an amine group.

[0525] WBD (US) 4897-9494-7440vl 56 Atty Docket No. P89339 2080WO (01275) “Optional” or “optionally” (e.g., optionally substituted) means that the subsequently described event of circumstances may or may not occur, and that the description includes instances where said event or circumstance occurs and instances in which it does not. For example, “optionally substituted alkyl” means that the alkyl radical may or may not be substituted and that the description includes both substituted alkyl radicals and alkyl radicals having no substitution.

[0526] Embodiments of the invention disclosed herein are also meant to encompass all pharmaceutically acceptable compounds of the compound of Formula (I) being isotopically- labelled by having one or more atoms replaced by an atom having a different atomic mass or mass number. Examples of isotopes that can be incorporated into the disclosed compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, chlorine, and iodine, such as2H,3H,nC,13C,14C,13N,15N,150,17O,180,31P,32P,35S,18F,36C1,123I, and125I, respectively. These radiolabeled compounds can be useful to help determine or measure the effectiveness of the compounds, by characterizing, for example, the site or mode of action, or binding affinity to pharmacologically important site of action. Certain isotopically-labelled compounds having a structure of Formula (I) or (II), for example, those incorporating a radioactive isotope, are useful in drug and / or substrate tissue distribution studies. The radioactive isotopes tritium, i.e.,3H, and carbon-14, i.e.,14C, are particularly useful for this purpose in view of their ease of incorporation and ready means of detection.

[0527] Substitution with heavier isotopes such as deuterium, i.e.,2H, may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements, and hence may be preferred in some circumstances.

[0528] Substitution with positron emitting isotopes, such asnC,18F,15O and13N, can be useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy. Isotopically-labeled compounds of Formula (I) of (II) can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the Preparations and Examples as set out below using an appropriate isotopically-labeled reagent in place of the non-labeled reagent previously employed.

[0529] “Pharmaceutically acceptable salt” includes both acid and base addition salts.

[0530] “Pharmaceutically acceptable acid addition salt” refers to those salts which retain the biological effectiveness and properties of the free bases, which are not biologically or otherwise undesirable, and which are formed with inorganic acids such as, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic WBD (US) 4897-9494-7440vl 57 Aty Docket No. P89339 2080WO (01275) acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor- 10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane- 1,2-disulfonic acid, ethanesulfonic acid, 2- hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-l,5-disulfonic acid, naphthalene-2-sulfonic acid, 1 -hydroxy -2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, -toluenesulfonic acid, trifluoroacetic acid, undecylenic acid, and the like.

[0531] “Pharmaceutically acceptable base addition salt” refers to those salts which retain the biological effectiveness and properties of the free acids, which are not biologically or otherwise undesirable. These salts are prepared from addition of an inorganic base or an organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, the sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Preferred inorganic salts are the ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purines, piperazine, piperidine, A-ethylpiperidine, polyamine resins and the like. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine.

[0532] Often crystallizations produce a solvate of the compound of the invention. As used herein, the term “solvate” refers to an aggregate that comprises one or more molecules of a compound of the invention with one or more molecules of solvent. The solvent may be water, in which case the solvate may be a hydrate. Alternatively, the solvent may be an organic solvent. Thus, the compounds of the present invention may exist as a hydrate, including a monohydrate, dihydrate, WBD (US) 4897-9494-7440vl 58 Aty Docket No. P89339 2080WO (01275) hemihydrate, sesquihydrate, trihydrate, tetrahydrate and the like, as well as the corresponding solvated forms. The compound of the invention may be true solvates, while in other cases, the compound of the invention may merely retain adventitious water or be a mixture of water plus some adventitious solvent.

[0533] A “pharmaceutical composition” refers to a formulation of a compound of the invention and a medium generally accepted in the art for the delivery of the biologically active compound to mammals, e.g., humans. Such a medium includes all pharmaceutically acceptable carriers, diluents or excipients therefor.

[0534] The lipid described herein, or their pharmaceutically acceptable salts may contain one or more asymmetric centers and may thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that may be defined, in terms of absolute stereochemistry, as (R)- or (5)- or, as (D)- or (L)- for amino acids. Embodiments of the present invention are meant to include all such possible isomers, as well as their racemic and optically pure forms. Optically active (+) and (-), (R)- and (5)-, or (D)- and (L)- isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, for example, chromatography and fractional crystallization. Conventional techniques for the preparation / isolation of individual enantiomers include chiral synthesis from a suitable optically pure precursor or resolution of the racemate (or the racemate of a salt or derivative) using, for example, chiral high pressure liquid chromatography (HPLC). When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers. Likewise, all tautomeric forms are also intended to be included.

[0535] A “stereoisomer” refers to a compound made up of the same atoms bonded by the same bonds but having different three-dimensional structures, which are not interchangeable. Embodiments of the present invention contemplates various stereoisomers and mixtures thereof and includes “enantiomers”, which refers to two stereoisomers whose molecules are nonsuperimposeable mirror images of one another.

[0536] A “tautomer” refers to a proton shift from one atom of a molecule to another atom of the same molecule. Embodiments of the present invention include tautomers of any said compounds.

[0537] Lipid Nanoparticles

[0538] In an aspect, the invention relates to an LNP encapsulating a nucleic acid sequence encoding an engineered meganuclease that binds and cleaves a recognition sequence. Such LNPs typically include components selected from cationic lipids (“Lipid 1”), neutral lipids, charged WBD (US) 4897-9494-7440vl 59 Aty Docket No. P89339 2080WO (01275) lipids, steroids and / or polymer conjugated-lipids (“Lipid 2”). Without wishing to be bound by theory, it is thought that these lipid nanoparticles shield oligonucleotides from degradation in the serum and provide for effective delivery of oligonucleotides to cells in vitro and in vivo.

[0539] In one embodiment, the cationic lipids for use in the LNPs have the structure of Formula (I):

[0540] I or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein:

[0541] L1and L2are each independently -O(C=O)-, -(C=O)O-, -C(=O)-, -O-,

[0542] -S(O)x-, -S-S-, -C(=O)S-, -SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, -NRaC(=O)NRa-,

[0543] -OC(=O)NRa-, -NRaC(=O)O- or a direct bond;

[0544] G1is C1-C2 alkylene, -(C=O)-, -O(C=O)-, -SC(=O)-, -NRaC(=O)- or a direct bond; G2is -C(=O)- , -(C=O)O-, -C(=O)S-, -C(=O)NRa- or a direct bond;

[0545] G3is Ci-Ce alkylene;

[0546] Rais H or C1-C12 alkyl;

[0547] Rlaand Rlbare, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) Rlais H or C1-C12 alkyl, and Rlbtogether with the carbon atom to which it is bound is taken together with an adjacent Rlband the carbon atom to which it is bound to form a carbon-carbon double bond;

[0548] R2aand R2bare, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R2ais H or C1-C12 alkyl, and R2btogether with the carbon atom to which it is bound is taken together with an adjacent R2band the carbon atom to which it is bound to form a carbon-carbon double bond;

[0549] R3aand R3bare, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R3ais H or C1-C12 alkyl, and R3btogether with the carbon atom to which it is bound is taken together with an adjacent R3band the carbon atom to which it is bound to form a carbon-carbon double bond;

[0550] WBD (US) 4897-9494-7440vl 60 Aty Docket No. P89339 2080WO (01275) R4aand R4bare, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R4ais H or C1-C12 alkyl, and R4btogether with the carbon atom to which it is bound is taken together with an adjacent R4band the carbon atom to which it is bound to form a carbon-carbon double bond;

[0551] R5and R6are each independently H or methyl;

[0552] R7is C4-C20 alkyl;

[0553] R8and R9are each independently C1-C12 alkyl; or R8and R9, together with the nitrogen atom to which they are attached, form a 5, 6 or 7-membered heterocyclic ring; a, b, c and d are each independently an integer from 1 to 24; and x is 0, 1 or 2.

[0554] In some embodiments, L1and L2are each independently -O(C=O)-, -(C=O)O- or a direct bond. In other embodiments, G1and G2are each independently -(C=O)- or a direct bond. In some different embodiments, L1and L2are each independently - O(C=O)-, -(C=O)O- or a direct bond; and G1and G2are each independently -(C=O)- or a direct bond.

[0555] In some different embodiments, L1and L2are each independently -C(=O)-, -O-, -S(O)X-, -S-S-, -C(=O)S-, -SC(=O)-, -NRa-, -NRaC(=O)-, -C(=O)NRa-, -NRaC(=O)NRa, -OC(=O)NRa-, -NRaC(=O)O-, -NRaS(O)xNRa-, -NRaS(O)x- or -S(O)xNRa-.

[0556] In other of the foregoing embodiments, the cationic lipid has one of the following structures (IA) or (IB):

[0557] In some embodiments, the c cationic lipid has structure (IA). In other embodiments, the cationic lipid has structure (IB).

[0558] In any of the foregoing embodiments, one of L1or L2is -O(C=O)-. For example, in some embodiments each of L1and L2are -O(C=O)-.

[0559] WBD (US) 4897-9494-7440vl 61 Aty Docket No. P89339 2080WO (01275) In some different embodiments of any of the foregoing, one of L1or L2is -(C=O)O-. For example, in some embodiments each of L1and L2is -(C=O)O-.

[0560] In different embodiments, one of L1or L2is a direct bond. As used herein, a “direct bond” means the group (e.g., L1or L2) is absent. For example, in some embodiments each of L1and L2is a direct bond.

[0561] In other different embodiments of the foregoing, for at least one occurrence of Rlaand Rlb, Rlais H or C1-C12 alkyl, and Rlbtogether with the carbon atom to which it is bound is taken together with an adjacent Rlband the carbon atom to which it is bound to form a carbon-carbon double bond.

[0562] In still other different embodiments, for at least one occurrence of R4aand R4b, R4ais H or C1-C12 alkyl, and R4btogether with the carbon atom to which it is bound is taken together with an adjacent R4band the carbon atom to which it is bound to form a carbon-carbon double bond.

[0563] In more embodiments, for at least one occurrence of R2aand R2b, R2ais H or C1-C12 alkyl, and R2btogether with the carbon atom to which it is bound is taken together with an adjacent R2band the carbon atom to which it is bound to form a carbon-carbon double bond.

[0564] In other different embodiments of any of the foregoing, for at least one occurrence of R3aand R3b, R3ais H or C1-C12 alkyl, and R3btogether with the carbon atom to which it is bound is taken together with an adjacent R3band the carbon atom to which it is bound to form a carboncarbon double bond.

[0565] It is understood that “carbon-carbon” double bond refers to one of the following structures: wherein Rcand Rdare, at each occurrence, independently H or a substituent. For example, in some embodiments Rcand Rdare, at each occurrence, independently H, C1-C12 alkyl or cycloalkyl, for example H or C1-C12 alkyl.

[0566] In various other embodiments, the cationic lipid has one of the following structures (IC) or (ID):

[0567] WBD (US) 4897-9494-7440vl 62 Aty Docket No. P89339 2080WO (01275) wherein e, f, g and h are each independently an integer from 1 to 12.

[0568] In some embodiments, the cationic lipid has structure (IC). In other embodiments, the cationic lipid has structure (ID).

[0569] In various embodiments of the c cationic lipid of structures (IC) or (ID), e, f, g and h are each independently an integer from 4 to 10.

[0570] In certain embodiments of the foregoing, a, b, c and d are each independently an integer from 2 to 12 or an integer from 4 to 12. In other embodiments, a, b, c and d are each independently an integer from 8 to 12 or 5 to 9. In some certain embodiments, a is 0. In some embodiments, a is 1. In other embodiments, a is 2. In more embodiments, a is 3. In yet other embodiments, a is 4. In some embodiments, a is 5. In other embodiments, a is 6. In more embodiments, a is 7. In yet other embodiments, a is 8. In some embodiments, a is 9. In other embodiments, a is 10. In more embodiments, a is 11. In yet other embodiments, a is 12. In some embodiments, a is 13. In other embodiments, a is 14. In more embodiments, a is 15. In yet other embodiments, a is 16.

[0571] In some embodiments, b is 1. In other embodiments, b is 2. In more embodiments, b is 3. In yet other embodiments, b is 4. In some embodiments, b is 5. In other embodiments, b is 6. In more embodiments, b is 7. In yet other embodiments, b is 8. In some embodiments, b is 9. In other embodiments, b is 10. In more embodiments, b is 11. In yet other embodiments, b is 12. In some embodiments, b is 13. In other embodiments, b is 14. In more embodiments, b is 15. In yet other embodiments, b is 16.

[0572] WBD (US) 4897-9494-7440vl 63 Atty Docket No. P89339 2080WO (01275) In some embodiments, c is 1. In other embodiments, c is 2. In more embodiments, c is 3. In yet other embodiments, c is 4. In some embodiments, c is 5. In other embodiments, c is 6. In more embodiments, c is 7. In yet other embodiments, c is 8. In some embodiments, c is 9. In other embodiments, c is 10. In more embodiments, c is 11. In yet other embodiments, c is 12. In some embodiments, c is 13. In other embodiments, c is 14. In more embodiments, c is 15. In yet other embodiments, c is 16.

[0573] In some certain embodiments, d is 0. In some embodiments, d is 1. In other embodiments, d is 2. In more embodiments, d is 3. In yet other embodiments, d is 4. In some embodiments, d is 5. In other embodiments, d is 6. In more embodiments, d is 7. In yet other embodiments, d is 8. In some embodiments, d is 9. In other embodiments, d is 10. In more embodiments, d is 11. In yet other embodiments, d is 12. In some embodiments, d is 13. In other embodiments, d is 14. In more embodiments, d is 15. In yet other embodiments, d is 16.

[0574] In some embodiments, e is 1. In other embodiments, e is 2. In more embodiments, e is 3. In yet other embodiments, e is 4. In some embodiments, e is 5. In other embodiments, e is 6. In more embodiments, e is 7. In yet other embodiments, e is 8. In some embodiments, e is 9. In other embodiments, e is 10. In more embodiments, e is 11. In yet other embodiments, e is 12.

[0575] In some embodiments, f is 1. In other embodiments, f is 2. In more embodiments, f is 3. In yet other embodiments, f is 4. In some embodiments, f is 5. In other embodiments, f is 6. In more embodiments, f is 7. In yet other embodiments, f is 8. In some embodiments, f is 9. In other embodiments, f is 10. In more embodiments, f is 11. In yet other embodiments, f is 12.

[0576] In some embodiments, g is 1. In other embodiments, g is 2. In more embodiments, g is 3. In yet other embodiments, g is 4. In some embodiments, g is 5. In other embodiments, g is 6. In more embodiments, g is 7. In yet other embodiments, g is 8. In some embodiments, g is 9. In other embodiments, g is 10. In more embodiments, g is 11. In yet other embodiments, g is 12.

[0577] In some embodiments, h is 1. In other embodiments, e is 2. In more embodiments, h is 3. In yet other embodiments, h is 4. In some embodiments, e is 5. In other embodiments, h is 6. In more embodiments, h is 7. In yet other embodiments, h is 8. In some embodiments, h is 9. In other embodiments, h is 10. In more embodiments, h is 11. In yet other embodiments, h is 12.

[0578] In some other various embodiments, a and d are the same. In some other embodiments, b and c are the same. In some other specific embodiments and a and d are the same and b and c are the same.

[0579] The sum of a and b and the sum of c and d are factors which may be varied to obtain a lipid having the desired properties. In one embodiment, a and b are chosen such that their sum is an

[0580] WBD (US) 4897-9494-7440vl 64 Atty Docket No. P89339 2080WO (01275) integer ranging from 14 to 24. In other embodiments, c and d are chosen such that their sum is an integer ranging from 14 to 24. In further embodiment, the sum of a and b and the sum of c and d are the same. For example, in some embodiments the sum of a and b and the sum of c and d are both the same integer which may range from 14 to 24. In still more embodiments, a. b, c and d are selected such that the sum of a and b and the sum of c and d is 12 or greater.

[0581] The substituents at Rla, R2a, R3aand R4aare not particularly limited. In some embodiments, at least one of Rla, R2a, R3aand R4ais H. In certain embodiments Rla, R2a, R3aand R4aare H at each occurrence. In certain other embodiments at least one of Rla, R2a, R3aand R4ais C1-C12 alkyl. In certain other embodiments at least one of Rla, R2a, R3aand R4ais Ci-Cs alkyl. In certain other embodiments at least one of Rla, R2a, R3aand R4ais Ci-Ce alkyl. In some of the foregoing embodiments, the Ci-Cs alkyl is methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n- hexyl or n-octyl.

[0582] In certain embodiments of the foregoing, Rla, Rlb, R4aand R4bare C1-C12 alkyl at each occurrence.

[0583] In further embodiments of the foregoing, at least one of Rlb, R2b, R3band R4bis H or Rlb, R2b, R3band R4bare H at each occurrence.

[0584] In certain embodiments of the foregoing, Rlbtogether with the carbon atom to which it is bound is taken together with an adjacent Rlband the carbon atom to which it is bound to form a carbon-carbon double bond. In other embodiments of the foregoing R4btogether with the carbon atom to which it is bound is taken together with an adjacent R4band the carbon atom to which it is bound to form a carbon-carbon double bond.

[0585] The substituents at R5and R6are not particularly limited in the foregoing embodiments. In certain embodiments one of R5or R6is methyl. In other embodiments each of R5or R6is methyl.

[0586] The substituents at R7are not particularly limited in the foregoing embodiments. In certain embodiments R7is Ce-Ci6 alkyl. In some other embodiments, R7is C6-C9 alkyl. In some of these embodiments, R7is substituted with -(C=O)ORb,

[0587] -O(C=O)Rb, -C(=O)Rb, -ORb, -S(O)xRb, -S-SRb, -C(=O)SRb, -SC(=O)Rb, -NRaRb, -NRaC(=O)Rb, -C(=O)NRaRb, -NRaC(=O)NRaRb, -OC(=O)NRaRb, -NRaC(=O)ORb, -NRaS(O)xNRaRb, -NRaS(O)xRbor -S(O)xNRaRb, wherein: Rais H or C1-C12 alkyl; Rbis Ci- C15 alkyl; and x is 0, 1 or 2. For example, in some embodiments R7is substituted with -(C=O)ORbor -O(C=O)Rb.

[0588] In various of the foregoing embodiments, Rbis branched C1-C15 alkyl. For example, in some embodiments Rbhas one of the following structures:

[0589] WBD (US) 4897-9494-7440vl 65 Aty Docket No. P89339 2080WO (01275)

[0590] In certain other of the foregoing embodiments, one of R8or R9is methyl. In other embodiments, both R8and R9are methyl.

[0591] In some different embodiments, R8and R9, together with the nitrogen atom to which they are attached, form a 5, 6 or 7-membered heterocyclic ring. In some embodiments of the foregoing, R8and R9, together with the nitrogen atom to which they are attached, form a 5-membered heterocyclic ring, for example a pyrrolidinyl ring. In some different embodiments of the foregoing, R8and R9, together with the nitrogen atom to which they are attached, form a 6-membered heterocyclic ring, for example a piperazinyl ring.

[0592] In still other embodiments of the foregoing cationic lipid, G3is C2-C4 alkylene, for example C3 alkylene.

[0593] In various different embodiments, the cationic lipid has one of the structures set forth in

[0594] Table 2 below.

[0595] Table 2

[0596] WBD (US) 4897-9494-7440vl 66 Aty Docket No. P89339 2080WO (01275)

[0597]

[0598] WBD (US) 4897-9494-7440vl 67 Atty Docket No. P89339 2080WO (01275)

[0599]

[0600] WBD (US) 4897-9494-7440vl 68 Atty Docket No. P89339 2080WO (01275)

[0601]

[0602] WBD (US) 4897-9494-7440vl 69 Atty Docket No. P89339 2080WO (01275)

[0603]

[0604] WBD (US) 4897-9494-7440vl Atty Docket No. P89339 2080WO (01275)

[0605]

[0606] WBD (US) 4897-9494-7440vl 71 Atty Docket No. P89339 2080WO (01275)

[0607]

[0608] The cationic lipids can be prepared according to procedures known in the art, including those set forth in WO 2017 / 004143, which is incorporated herein by reference.

[0609] In some embodiments, the LNPs include a neutral lipid. In some embodiments, the neutral lipid is selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM. In some embodiments, the neutral lipid is DSPC. In various embodiments, the molar ratio of the cationic lipid to the neutral lipid ranges from about 2: 1 to about 8: 1.

[0610] WBD (US) 4897-9494-7440vl 72 Atty Docket No. P89339 2080WO (01275) In various embodiments, the LNPs further comprise a steroid or steroid analogue. In certain embodiments, the steroid or steroid analogue is cholesterol. In some of these embodiments, the molar ratio of the cationic lipid to cholesterol ranges from about 2: 1 to 1 : 1.

[0611] In various embodiments, the LNPs include a polymer conjugated lipid. In some embodiments, the polymer conjugated lipid is a pegylated lipid. For example, some embodiments include a pegylated diacylglycerol (PEG-DAG) such as l-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG), a pegylated phosphatidylethanoloamine (PEG-PE), a PEG succinate diacylglycerol (PEG-S-DAG) such as 4-0- (2’ ,3 ’ -di(tetradecanoyloxy)propyl- 1 -0-(o -methoxy(polyethoxy)ethyl)butanedioate (PEG-S- DMG), a pegylated ceramide (PEG-cer), or a PEG dialkoxypropylcarbamate such as o- methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxy)propyl)carbamate or 2,3- di(tetradecanoxy)propyl-N-(o-methoxy(polyethoxy)ethyl)carbamate. In various embodiments, the molar ratio of the cationic lipid to the pegylated lipid ranges from about 100: 1 to about 25: 1.

[0612] In some embodiments, the LNPs comprise a pegylated lipid having the following structure (II): or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein:

[0613] R10and R11are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds; and z has a mean value ranging from 30 to 60.

[0614] In some embodiments, R10and R11are each independently straight, saturated alkyl chains containing from 12 to 16 carbon atoms. In other embodiments, the average z is about 45.

[0615] In some of the foregoing embodiments of the pegylated lipid (II), R10and R11are not both n-octadecyl when z is 42. In some embodiments, R10and R11are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10 to 18 carbon atoms. In some embodiments, R10and R11are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 12 to 16 carbon atoms. In some embodiments, R10and R11are each independently a straight or branched, saturated or unsaturated alkyl chain containing 12 carbon

[0616] WBD (US) 4897-9494-7440vl 73 Aty Docket No. P89339 2080WO (01275) atoms. In some embodiments, R10and R11are each independently a straight or branched, saturated or unsaturated alkyl chain containing 14 carbon atoms. In other embodiments, R10and R11are each independently a straight or branched, saturated or unsaturated alkyl chain containing 16 carbon atoms. In still more embodiments, R10and R11are each independently a straight or branched, saturated or unsaturated alkyl chain containing 18 carbon atoms. In still other embodiments, R10is a straight or branched, saturated or unsaturated alkyl chain containing 12 carbon atoms and R11is a straight or branched, saturated or unsaturated alkyl chain containing 14 carbon atoms.

[0617] In various embodiments, z spans a range that is selected such that the PEG portion of (II) has an average molecular weight of about 400 to about 6000 g / mol. In some embodiments, the average z is about 45.

[0618] In other embodiments, the pegylated lipid has one of the following structures: wherein n spans a range such that the average molecular weight of the pegylated lipid is about 2500 g / mol.

[0619] The pegylated lipids can be prepared according to procedures known in the art, including those set forth in WO 2015 / 199952, which is incorporated herein by reference.

[0620] In certain embodiments, the LNP provided herein comprises: a) Bis(2-butyloctyl) 10-(N-(3- (pyrrolidin-l-yl)propyl)nonanamido)nonadecanedioate (“Lipid 1”; Compound of Structure 15 in Table 2); b) 2- [2-(o -methoxy (polyethyleneglycol2000) ethoxy]-N,N-ditetradecylacetamide (“Lipid 2”; Compound of Structure Ila); c) l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); and d) cholesterol. Lipid 1 is an ionizable lipid. Lipid 2 is a structural lipid (non-ionizable lipid). 1,2- distearoyl-sn-glycero-3-phosphocholine (DSPC) is a PEG lipid. In certain embodiments, the LNP formulation provided herein enhances stability and delivery of the polynucleotide to the target cells.

[0621] WBD (US) 4897-9494-7440vl 74 Aty Docket No. P89339 2080WO (01275) In specific embodiments, the LNP provided herein has the molar ratio of Lipid 1 : Lipid 2 : DSPC : cholesterol of about 47.5:2.5: 10:40, an LNP particle size of between about 55-75 nm, and an N:P ratio of about 6: 1. An “N:P ratio” refers to the ratio between the amine group (N) of the ionizable lipid of the LNP and the phosphate group (P) of the cargo (e.g., polynucleotide encoding a meganuclease). An example LNP formulation of the present disclosure is listed in Table 3 below.

[0622] The LNP formulations provided herein can contain Lipid 1, Lipid 2, DSPC, and / or cholesterol. In some embodiments, the molar concentration of Lipid 1 in the LNP is between about 40% to about 55% of the total molar lipid concentration. For example, the molar concentration of Lipid 1 in the LNP can be about 40-55%, 40-45%, 40-50%, 40-45%, 45-50%, 45-55%, or 50-55%; about 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, or 55% of the total molar lipid concentration. In specific embodiments, the molar concentration of Lipid 1 in the LNP is 45% to about 50%, or about 47.5% of the total molar lipid concentration.

[0623] In some embodiments, the molar concentration of Lipid 2 in the LNP is between about 0.5% to about 4.5% of the total molar lipid concentration. For example, the molar concentration of Lipid 2 in the LNP can be about 0.5-4.5%, 0.5-4.0%, 0.5-3.5%, 0.5-3.0%, 0.5-2.5%, 0.5-2.0%, 0.5-1.5%, 0.5-1.0%, 1.0-4.0%, 1.0-3.5%, 1.0-3.0%, LO-2.5%, L0-2.0%, 1.0-1.5%, 1.5-4.0%, 1.5-3.5%, 1.5- 3.0%, 1.5-2.5%, 1.5-2.0%, 2.0-4.0%, 2.0-3.5%, 2.0-3.0%, 2.0-2.5%, 2.5-4.0%, 2.5-3.5%, 2.5-3.0%, 3.0-4.0%, 3.0-3.5%, or 3.5-4.0%; about 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, or 4.5% of the total molar lipid concentration. In specific embodiments, the molar concentration of Lipid 2 in the LNP is between about 1.5% to about 3.5%, or about 2.5% of the total molar lipid concentration.

[0624] In some embodiments, the molar concentration of DSPC in the LNP is between about 5.0% to about 15.0% of the total molar lipid concentration. For example, the molar concentration of DSPC in the LNP can be about 5.0-15.0%, 5.0-12.5%, 5.0-10.0%, 5.0-7.5%, 7.5-15.0%, 7.5-12.5%, 7.5-10.0%, 10.0-15.0%, 10.0-12.5%, or 12.5-15.0%; or about 5.0%, 7.5%, 10.0%, 12.5%, or 15.0% of the total molar lipid concentration. In specific embodiments, the molar concentration of DSPC in the LNP is between about 7.5% to about 12.5%, or about 10% of the total molar lipid concentration.

[0625] In some embodiments, the molar concentration of cholesterol in the LNP is between about 30% to about 50% of the total molar lipid concentration. For example, the molar concentration of cholesterol in the LNP can be about 30-50%, 30-45%, 30-42.5%, 30-40%, 30-37.5%, 30-35%, 35- 50%, 35-45%, 35-42.5%, 35-40%, 35-37.5%, 37.5-50%, 37.5-45%, 37.5-42.5%, 37.5-40%, 40- 50%, 40-45%, 40-42.5%, 42.5-50%, 42.5-45%, or 45-50%; about 30%, 35%, 37.5%, 40%, 42.5%, 45%, or 50% of the total molar lipid concentration. In specific embodiments, the molar

[0626] WBD (US) 4897-9494-7440vl 75 Atty Docket No. P89339 2080WO (01275) concentration of cholesterol in the LNP is between about 37.5% to about 42.5%, or about 40% of the total molar lipid concentration.

[0627] In specific embodiments, the molar ratio of Lipid 1 : Lipid 2 : DSPC : cholesterol in the LNP is about 47.5:2.5: 10:40.

[0628] For example the LNP provided herein can contain about 0.5-3.0 mg polynucleotide (e.g., about 0.5-3.0, 0.5-2.0, 0.5-1.2, 0.5-1.1, 0.5-1.0, 0.5-0.95, 0.5-0.8, 0.8-3.0, 0.8-2.0, 0.8-1.2, 0.8-1.1, 0.8-1.0, 0.8-0.95, 0.95-3.0, 0.95-2.0, 0.95-1.2, 0.95-1.1, 0.95-1.0, 1.0-3.0, 1.0-2.0, 1.0-1.2, 1.0-1.1,

[0629] 1.1-3.0, 1.1-2.0, 1.1-1.2, 1.2-3.0, 1.2-2.0, or 2.0-3.0 mg, or about 0.5, 0.8, 0.95, 1.0, 1.1, 1.2, 1.5, 2.0, 2.5, or 3.0 mg polynucleotide), about 3.5-9.0 mg / ml cholesterol (e.g., about 3.5-9.0, 3.5-8.0,

[0630] 3.5-7.0, 3.5-6.6, 3.5-6.4, 3.5-6.0, 3.5-5.5, 3.5-5.0, 3.5-4.0, 4.0-9.0, 4.0-8.0, 4.0-7.0, 4.0-6.6, 4.0-6.4,

[0631] 4.0-6.0, 4.0-5.5, 4.0-5.0, 5.0-9.0, 5.0-8.0, 5.0-7.0, 5.0-6.6, 5.0-6.4, 5.0-6.0, 5.0-5.5, 5.5-9.0, 5.5-8.0,

[0632] 5.5-7.0, 5.5-6.6, 5.5-6.4, 5.5-6.0, 6.0-9.0, 6.0-8.0, 6.0-7.0, 6.0-6.6, 6.0-6.4, 6.4-9.0. 6.4-8.0, 6.4-7.0,

[0633] 6.4-6.6, 6.6-9.0, 6.6-8.0, 6.6-7.0, 7.0-9.0, 7.0-8.0, or 8.0-9.0 mg / ml, or about 3.5, 4.0. 5.0, 5.5, 6.0,

[0634] 6.4, 6.6, 7.0. 8.0, or 9.0 mg / ml cholesterol), about 10-25 mg / ml Lipid 1 (e.g., about 10-25, 10-20, 10-19.2, 10-17.4, 10-18.5,10-15, 15-25, 15-20, 15-19.2, 15-17.4, 15-18.5, 15.7-25, 15.7-20, 15.7- 19.2, 15.7-18.5, 15.7-17.4, 17.4-25, 17.4-20, 17.4-18.5, 18.5-25, 18.5-20, 20-25, or about 10, 15, 15.7, 17.4, 18.5, 19.2, 20, or 25 mg / ml Lipid 1), about 1.5-4.0 mg / ml Lipid 2 (e.g., about 1.5-4.0,

[0635] 1.5-3.5, 1.5-3.0, 1.5-2.8, 1.5-2.6, 1.5-2.5, 1.5-2.0, 2.0-4.0, 2.0-3.5, 2.0-3.0, 2.0-2.8, 2.0-2.6, 2.0-2.5,

[0636] 2.2-4.0, 2.2-3.5, 2.2-3.0, 2.2-2.8, 2.2-2.6, 2.5-4.0, 2.5-3.5, 2.5-3.0, 2.5-2.8, 2.5-2.6, 2.6-4.0, 2.6-3.5,

[0637] 2.6-3.0, 2.6-2.8, 2.8-4.0, 2.8-3.5, 2.8-3.0, 3.0-4.0, 3.0-3.5, or 3.5-4.0 mg / ml Lipid 2), and about 1.5-

[0638] 4.5 mg / ml DSPC (e.g., about 1.5-4.5, 1.5-4.0, 1.5-3.5, 1.5-3.4, 1.5-3.3, 1.5-3.0, 1.5-2.5, 1.5-2.0, 2.0-4.5, 2.0-4.0, 2.0-3.5, 2.0-3.4, 2.0-3.3, 2.0-3.0, 2.0-2.5, 2.5-4.5, 2.5-4.0, 2.5-3.5, 2.5-3.4, 2.5-3.3,

[0639] 2.5-3.0, 3.0-4.5, 3.0-4.0, 3.0-3.5, 3.0-3.4, 3.0-3.3, 3.3-4.5, 3.3-4.0, 3.3-3.5, 3.3-3.4, 3.4-4.5, 3.4-4.0,

[0640] 3.4-3.5, 3.5-4.5, 3.5-4.0, or 4.5-5.0 mg / ml, or about 1.5, 2.0, 2.5, 3.0, 3.3, 3.4, 3.5, 4.0, 4.5, or 5.0 mg / ml DSPC).

[0641] In specific embodiments, the LNP provided herein contains about 0.8-1.2 mg / ml polynucleotide (e.g., about 1.0 mg polynucleotide), about 5.5-7.0 mg / ml cholesterol (e.g., about

[0642] 6.4-6.6 mg / ml cholesterol), about 15.7-19.2 mg / ml Lipid 1 (e.g., about 17.4-18.5 mg / ml Lipid 1), about 2.2-3.0 mg / ml Lipid 2 (about 2.6-2.8 mg / ml Lipid 2), and about 2.5-3.4 mg / ml DSPC (e.g., about 3.3-3.4 mg / ml DSPC).

[0643] The LNP of the present disclosure has any particle size that is suitable for efficient delivery of cargo (e.g., polynucleotide encoding the meganuclease of the present disclosure) to target cells, e.g., in vitro or in vivo. In embodiments, the LNP has a particle size of between about 50-80 nm. For example, the LNP can have a particle size of about 50-80 nm, 50-75 nm, 50-73 nm, 50-70 nm, 50-65 nm, 50-62 nm, 50-60 nm, 50-55 nm, 55-80 nm, 55-75 nm, 55-73 nm, 55-70 nm, 55-65 nm, WBD (US) 4897-9494-7440vl 76 Aty Docket No. P89339 2080WO (01275) 55-62 nm, 55-60 nm, 60-80 nm, 60-75 nm, 60-73 nm, 60-70 nm, 60-65 nm, 60-62 nm, 62-80 nm, 62-75 nm, 62-73 nm, 62-70 nm, 62-65 nm, 65-80 nm, 65-75 nm, 65-73 nm, 65-70 nm, 70-80 nm, 70-75 nm, 70-73 nm, 73-80 nm, 73-75 nm, or 75-80 nm; or about 50 nm, 55 nm, 60 nm, 62 nm, 65 nm, 70 nm, 73 nm, 75 nm, or 80 nm. In specific embodiments, the LNP has a particle size of between about 55-75 nm, between about 62-73 nm, or about 65 nm.

[0644] The LNP of the present disclosure has any N:P ratio that is suitable for efficient delivery of cargo (e.g., polynucleotide encoding the meganuclease of the present disclosure) to target cells, e.g., in vitro or in vivo. For example, the LNP can have an N:P ratio of about 2: 1 -9: 1, 2:1 -8: 1, 2: 1- 7: 1, 2: 1-6: 1, 2.1-5: 1, 2: 1-4: 1, 2: 1-3: 1, 3: 1-9: 1, 3: 1-8: 1, 3: 1-7: 1, 3: 1-6: 1, 3: 1-5: 1, 3: 1-4: 1, 4: 1-9: 1, 4: 1-8: 1, 4: 1-7: 1, 4: 1-6: 1, 4: 1-5: 1, 5: 1-9: 1, 5: 1-8: 1, 5: 1-7: 1, 5: 1-6: 1, 6: 1-9: 1, 6: 1-8: 1, 6: 1-7: 1, 7: 1- 9: 1, 7: 1-8: 1, or 8: 1-9: 1, or about 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, or 9: 1. In specific embodiments, the LNP has an N:P ratio of between about 4 : 1 -8 : 1 , about 5 : 1 -7 : 1 , or about 6: 1.

[0645] Meganuclease Sequences in the polynucleotide

[0646] The LNP of the present disclosure can contain a polynucleotide that comprises a nucleic acid sequence encoding an engineered meganuclease that binds and cleaves a recognition sequence comprising SEQ ID NO: 3 in a polymerase (pol) gene of a hepatitis B virus (HBV) genome or HBV genome fragment. Any engineered meganucleases provided herein, e.g., any HBV 11-12 meganucleases provided herein, e.g., any HBV 11-12L.1090 E80 meganuclease or variants thereof provided herein, may be used in the LNP of the present disclosure. For example, the engineered meganuclease can comprise an amino acid sequence having at least 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 5 or SEQ ID NO: 6. In some embodiments, the engineered meganuclease comprises an amino acid sequence set forth in SEQ ID NO: 5 or SEQ ID NO: 6.

[0647] In some embodiments, the nucleic acid sequence encoding the engineered meganuclease comprises a sequence having at least 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 7 or SEQ ID NO: 8. In some embodiments, the nucleic acid (mRNA) sequence encoding the engineered meganuclease comprises a sequence set forth in SEQ ID NO: 7 or SEQ ID NO: 8.

[0648] In some embodiments, the nucleic acid sequence encoding the engineered meganuclease comprises a sequence having at least 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 9 or SEQ ID NO: 10. In some embodiments, the nucleic acid sequence encoding the engineered meganuclease comprises a sequence set forth in SEQ ID NO: 9 or SEQ ID NO: 10.

[0649] WBD (US) 4897-9494-7440vl 77 Atty Docket No. P89339 2080WO (01275) The engineered meganuclease may further comprise one or more NLSs. For example, the engineered meganuclease can comprise a 5’ NLS at its N-terminus. In some embodiments, the 5’ NLS comprises an amino acid sequence having at least 80% sequence identity (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 11. In some embodiments, the 5’ NLS comprises an amino acid sequence set forth in SEQ ID NO: 11.

[0650] The engineered meganuclease can comprise a 3’ NLS at its C-terminus. In some embodiments, the 3’ NLS comprises an amino acid sequence having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 12. In some embodiments, the 3’ NLS comprises an amino acid sequence set forth in SEQ ID NO: 12.

[0651] In some embodiments, the engineered meganuclease comprises a 5’ NLS at its N-terminus comprising an amino acid sequence having at least 80% sequence identity (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 11, and a 3’ NLS at its C-terminus comprising an amino acid sequence having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 12. For example, the engineered meganuclease can comprise a 5’ NLS at its N-terminus comprising an amino acid sequence set forth in SEQ ID NO: 11, and a 3’ NLS at its C-terminus comprising an amino acid sequence set forth in SEQ ID NO: 12.

[0652] Additional Components of the Polynucleotide

[0653] The polynucleotide contained in the LNP of the present disclosure may include components in addition to the nucleic acid sequence encoding an engineered meganuclease that binds and cleaves a recognition sequence comprising SEQ ID NO: 3 in a polymerase (pol) gene of a hepatitis B virus (HBV) genome or HBV genome fragment (e.g., any HBV 11-12 meganucleases provided herein, e.g., any HBV 11-12L 1090 E80 meganuclease or variants thereof). Such additional components may facilitate stability and / or delivery of the polynucleotide to the target cell, e.g., in vivo or in vitro.

[0654] For example, the polynucleotide can comprise a 5’ UTR sequence. The 5’ UTR sequence can comprise a nucleic acid sequence having at least 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 13. In some embodiments, the 5’ UTR sequence comprises a nucleic acid sequence set forth in SEQ ID NO: 13. The 5’ UTR sequence can also comprise a nucleic acid sequence having at least 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity)

[0655] WBD (US) 4897-9494-7440vl 78 Atty Docket No. P89339 2080WO (01275) to SEQ ID NO: 14. In some embodiments, the 5’ UTR sequence comprises a nucleic acid sequence set forth in SEQ ID NO: 14.

[0656] For example, the polynucleotide can comprise a 3’ UTR sequence. The 3’ UTR sequence can comprise a nucleic acid sequence having at least 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 15. In some embodiments, the 3’ UTR sequence comprises a nucleic acid sequence set forth in SEQ ID NO: 15. The 3’ UTR sequence can also comprise a nucleic acid sequence having at least 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 16. In some embodiments, the 3’ UTR sequence comprises a nucleic acid sequence set forth in SEQ ID NO: 16.

[0657] In some embodiments, the polynucleotide comprises a 5’ UTR sequence that comprises a nucleic acid sequence having at least 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 13, and a 3’ UTR sequence that comprises a nucleic acid sequence having at least 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 15. For example, the polynucleotide can comprise a 5’ UTR sequence that comprises a nucleic acid sequence set forth in SEQ ID NO: 13, and a 3’ UTR sequence that comprises a nucleic acid sequence set forth in SEQ ID NO: 15.

[0658] In some embodiments, the polynucleotide comprises a 5’ UTR sequence that comprises a nucleic acid sequence having at least 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 14, and a 3’ UTR sequence that comprises a nucleic acid sequence having at least 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 16. For example, the polynucleotide can comprise a 5’ UTR sequence that comprises a nucleic acid sequence set forth in SEQ ID NO: 14, and a 3’ UTR sequence that comprises a nucleic acid sequence set forth in SEQ ID NO: 16.

[0659] The polynucleotide can also comprise a Kozak sequence. A Kozak sequence, also referred to as a Kozal consensus or Kozak consensus sequence, is a nucleic acid motif that functions as the protein translation initiation site in most eukaryotic mRNA transcripts. The Kozak sequence ensures that a protein is correctly translated from the genetic message, mediating ribosome assembly and translation initiation. For example, the polynucleotide can comprise a Kozak sequence comprising a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 17. In some embodiments, the polynucleotide comprises a Kozak sequence comprising a nucleic acid sequence set forth in SEQ ID NO: 17. The polynucleotide can comprise a Kozak sequence comprising a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 18. In WBD (US) 4897-9494-7440vl 79 Aty Docket No. P89339 2080WO (01275) some embodiments, the polynucleotide comprises a Kozak sequence comprising a nucleic acid sequence set forth in SEQ ID NO: 18.

[0660] The polynucleotide can also comprise a polyA sequence. In some embodiments, the polyA sequence comprises a nucleic acid sequence having at least 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 19. In some embodiments, the polyA sequence comprises a nucleic acid sequence set forth in SEQ ID NO: 19.

[0661] In some embodiments, the polynucleotide comprises a nucleic acid sequence having at least 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 20 or SEQ ID NO: 21. In some embodiments, the polynucleotide comprises a nucleic acid sequence set forth SEQ ID NO: 20 or SEQ ID NO: 21.

[0662] In some embodiments, the polynucleotide comprises a 5’ UTR from the albumin gene (ALB), an ORF containing the coding sequence of the meganuclease, sequences encoding SV40 NLS sequences at the 5’ and 3’ extremes of the ORF, a 3’ UTR from the small nuclear ribonucleoprotein polypeptides B and Bl genes (SNRPB) and a poly A tail. For example, the polynucleotide can be a DNA and can comprise a nucleic acid sequence having at least 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 22 or SEQ ID NO: 23. In some embodiments, the polynucleotide comprises a nucleic acid sequence set forth SEQ ID NO: 22 or SEQ ID NO: 23.

[0663] In some embodiments, the polynucleotide is a messenger RNA (mRNA). When the polynucleotide is a mRNA, the mRNA can comprise a 5’ cap structure. In some embodiments, the 5’ cap structure comprises (m7G(5’)ppp(5’)(2’OMeA)pG), e.g., a CleanCap® Cap-1 structure. One or more, or all uridine bases in the mRNA can be substituted with a modified nucleotide such as, for example, pseudouridine, Nl-methyl-pseudouridine, 5-methoxyuridine, or 2-thiouridine. One or more, or all cytosine bases in the mRNA can be substituted with a modified nucleotide such as, for example, 5-methylcytidine, 2'-O-methylcytidine, or N4-acetyl-cytosine. One or more, or all adenine bases in the mRNA can be substituted with a modified nucleotide such as, for example, N6- methyladenosine or Nl-methyl-adenine.

[0664] The mRNA can comprise a 5’ cap structure, a 5’ UTR from the albumin gene (ALB), an ORF containing the coding sequence of the meganuclease, sequences encoding SV40 NLS sequences at the 5’ and 3’ extremes of the ORF, a 3’ UTR from the small nuclear ribonucleoprotein polypeptides B and Bl genes (SNRPB), and a poly-Atail, as shown in Figure 2. In specific embodiments, the mRNA sequence encoding the HBV 11-12L.1090 E80 meganuclease provided herein includes a Cap-1 structure at the 5’ terminus (m7G(5’)ppp(5’)(2’OMeA)pG), a 5’ UTR from the albumin gene (ALB) (e.g., SEQ ID NO: 13), an ORF containing the coding sequence of the WBD (US) 4897-9494-7440vl 80 Aty Docket No. P89339 2080WO (01275) meganuclease (e.g., SEQ ID NO: 7 or 8), sequences encoding SV40 NLS sequences at the 5’ and 3’ extremes of the ORF (e.g., SEQ ID NOs: 11 and 12 at the 5’ and 3’ ends, respectively), a 3’ UTR from the small nuclear ribonucleoprotein polypeptides B and Bl genes (SNRPB) (e.g., SEQ ID NO: 15), and finally a poly-Atail (e.g., SEQ ID NO: 19), as schematically depicted in Figure 2. In some embodiments, the mRNAthat is a polynucleotide provided herein comprises a nucleic acid sequence having at least 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 20 or SEQ ID NO: 21. In some embodiments, the mRNA comprises a nucleic acid sequence set forth SEQ ID NO: 20 or SEQ ID NO: 21.

[0665] In specific embodiments, the engineered meganuclease provided herein is encoded by an mRNA sequence set forth in SEQ ID NO: 20 or 21, or a DNA sequence set forth in SEQ ID NO: 22 or 23.

[0666] 2,5 Pharmaceutical Compositions

[0667] A pharmaceutical composition is provided herein that comprises a pharmaceutically acceptable carrier and an engineered meganuclease described herein, or a pharmaceutically acceptable carrier and a polynucleotide described herein that comprises a nucleic acid sequence encoding an engineered meganuclease described herein. Such polynucleotides can be, for example, mRNA as described herein. In some such examples, the polynucleotide in the pharmaceutical composition can be comprised by a lipid nanoparticle. Pharmaceutical compositions of the disclosure can be useful for treating a subject having HBV or an HBV-associated condition (e.g., CHB, HCC), reducing the level or proliferation of HBV, or reducing at least one symptom of a disease associated with HBV infection (e.g., CHB, HCC).

[0668] Pharmaceutical compositions can be designed or selected according to the genotype of the target HBV strain. As described in detail herein, meganucleases described herein have been engineered to recognize and cleave a recognition sequence in specific genotypes of HBV. The presently disclosed HBV 11-12 meganucleases (e.g., SEQ ID NOs: 5 and 6 or variants thereof), recognize and cleave the HBV 11-12 recognition sequence that is at least found in the genome of HBV genotypes A, B, C, D, E, F, and G. In some embodiments, the pharmaceutical compositions described herein can be administered to a subject having any genotype of HBV comprising a recognition sequence set forth in SEQ ID NO: 3.

[0669] In certain embodiments, provided is a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a plurality of LNP provided herein. The LNP comprises a polynucleotide encoding an engineered meganuclease that binds and cleaves a recognition sequence

[0670] WBD (US) 4897-9494-7440vl 81 Atty Docket No. P89339 2080WO (01275) comprising SEQ ID NO: 3 in a polymerase (pol) gene of a hepatitis B virus (HBV) genome or HBV genome fragment, and an LNP component containing ionizable lipid, non-ionizable lipid, PEG lipid, and cholesterol as provided in the present disclosure.

[0671] The pharmaceutical composition can contain about 10-25 mg / ml Lipid 1. For example, the pharmaceutical composition can contain about 10-25, 10-20, 10-19.2, 10-17.4, 10-18.5,10-15, 15- 25, 15-20, 15-19.2, 15-17.4, 15-18.5, 15.7-25, 15.7-20, 15.7-19.2, 15.7-18.5, 15.7-17.4, 17.4-25,

[0672] 17.4-20, 17.4-18.5, 18.5-25, 18.5-20, 20-25, or about 10, 15, 15.7, 17.4, 18.5, 19.2, 20, or 25 mg / ml Lipid 1. In specific embodiments, the pharmaceutical composition comprises between about 15.7- 19.2 mg / mL or about 17.4-18.5 mg / mL of Lipid 1.

[0673] The pharmaceutical composition can contain about 1.5-4.0 mg / ml Lipid 2. For example, the pharmaceutical composition can contain about 1.5-4.0, 1.5-3.5, 1.5-3.0, 1.5-2.8, 1.5-2.6, 1.5-2.5,

[0674] 1.5-2.0, 2.0-4.0, 2.0-3.5, 2.0-3.0, 2.0-2.8, 2.0-2.6, 2.0-2.5, 2.2-4.0, 2.2-3.5, 2.2-3.0, 2.2-2.8, 2.2-2.6,

[0675] 2.5-4.0, 2.5-3.5, 2.5-3.0, 2.5-2.8, 2.5-2.6, 2.6-4.0, 2.6-3.5, 2.6-3.0, 2.6-2.8, 2.8-4.0, 2.8-3.5, 2.8-3.0, 3.0-4.0, 3.0-3.5, or 3.5-4.0 mg / ml, or about 1.5, 2.0. 2.2, 2.5, 2.6, 2.8, 3.0. 3.5, or 4.0 mg / ml Lipid 2. In specific embodiments, the pharmaceutical composition comprises between about 2.2-3.0 mg / mL or about 2.6-2.8 mg / mL of Lipid 2.

[0676] The pharmaceutical composition can contain about 1.5-4.5 mg / ml DSPC. For example, the pharmaceutical composition can contain about 1.5-4.5, 1.5-4.0, 1.5-3.5, 1.5-3.4, 1.5-3.3, 1.5-3.0,

[0677] 1.5-2.5, 1.5-2.0, 2.0-4.5, 2.0-4.0, 2.0-3.5, 2.0-3.4, 2.0-3.3, 2.0-3.0, 2.0-2.5, 2.5-4.5, 2.5-4.0, 2.5-3.5,

[0678] 2.5-3.4, 2.5-3.3, 2.5-3.0, 3.0-4.5, 3.0-4.0, 3.0-3.5, 3.0-3.4, 3.0-3.3, 3.3-4.5, 3.3-4.0, 3.3-3.5, 3.3-3.4,

[0679] 3.4-4.5, 3.4-4.0, 3.4-3.5, 3.5-4.5, 3.5-4.0, or 4.5-5.0 mg / ml, or about 1.5, 2.0, 2.5, 3.0, 3.3, 3.4, 3.5, 4.0, 4.5, or 5.0 mg / ml DSPC. In specific embodiments, the pharmaceutical composition comprises between about 2.5-3.4 mg / mL or about 3.3. -3.4 mg / mL of DSPC.

[0680] The pharmaceutical composition can contain about 3.5-9.0 mg / ml cholesterol. For example, the pharmaceutical composition can contain about 3.5-9.0, 3.5-8.0, 3.5-7.0, 3.5-6.6, 3.5-6.4, 3.5- 6.0, 3.5-5.5, 3.5-5.0, 3.5-4.0, 4.0-9.0, 4.0-8.0, 4.0-7.0, 4.0-6.6, 4.0-6.4, 4.0-6.0, 4.0-5.5, 4.0-5.0, 5.0-

[0681] 9.0, 5.0-8.0, 5.0-7.0, 5.0-6.6, 5.0-6.4, 5.0-6.0, 5.0-5.5, 5.5-9.0, 5.5-8.0, 5.5-7.0, 5.5-6.6, 5.5-6.4, 5.5-

[0682] 6.0, 6.0-9.0, 6.0-8.0, 6.0-7.0, 6.0-6.6, 6.0-6.4, 6.4-9.0. 6.4-8.0, 6.4-7.0, 6.4-6.6, 6.6-9.0, 6.6-8.0, 6.6-

[0683] 7.0, 7.0-9.0, 7.0-8.0, or 8.0-9.0 mg / ml, or about 3.5, 4.0. 5.0, 5.5, 6.0, 6.4, 6.6, 7.0. 8.0, or 9.0 mg / ml cholesterol). In specific embodiments, the pharmaceutical composition comprises between about 5.5-7.0 mg / mL or about 6.4-6.6 mg / mL of cholesterol.

[0684] The pharmaceutical composition can contain about 0.5-3.0 mg polynucleotide (e.g., about 0.5-3.0, 0.5-2.0, 0.5-1.2, 0.5-1.1, 0.5-1.0, 0.5-0.95, 0.5-0.8, 0.8-3.0, 0.8-2.0, 0.8-1.2, 0.8-1.1, 0.8-1.0, 0.8- 0.95, 0.95-3.0, 0.95-2.0, 0.95-1.2, 0.95-1.1, 0.95-1.0, 1.0-3.0, 1.0-2.0, 1.0-1.2, 1.0-1.1, 1.1-3.0, 1.1- 2.0, 1.1-1.2, 1.2-3.0, 1.2-2.0, or 2.0-3.0 mg, or about 0.5, 0.8, 0.95, 1.0, 1.1, 1.2, 1.5, 2.0, 2.5, or 3.0 WBD (US) 4897-9494-7440vl 82 Aty Docket No. P89339 2080WO (01275) mg polynucleotide). In specific embodiments, the pharmaceutical composition comprises between about 0.8-1.2 mg / mL, about 0.95-1.1 mg / mL, or about 1.0 mg / mL of the polynucleotide.

[0685] In some embodiments, the pharmaceutical composition comprises: a) between about 15.7- 19.2 mg / mL of Lipid 1; b) between about 2.2-3.0 mg / mL of Lipid 2; c) between about 2.5-3.4 mg / mL of DSPC; d) between about 5.5-7.0 mg / mL of cholesterol; and e) between about 0.8-1.2 mg / mL of the polynucleotide.

[0686] The LNPs can be formulated in any suitable solution for delivery of the pharmaceutical composition to target cells, e.g., in vitro or in vivo. For example, the LNPs can be formulated in as a buffered solution or an unbuffered solution. For example, the plurality of LNPs are formulated in phosphate-buffered saline (e.g., Dulbecco’s phosphate-buffered saline) and sucrose. The sucrose in the formulation can be at a concentration of about 250-350 mM, such as about 250-350, 250-300, 300-350, or about 250, 275, 300, 325, or 350 mM. In specific embodiments, the sucrose in the formulation is about 300 mM.

[0687] The pharmaceutical composition can have any suitable pH for delivery of the pharmaceutical composition to target cells, e.g., in vitro or in vivo. For example, the pharmaceutical composition can have a pH of about 6.5-7.8, such as about 6.5-7.8, 6.5-7.6, 6.5-7.5, 6.5-7.4, 6.5- 7.2, 6.5-7.0, 6.5-6.7, 6.7-7.8, 6.7-7.6, 6.7-7.5, 6.7-7.4, 6.7-7.2, 6.7-7.0, 7.0-7.8, 7.0-7.6, 7.0-7.5, 7.0-

[0688] 7.4, 7.0-7.2, 7.2-7.8, 7.2-7.6, 7.2-7.5, 7.2-7.4, 7.4-7.8, 7.4-7.6, 7.4-7.5, 7.5-7.8, 7.5-7.6, 7.6-8.0, 7.6-

[0689] 7.8, or 7.8-8.0, or about 6.5, 6.7, 6.92, 7.0, 7,2, 7.4, 7.42, 7.5, 7.6, 7.8, or 8.0. In specific embodiments, the pharmaceutical composition has a pH between about 6.7-7.6, about 6.92-7.42, or about 7.4-7.5.

[0690] The pharmaceutical composition can have any suitable poly dispersity index for delivery of the pharmaceutical composition to target cells, e.g., in vitro or in vivo. For example, the pharmaceutical composition can have a poly dispersity index no greater than about 0.30, 0.20, 0.10; about 0.10-0.30, 0.10-0.20, 0.20-0.30; or about 0.30, 0.20, or 0.10. In specific embodiments, the pharmaceutical composition has a poly dispersity index equal to, or less than, 0.20.

[0691] The pharmaceutical composition can have any suitable osmolality for delivery of the pharmaceutical composition to target cells, e.g., in vitro or in vivo. For example, the pharmaceutical composition can have osmolality of about 450-650 mOsm / kg, such as about 450-650, 450-605, 450-603, 450-600, 450-564, 450-550, 450-500, 450-495, 495-650, 495-605, 495-603, 495-600,

[0692] 495-564, 495-550, 495-500, 495-650, 500-605, 500-603, 500-600, 500-564, 500-550, 550-605,

[0693] 550-603, 550-600, 550-564, 564-605, 564-603, 564-600, 600-650, 600-605, 600-603, 603-650,

[0694] 603-605, 605-600, or about 450, 495, 500, 550, 564, 600, 603, 605, or 650 mOsm / kg. In specific embodiments, the osmolality of the pharmaceutical composition is between about 495-605 mOsm / kg, or about 564-603 mOsm / kg.

[0695] WBD (US) 4897-9494-7440vl 83 Aty Docket No. P89339 2080WO (01275) The polynucleotide contained in the LNP of the pharmaceutical composition can be an mRNA. In some embodiments, about 85% or more (e.g., about e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) of the plurality of LNPs comprise the polynucleotide (e.g., mRNA).

[0696] In specific embodiments, the pharmaceutical composition comprising the LNP comprising the polynucleotides encoding the meganuclease (“meganuclease pharmaceutical composition”) provided herein has a pH of between 6.7-7.6, a polydispersity index of equal to, or less than, 0.20, an osmolality of between about 495-605 mOsm / kg, and an encapsulation percentage that exceeds 90%. In specific embodiments, the meganuclease pharmaceutical composition is diluted into normal saline to the desired concentration prior to administration by intravenous infusion.

[0697] The pharmaceutical compositions provided herein can be prepared in accordance with known techniques. See, e.g., Remington, The Science And Practice of Pharmacy (21st ed., Philadelphia, Lippincott, Williams & Wilkins, 2005). In the manufacture of a pharmaceutical formulation according to the disclosure, engineered meganucleases described herein, polynucleotides encoding the same, or cells expressing the same, are typically admixed with a pharmaceutically acceptable carrier and the resulting composition is administered to a subject. The carrier must be acceptable in the sense of being compatible with any other ingredients in the formulation and must not be deleterious to the subject. The carrier can be a solid or a liquid, or both, and can be formulated with the compound as a unit-dose formulation.

[0698] The presently disclosed engineered meganucleases can have improved (i.e., increased) specificity resulting in reduced off-target cutting, and enhanced (i.e., increased) efficiency of cleavage and indel formation, particularly in cells comprising an integrated copy of the HBV genome, as compared to previously available meganucleases (e.g., the HBV 11-12L.1090QQ Linkerl meganuclease). As such, the presently disclosed pharmaceutical compositions comprising optimized engineered meganucleases, nucleic acid sequences encoding the same, or cells expressing the same, cam also have improved (i.e., increased) efficacy in treating HBV infection, reducing the level or proliferation of HBV, reducing at least one symptom of a disease associated with HBV infection, or treating a disease associated with HBV infection in a subject, when compared to the administration of pharmaceutical compositions comprising previously available meganucleases (e.g., the HBV 11-12L.1090QQ Linkerl meganuclease).

[0699] The pharmaceutical compositions described herein can include a therapeutically effective amount of any engineered meganuclease described herein, or any polynucleotide described herein encoding any engineered meganuclease described herein. For example, in some embodiments, the pharmaceutical composition can include polynucleotides described herein at any of the doses (e.g., gc / kg of an encoding nucleic acid sequence or mg / kg of mRNA) described herein.

[0700] WBD (US) 4897-9494-7440vl 84 Aty Docket No. P89339 2080WO (01275) The present disclosure also provides engineered meganucleases described herein (or nucleic acids encoding the same or cells expressing the engineered meganucleases) for use as a medicament. The present disclosure further provides the use of an engineered meganuclease described herein (or a nucleic acid encoding the same or cells expressing an engineered meganuclease) in the manufacture of a medicament for treating an HBV infection, for reducing the level or proliferation of HBV, reducing the symptoms associated with a disease associated with HBV infection, or treating a disease associated with HBV infection.

[0701] 3 , 1 Methods of Inactivating an HBV pol Gene in a Cell

[0702] Methods for inactivating a polymerase (pol) gene of an HBV genome or an HBV genome fragment are provided. The methods include introducing into a eukaryotic cell comprising the HBV genome or fragment thereof an engineered meganuclease described herein or a nucleic acid encoding the engineered meganuclease, wherein the engineered meganuclease produces a cleavage site at a recognition sequence comprising SEQ ID NO: 3 (i.e., HBV 11-12) and the pol gene is inactivated by introduction of an indel at the cleavage site or by elimination of the HBV genome or fragment thereof. In the methods described herein, an engineered meganuclease of the disclosure can be delivered to and / or expressed from DNA / RNA in target cells that can provide the engineered meganuclease to the HBV genome.

[0703] Introducing Engineered Meganucleases into Cells

[0704] Engineered meganuclease proteins described herein, or polynucleotides encoding the same, can be delivered into cells to cleave genomic DNA or an HBV genome fragment by a variety of different mechanisms known in the art, including those further detailed herein below.

[0705] Provided is a method for inactivating a pol gene of an HBV genome or an HBV genome fragment. The method comprises introducing into a cell (e.g., comprising the HBV genome or HBV genome fragment) the LNP provided herein, wherein the engineered meganuclease is expressed in the cell and produces a cleavage site at a recognition sequence comprising SEQ ID NO: 3 in the pol gene.

[0706] In some embodiments, the pol gene is inactivated by introduction of an indel at the cleavage site, or wherein the pol gene is inactivated by elimination of the HBV genome or the HBV genome fragment.

[0707] In some embodiments, the HBV genome or the HBV genome fragment is comprised by covalently closed circular DNA (cccDNA). The cccDNA can be eliminated by degradation following generation of the cleavage site, leading to inactivation of the pol gene. Alternatively, the pol gene can also be inactivated in cccDNA by introduction of an indel at the cleavage site. In some

[0708] WBD (US) 4897-9494-7440vl 85 Atty Docket No. P89339 2080WO (01275) embodiments, the indel is introduced by non-homologous end joining (NHEJ). In some embodiments, the inactivated pol gene does not encode an active and / or full-length HBV polymerase protein.

[0709] In some embodiments, the HBV genome or the HBV genome fragment is comprised in the genome of the target cell. In some embodiments, the pol gene is inactivated in the HBV genome or the HBV genome fragment by introduction of an indel at the cleavage site in the pol gene. In some embodiments, the genome is the nuclear genome. In some embodiments, the genome is the mitochondrial genome. In some embodiments, the indel is introduced by NHEJ. In some embodiments, the inactivated pol gene does not encode an active and / or full-length HBV polymerase protein.

[0710] In some embodiments, the method inactivates an HBsAg gene in the HBV genome or the HBV genome fragment.

[0711] In some embodiments, the cell is a liver cell, such a hepatocyte. The cell can be located within or outside a subject. Accordingly, the methods provided herein can be conducted in vitro or in vivo.

[0712] In some embodiments, the polynucleotide comprised by the LNP is an mRNA. The mRNA may have any features provided in the present disclosure. For example, the mRNA can contain a 5’ cap structure, a 5’ UTR from the albumin gene (ALB), an ORF containing the coding sequence of the meganuclease, sequences encoding SV40 NLS sequences at the 5’ and 3’ extremes of the ORF, a 3’ UTR from the small nuclear ribonucleoprotein polypeptides B and Bl genes (SNRPB), and a poly-Atail, as shown in Figure 2. In specific embodiments, the mRNA sequence encoding the HBV 11-12L.1090 E80 meganuclease provided herein includes a Cap-1 structure at the 5’ terminus (m7G(5’)ppp(5’)(2’OMeA)pG), a 5’ UTR from the albumin gene (ALB) (e.g., SEQ ID NO: 13), an ORF containing the coding sequence of the meganuclease (e.g., SEQ ID NO: 7 or 8), sequences encoding SV40 NLS sequences at the 5’ and 3’ extremes of the ORF (e.g., SEQ ID NOs: 11 and 12 at the 5’ and 3’ ends, respectively), a 3’ UTR from the small nuclear ribonucleoprotein polypeptides B and Bl genes (SNRPB) (e.g., SEQ ID NO: 15), and finally a poly-Atail (e.g., SEQ ID NO: 19), as schematically depicted in Figure 2. In some embodiments, the mRNA that is a polynucleotide provided herein comprises a nucleic acid sequence having at least 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 20 or SEQ ID NO: 21. In some embodiments, the mRNA comprises a nucleic acid sequence set forth SEQ ID NO: 20 or SEQ ID NO: 21.

[0713] Engineered meganucleases described herein can be delivered into a cell in the form of protein or, preferably, as a polynucleotide comprising a nucleic acid sequence encoding the

[0714] WBD (US) 4897-9494-7440vl 86 Atty Docket No. P89339 2080WO (01275) engineered meganuclease. Such polynucleotides can be, for example, DNA (e.g., circular or linearized plasmid DNA, PCR products, or viral genomes) or RNA (e.g., mRNA).

[0715] For embodiments in which the engineered meganuclease coding sequence is delivered in DNA form, it should be operably linked to a promoter to facilitate transcription of the meganuclease gene. Mammalian promoters suitable for use include constitutive promoters such as the cytomegalovirus early (CMV) promoter (Thomsen et al. (1984), Proc Natl Acad Sci USA. 81(3):659-63) or the SV40 early promoter (Benoist and Chambon (1981), Nature. 290(5804):304- 10) as well as inducible promoters such as the tetracycline-inducible promoter (Dingermann et al. (1992), Mol Cell Biol. 12(9):4038-45). An engineered meganuclease of the disclosure can also be operably linked to a synthetic promoter. Synthetic promoters can include, without limitation, the JeT promoter (WO 2002 / 012514). In specific embodiments, a nucleic acid sequence encoding an engineered meganuclease as described herein can be operably linked to a liver-specific promoter. Examples of liver-specific promoters include, without limitation, human alpha-1 antitrypsin promoter, hybrid liver-specific promoter (hepatic locus control region from ApoE gene (ApoE- HCR) and a liver-specific alphal -antitrypsin promoter), human thyroxine binding globulin (TBG) promoter, and apolipoprotein A-II promoter.

[0716] In specific embodiments, a polynucleotide comprising a nucleic acid sequence encoding at least one engineered meganuclease described herein is delivered on a recombinant DNA construct or expression cassette. For example, the recombinant DNA construct can comprise an expression cassette (i.e., “cassette”) comprising a promoter and a nucleic acid sequence encoding an engineered meganuclease described herein.

[0717] In another particular embodiment, a polynucleotide comprising a nucleic acid sequence encoding an engineered meganuclease described herein is introduced into the cell using a singlestranded DNA template. The single-stranded DNA can further comprise a 5’ and / or a 3’ AAV inverted terminal repeat (ITR) upstream and / or downstream of the sequence encoding the engineered nuclease. The single-stranded DNA can further comprise a 5’ and / or a 3’ homology arm upstream and / or downstream of the sequence encoding the engineered meganuclease.

[0718] In another particular embodiment, a polynucleotide comprising a nucleic acid sequence encoding an engineered meganuclease described herein can be introduced into a cell using a linearized DNA template. Such linearized DNA templates can be produced by methods known in the art. For example, a plasmid DNA encoding a nuclease can be digested by one or more restriction enzymes such that the circular plasmid DNA is linearized prior to being introduced into a cell.

[0719] WBD (US) 4897-9494-7440vl 87 Atty Docket No. P89339 2080WO (01275) In some embodiments, mRNA encoding an engineered meganuclease is delivered to a cell because this reduces the likelihood that the gene encoding the engineered meganuclease will integrate into the genome of the cell.

[0720] Such mRNA can be produced using methods known in the art such as in vitro transcription. In some embodiments, the mRNA is 5’ capped using 7-methyl-guanosine, anti-reverse cap analogs (ARCA) (US 7,074,596), CleanCap® analogs such as Cap 1 analogs (Trilink, San Diego, CA), or enzymatically capped using vaccinia capping enzyme or similar. In some embodiments, the mRNA may be polyadenylated. The mRNA may contain various 5’ and 3’ untranslated sequence elements to enhance expression of the encoded engineered meganuclease and / or stability of the mRNA itself. Non-limiting examples of such elements include a 5’ UTR for the albumin (ALB) gene having the sequence set forth as SEQ ID NO: 13 (encoded by SEQ ID NO: 14), a Kozak sequence having the sequence set forth as SEQ ID NO: 17 (encoded by SEQ ID NO: 18), a 3’ UTR for the small nuclear ribonucleoprotein B (SNRPB) gene having the sequence set forth as SEQ ID NO: 15 (encoded by SEQ ID NO: 16), and a polyA termination sequence such as the sequence set forth as SEQ ID NO: 19. Such elements can include, for example, posttranslational regulatory elements such as a woodchuck hepatitis virus posttranslational regulatory element. The mRNA may contain nucleoside analogs or naturally-occurring nucleosides, such as pseudouridine, 5-methylcytidine, N6- methyladenosine, 5-methyluridine, or 2-thiouridine. Additional nucleoside analogs include, for example, those described in US 8,278,036. As uridine-rich RNA sequences can trigger the innate immune response, the mRNA can be uridine depleted. The polynucleotide comprising a nucleic acid sequence encoding an engineered meganuclease described herein can be codon-optimized for expression in mammalian cells and more specifically, for expression in particular tissues, such as liver.

[0721] Purified meganuclease proteins can be delivered into cells to cleave genomic DNAby a variety of different mechanisms known in the art, including those further detailed herein.

[0722] Delivering Engineered Meganucleases in Target Cells in a Subject

[0723] In an in vivo aspect of the methods described herein, the present disclosure provides a method for inactivating a pol gene of an HB V genome or an HB V genome fragment in a target cell in a subject. The method includes delivering to the target cell (comprising the HBV genome or HBV genome fragment) the LNP provided herein, wherein the engineered meganuclease is expressed in the target cell and produces a cleavage site at a recognition sequence comprising SEQ ID NO: 3 in the pol gene.

[0724] The target tissue(s) for delivery of engineered meganucleases of the disclosure include, without limitation, cells of the liver, such as a hepatocyte cell or preferably a primary hepatocyte,

[0725] WBD (US) 4897-9494-7440vl 88 Atty Docket No. P89339 2080WO (01275) more preferably a human hepatocyte or a human primary hepatocyte, a HepG2.2.15 or a HepG2- hNTCP cell. Meganucleases disclosed herein can be delivered as purified protein or as RNA or DNA encoding the meganuclease. In one embodiment, meganuclease protein, mRNA, DNA, or cells expressing meganucleases can be delivered systemically via the circulatory system. Alternatively, meganuclease proteins, or mRNA, or DNA vectors encoding meganucleases, can be supplied to target cells (e.g., cells in the liver) via injection directly to the target tissue.

[0726] In some embodiments, the pol gene is inactivated by introduction of an indel at the cleavage site, or wherein the pol gene is inactivated by elimination of the HBV genome or the HBV genome fragment.

[0727] In some embodiments, the HBV genome or the HBV genome fragment is comprised by cccDNA. The cccDNA can be eliminated by degradation following generation of the cleavage site. Alternatively, the pol gene can be inactivated in the cccDNA by introduction of an indel at the cleavage site. In some embodiments, the indel is introduced by non-homologous end joining (NHEJ). In some embodiments, the inactivated pol gene does not encode an active and / or full- length HBV polymerase protein.

[0728] In some embodiments, the HBV genome or the HBV genome fragment is comprised in the genome of the target cell. In some embodiments, the pol gene is inactivated in the HBV genome or the HBV genome fragment by introduction of an indel at the cleavage site.

[0729] In some embodiments, the genome is the nuclear genome. In some embodiments, the genome is the mitochondrial genome. In some embodiments, the indel is introduced by NHEJ. In some embodiments, the inactivated pol gene does not encode an active and / or full-length HBV polymerase protein.

[0730] In some embodiments, the method inactivates an HBsAg gene in the HBV genome or the HBV genome fragment.

[0731] In some embodiments, the cell is a liver cell, such a hepatocyte, which is in the subject.

[0732] In some embodiments, the polynucleotide comprised by the LNP is an mRNA. The mRNA may have any features provided in the present disclosure. For example, the mRNA can contain a 5’ cap structure, a 5’ UTR from the albumin gene (ALB), an ORF containing the coding sequence of the meganuclease, two SV40 NLS sequences at 5’ and 3’ extremes of the ORF, a 3’ UTR from the small nuclear ribonucleoprotein polypeptides B and Bl genes (SNRPB), and a poly-Atail, as shown in Figure 2. In specific embodiments, the mRNA sequence encoding the HBV 11- 12L.1090QE 1923(1+2) meganuclease provided herein consists of a Cap-1 structure at the 5’ terminus (m7G(5’)ppp(5’)(2’OMeA)pG), a 5’ UTR from the albumin gene (ALB) (e.g., SEQ ID NO: 13), an ORF containing the coding sequence of the meganuclease (e.g., SEQ ID NO: 7 or 8), sequence encoding SV40 NLS sequences at 5’ and 3’ extremes of the ORF (e.g., SEQ ID NOs: 11 WBD (US) 4897-9494-7440vl 89 Aty Docket No. P89339 2080WO (01275) and 12 at the 5’ and 3’ ends, respectively), a 3’ UTR from the small nuclear ribonucleoprotein polypeptides B and Bl genes (SNRPB) (e.g., SEQ ID NO: 15), and finally a poly-Atail (e.g., SEQ ID NO: 19), as schematically depicted in Figure 2. In some embodiments, the mRNAthat is a polynucleotide provided herein comprises a nucleic acid sequence having at least 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 20 or SEQ ID NO: 21. In some embodiments, the mRNA comprises a nucleic acid sequence set forth SEQ ID NO: 20 or SEQ ID NO: 21.

[0733] 3 ,2 Methods of Treating CHB or a Condition Related to HB V

[0734] Methods are provided for treating HBV infection or a disease associated with HBV infection (e.g., CHB) in a subject. Likewise, methods are provided for reducing the symptoms of an HBV infection and / or a disease associated with HBV infection, reducing the amount of HBV, and / or reducing the rate of proliferation of HBV in a subject. The methods include administering a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an engineered meganuclease described herein, or a nucleic acid encoding the engineered meganuclease.

[0735] In one aspect, the methods comprise administering to an HBV-infected subject a therapeutically effective dose of a pharmaceutical composition comprising a plurality of LNPs provided herein, wherein the plurality of LNPs are delivered to target cells in the subject that comprise an HBV genome or HBV genome fragment, wherein the engineered meganuclease is expressed in the target cells and produces a cleavage site at a recognition sequence comprising SEQ ID NO: 3 in a pol gene in the HBV genome or the HBV genome fragment. The pharmaceutical composition and / or the LNPs have any features of the pharmaceutical composition and / or the LNPs comprising a polynucleotide encoding a meganuclease, as provided elsewhere herein

[0736] In some embodiments, the pol gene is inactivated by introduction of an indel at the cleavage site, or wherein the pol gene is inactivated by elimination of the HBV genome or the HBV genome fragment.

[0737] In some embodiments, the polynucleotide is an mRNA. For example, the mRNA comprises a 5’ cap structure, a 5’ UTR from the albumin gene (ALB), an ORF containing the coding sequence of the meganuclease, sequences encoding SV40 NLS sequences at the 5’ and 3’ extremes of the ORF, a 3’ UTR from the small nuclear ribonucleoprotein polypeptides B and Bl genes (SNRPB), and a poly-Atail, as shown in Figure 2. In specific embodiments, the mRNA sequence encoding the HBV 11-12L.1090 E80 meganuclease provided herein includes a Cap-1 structure at the 5’ terminus (m7G(5’)ppp(5’)(2’OMeA)pG), a 5’ UTR from the albumin gene (ALB) (e.g., SEQ ID NO: 13), an ORF containing the coding sequence of the meganuclease (e.g., SEQ ID NO: 7 or 8), sequences

[0738] WBD (US) 4897-9494-7440vl 90 Atty Docket No. P89339 2080WO (01275) encoding SV40 NLS sequences at the 5’ and 3’ extremes of the ORF (e.g., SEQ ID NOs: 11 and 12 at the 5’ and 3’ ends, respectively), a 3’ UTR from the small nuclear ribonucleoprotein polypeptides B and Bl genes (SNRPB) (e.g., SEQ ID NO: 15), and finally a poly-Atail (e.g., SEQ ID NO: 19), as schematically depicted in Figure 2. In some embodiments, the mRNAthat is a polynucleotide provided herein comprises a nucleic acid sequence having at least 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 20 or SEQ ID NO: 21. In some embodiments, the mRNA comprises a nucleic acid sequence set forth SEQ ID NO: 20 or SEQ ID NO: 21.

[0739] In some embodiments, the HBV genome or the HBV genome fragment is comprised by cccDNA. The cccDNA can be eliminated by degradation following generation of the cleavage site. Alternatively, the pol gene can be inactivated in the cccDNA by introduction of an indel at the cleavage site. The indel can be introduced by NHEJ. In some embodiments, the inactivated pol gene does not encode an active and / or full-length HBV polymerase protein.

[0740] In some embodiments, the HBV genome or the HBV genome fragment is comprised in the genome of the target cells. The pol gene can be inactivated in the HBV genome or the HBV genome fragment by introduction of an indel at the cleavage site. The genome can be the nuclear genome and / or the mitochondrial genome. The indel can be introduced by NHEJ. In some embodiments, the inactivated pol gene does not encode an active and / or full-length HBV polymerase protein.

[0741] In some embodiments, the method inactivates an HBsAg gene in the HBV genome or the HBV genome fragment.

[0742] The target cells can be liver cells, such as hepatocytes.

[0743] Route of Administration

[0744] In various embodiments of the methods and compositions described herein, the engineered meganucleases described herein, polynucleotides encoding the same, LNPs comprising such polynucleotides, or pharmaceutical compositions comprising such LNPs can be administered via any suitable route of administration known in the art. Such routes of administration can include, for example, intravenous, oral, intramuscular, intraperitoneal, subcutaneous, intrahepatic, transmucosal, transdermal, intraarterial, and sublingual. Other suitable routes of administration can be readily determined by the treating physician as necessary.

[0745] In some embodiments, LNPs comprising polynucleotides described herein or pharmaceutical compositions comprising such LNPs are administered to the subject by intravenous (IV) administration for delivery to target cells (e.g., liver cells, e.g., hepatocytes). Administration to the subject by IV administration can be, for example, over about 1-3 hours, such as 1-2 or 2-3

[0746] WBD (US) 4897-9494-7440vl 91 Atty Docket No. P89339 2080WO (01275) hours, or about 1, 2, or 3 hours per administration. In specific embodiments, administration by IV administration can be over about 2 hours per administration.

[0747] Dosing of LNP Composition

[0748] The methods provided herein comprise administering to an HBV-infected subject a therapeutically effective dose of a pharmaceutical composition comprising a plurality of LNPs provided herein. The therapeutically effective dose of the pharmaceutical composition can be determined based on multiple clinical and pharmaceutical factors, such as age, body weight, sex, severity or stage of HBV-associated disease, and therapeutic effects in corresponding population, for each subject.

[0749] In some embodiments, a subject is administered a pharmaceutical composition comprising LNPs comprising a polynucleotide (e.g., mRNA) comprising a nucleic acid sequence encoding an engineered meganuclease described herein. In some such embodiments, the dose of the polynucleotide (e.g., mRNA) is about 0.01 mg / kg to about 3 mg / kg. In some embodiments, the dose of the polynucleotide (e.g., mRNA) is about 0.01 mg / kg, about 0.1 mg / kg, about 0.2 mg / kg, about 0.25 mg / kg, about 0.3 mg / kg, about 0.4 mg / kg, about 0.5 mg / kg, about 0.6 mg / kg, about 0.7 mg / kg, about 0.75 mg / kg, about 0.8 mg / kg, about 0.9 mg / kg, about 1.0 mg / kg, about 1.1 mg / kg, about 1.2 mg / kg, about 1.3 mg / kg, about 1.4 mg / kg, about 1.5 mg / kg, about 2.0 mg / kg, about 2.5 mg / kg, or about 3.0 mg / kg. In some embodiments, the dose of the polynucleotide (e.g., mRNA) is about 0.01 mg / kg to about 0.1 mg / kg, about 0.1 mg / kg to about 0.25 mg / kg, about 0.25 mg / kg to about 0.4 mg / kg, about 0.4 mg / kg to about 0.5 mg / kg, about 0.5 mg / kg to about 0.75 mg / kg, about 0.75 mg / kg to about 1.0 mg / kg, about 1.0 mg / kg to about 1.5 mg / kg, about 1.5 mg / kg to about 2.0 mg / kg, about 2.0 mg / kg to about 2.5 mg / kg, or about 2.5 mg / kg to about 3.0 mg / kg. In specific embodiments, the pharmaceutical composition is administered at a dose of between about 0.01-2.0 mg / kg, about 0.1-1.5 mg / kg, about 0.1-0.4 mg / kg, about 0.4-0.7 mg / kg, about 0.7-1.0 mg / kg, about 1.0-1.3 mg / kg, about 1.3-1.5 mg / kg, about 1.5-2.0 mg / kg, about 2.0-2.5 mg / kg, about 2.5-3.0 mg / kg, about 0.1 mg / kg, about 0.2 mg / kg, about 0.3 mg / kg, about 0.4 mg / kg, about 0.5 mg / kg, about 0.6 mg / kg, about 0.7 mg / kg, about 0.8 mg / kg, about 0.9 mg / kg, about 1.0 mg / kg, about 1.1 mg / kg, about 1.2 mg / kg, about 1.3 mg / kg, about 1.4 mg / kg, about 1.5 mg / kg, about 1.6 mg / kg, about 1.7 mg / kg, about 1.8 mg / kg, about 1.9 mg / kg, about 2.0 mg / kg, about 2.1 mg / kg, about 2.2 mg / kg, about 2.3 mg / kg, about 2.4 mg / kg, about 2.5 mg / kg, about 2.6 mg / kg, about 2.7 mg / kg, about 2.8 mg / kg, about 2.9 mg / kg, or about 3.0 mg / kg of the polynucleotide (e.g., mRNA). In specific embodiments, the pharmaceutical composition is administered at a dose of about 0.1 mg / kg of the polynucleotide (e.g., mRNA). In specific embodiments, the pharmaceutical composition is administered at a dose of about 0.2 mg / kg of the polynucleotide (e.g., mRNA). In specific

[0750] WBD (US) 4897-9494-7440vl 92 Atty Docket No. P89339 2080WO (01275) embodiments, the pharmaceutical composition is administered at a dose of about 0.3 mg / kg of the polynucleotide (e.g., mRNA). In specific embodiments, the pharmaceutical composition is administered at a dose of about 0.4 mg / kg of the polynucleotide (e.g., mRNA). In specific embodiments, the pharmaceutical composition is administered at a dose of about 0.5 mg / kg of the polynucleotide (e.g., mRNA). In specific embodiments, the pharmaceutical composition is administered at a dose of about 0.6 mg / kg of the polynucleotide (e.g., mRNA). In specific embodiments, the pharmaceutical composition is administered at a dose of about 0.7 mg / kg of the polynucleotide (e.g., mRNA). In specific embodiments, the pharmaceutical composition is administered at a dose of about 0.8 mg / kg of the polynucleotide (e.g., mRNA). In specific embodiments, the pharmaceutical composition is administered at a dose of about 0.9 mg / kg of the polynucleotide (e.g., mRNA). In specific embodiments, the pharmaceutical composition is administered at a dose of about 1.0 mg / kg of the polynucleotide (e.g., mRNA). In specific embodiments, the pharmaceutical composition is administered at a dose of about 1.1 mg / kg of the polynucleotide (e.g., mRNA). In specific embodiments, the pharmaceutical composition is administered at a dose of about 1.2 mg / kg of the polynucleotide (e.g., mRNA). In specific embodiments, the pharmaceutical composition is administered at a dose of about 1.3 mg / kg of the polynucleotide (e.g., mRNA). In specific embodiments, the pharmaceutical composition is administered at a dose of about 1.4 mg / kg of the polynucleotide (e.g., mRNA). In specific embodiments, the pharmaceutical composition is administered at a dose of about 1.5 mg / kg of the polynucleotide (e.g., mRNA).

[0751] Any suitable dosing regimen may be employed to effect the treatment in the subject. In some embodiments, the subject is administered a single dose of the pharmaceutical composition on day 0.

[0752] In some embodiments, the subject is further administered a second dose of the pharmaceutical composition following administration of the first dose. In some embodiments, the subject is further administered a second dose of the pharmaceutical composition about 8 weeks following administration of the first dose. In some embodiments, the subject is further administered a second dose of the pharmaceutical composition about 4 weeks following administration of the first dose. In some embodiments, the subject is further administered a second dose of the pharmaceutical composition about 5 weeks following administration of the first dose. In some embodiments, the subject is further administered a second dose of the pharmaceutical composition about 6 weeks following administration of the first dose. In some embodiments, the subject is further administered a second dose of the pharmaceutical composition about 7 weeks following administration of the first dose. In some embodiments, the subject is further administered a second dose of the pharmaceutical composition about 9 weeks following administration of the first dose. In WBD (US) 4897-9494-7440vl 93 Aty Docket No. P89339 2080WO (01275) some embodiments, the subject is further administered a second dose of the pharmaceutical composition about 10 weeks following administration of the first dose. In some embodiments, the subject is further administered a second dose of the pharmaceutical composition about 11 weeks following administration of the first dose. In some embodiments, the subject is further administered a second dose of the pharmaceutical composition about 12 weeks following administration of the first dose. In some embodiments, the subject is further administered a second dose of the pharmaceutical composition up to about 52 weeks following administration of the first dose.

[0753] In some embodiments, the first dose and the second dose are equivalent. In some embodiments, the second dose is higher than the first dose. In some embodiments, the second dose is lower than the first dose.

[0754] For example, the second dose of the polynucleotide (e.g., mRNA) can be about 0.01 mg / kg to about 3 mg / kg, for example, about 0.01-2.0 mg / kg, about 0.1-1.5 mg / kg, about 0.1-0.4 mg / kg, about 0.4-0.7 mg / kg, about 0.7-1.0 mg / kg, about 1.0-1.3 mg / kg, about 1.3-1.5 mg / kg, about 1.5-2.0 mg / kg, about 2.0-2.5 mg / kg, about 2.5-3.0 mg / kg, about 0.01 mg / kg, about 0.1 mg / kg, about 0.2 mg / kg, about 0.25 mg / kg, about 0.3 mg / kg, about 0.4 mg / kg, about 0.5 mg / kg, about 0.6 mg / kg, about 0.7 mg / kg, about 0.75 mg / kg, about 0.8 mg / kg, about 0.9 mg / kg, about 1.0 mg / kg, about 1.1 mg / kg, about 1.2 mg / kg, about 1.3 mg / kg, about 1.4 mg / kg, about 1.5 mg / kg, about 1.6 mg / kg, about 1.7 mg / kg, about 1.8 mg / kg, about 1.9 mg / kg, about 2.0 mg / kg, about 2.5 mg / kg, or about 3.0 mg / kg; about 0.01 mg / kg to about 0.1 mg / kg, about 0.1 mg / kg to about 0.25 mg / kg, about 0.25 mg / kg to about 0.5 mg / kg, about 0.5 mg / kg to about 0.75 mg / kg, about 0.75 mg / kg to about 1.0 mg / kg, about 1.0 mg / kg to about 1.5 mg / kg, about 1.5 mg / kg to about 2.0 mg / kg, about 2.0 mg / kg to about 2.5 mg / kg, or about 2.5 mg / kg to about 3.0 mg / kg. In specific embodiments, the second dose of the polynucleotide (e.g., mRNA) is between about 0.01-2.0 mg / kg, about 0.1-1.5 mg / kg, about 0.1-0.4 mg / kg, about 0.4-0.7 mg / kg, about 0.7-1.0 mg / kg, about 0.2 mg / kg, about 0.25 mg / kg, about 0.3 mg / kg, about 0.4 mg / kg, about 0.5 mg / kg, about 0.6 mg / kg, about 0.7 mg / kg, about 0.75 mg / kg, about 0.8 mg / kg, about 0.9 mg / kg, about 1.0 mg / kg, about 1.1 mg / kg, about 1.2 mg / kg, about 1.3 mg / kg, about 1.4 mg / kg, or about 1.5 mg / kg of the polynucleotide (e.g., mRNA). In specific embodiments, the second dose is about 0.1 mg / kg of the polynucleotide (e.g., mRNA). In specific embodiments, the second dose is about 0.2 mg / kg of the polynucleotide (e.g., mRNA). In specific embodiments, the second dose is about 0.3 mg / kg of the polynucleotide (e.g., mRNA). In specific embodiments, the second dose is about 0.4 mg / kg of the polynucleotide (e.g., mRNA). In specific embodiments, the second dose is about 0.5 mg / kg of the polynucleotide (e.g., mRNA). In specific embodiments, the second dose is about 0.6 mg / kg of the polynucleotide (e.g., mRNA). In specific embodiments, the second dose is about 0.7 mg / kg of the polynucleotide (e.g., mRNA). In specific embodiments, the second dose is about 0.8 mg / kg of the polynucleotide (e.g., mRNA). WBD (US) 4897-9494-7440vl 94 Aty Docket No. P89339 2080WO (01275) In specific embodiments, the second dose is about 0.9 mg / kg of the polynucleotide (e.g., mRNA). In specific embodiments, the second dose is about 1.0 mg / kg of the polynucleotide (e.g., mRNA). In specific embodiments, the second dose is about 1.1 mg / kg of the polynucleotide (e.g., mRNA). In specific embodiments, the second dose is about 1.2 mg / kg of the polynucleotide (e.g., mRNA). In specific embodiments, the second dose is about 1.3 mg / kg of the polynucleotide (e.g., mRNA). In specific embodiments, the second dose is about 1.4 mg / kg of the polynucleotide (e.g., mRNA). In specific embodiments, the second dose is about 1.5 mg / kg of the polynucleotide (e.g., mRNA).

[0755] In some embodiments, the first dose is about 0.1 mg / kg of the polynucleotide (e.g., mRNA) and the second dose is about 0.1 mg / kg of the polynucleotide (e.g., mRNA).

[0756] In some embodiments, the first dose is about 0.2 mg / kg of the polynucleotide (e.g., mRNA) and the second dose is about 0.2 mg / kg of the polynucleotide (e.g., mRNA).

[0757] In some embodiments, the first dose is about 0.3 mg / kg of the polynucleotide (e.g., mRNA) and the second dose is about 0.3 mg / kg of the polynucleotide (e.g., mRNA).

[0758] In some embodiments, the first dose is about 0.4 mg / kg of the polynucleotide (e.g., mRNA) and the second dose is about 0.4 mg / kg of the polynucleotide (e.g., mRNA).

[0759] In some embodiments, the first dose is about 0.5 mg / kg of the polynucleotide (e.g., mRNA)and the second dose is about 0.5 mg / kg of the polynucleotide (e.g., mRNA).

[0760] In some embodiments, the first dose is about 0.6 mg / kg of the polynucleotide (e.g., mRNA) and the second dose is about 0.6 mg / kg of the polynucleotide (e.g., mRNA).

[0761] In some embodiments, the first dose is about 0.7 mg / kg of the polynucleotide (e.g., mRNA) and the second dose is about 0.7 mg / kg of the polynucleotide (e.g., mRNA).

[0762] In some embodiments, the first dose is about 0.8 mg / kg of the polynucleotide (e.g., mRNA) and the second dose is about 0.8 mg / kg of the polynucleotide (e.g., mRNA).

[0763] In some embodiments, the first dose is about 0.9 mg / kg of the polynucleotide (e.g., mRNA) and the second dose is about 0.9 mg / kg of the polynucleotide (e.g., mRNA).

[0764] In some embodiments, the first dose is about 1.0 mg / kg of the polynucleotide (e.g., mRNA) and the second dose is about 1.0 mg / kg of the polynucleotide (e.g., mRNA).

[0765] In some embodiments, the subject is further administered a third dose of the pharmaceutical composition following administration of the second dose.

[0766] In some embodiments, the subject is further administered a third dose of the pharmaceutical composition about 8 weeks following administration of the second dose. In some embodiments, the subject is further administered a third dose of the pharmaceutical composition about 4 weeks following administration of the second dose. In some embodiments, the subject is further administered a third dose of the pharmaceutical composition about 5 weeks following administration of the second dose. In some embodiments, the subject is further administered a third

[0767] WBD (US) 4897-9494-7440vl 95 Aty Docket No. P89339 2080WO (01275) dose of the pharmaceutical composition about 6 weeks following administration of the second dose. In some embodiments, the subject is further administered a third dose of the pharmaceutical composition about 7 weeks following administration of the second dose. In some embodiments, the subject is further administered a third dose of the pharmaceutical composition about 9 weeks following administration of the second dose. In some embodiments, the subject is further administered a third dose of the pharmaceutical composition about 10 weeks following administration of the second dose. In some embodiments, the subject is further administered a third dose of the pharmaceutical composition about 11 weeks following administration of the second dose. In some embodiments, the subject is further administered a third dose of the pharmaceutical composition about 12 weeks following administration of the second dose. In some embodiments, the subject is further administered a third dose of the pharmaceutical composition up to about 52 weeks following administration of the second dose.

[0768] In some embodiments, the first dose, the second dose, and the third dose are equivalent.

[0769] In some embodiments, the third dose is higher than the second dose.

[0770] In some embodiments, the third dose is lower than the second dose.

[0771] For example, the third dose of the polynucleotide (e.g., mRNA) can be about 0.01 mg / kg to about 3 mg / kg, for example, about 0.01 mg / kg, about 0.1 mg / kg, about 0.2 mg / kg, about 0.25 mg / kg, about 0.3 mg / kg, about 0.4 mg / kg, about 0.5 mg / kg, about 0.6 mg / kg, about 0.7 mg / kg, about 0.75 mg / kg, about 0.8 mg / kg, about 0.9 mg / kg, about 1.0 mg / kg, about 1.1 mg / kg, about 1.2 mg / kg, about 1.3 mg / kg, about 1.4 mg / kg, about 1.5 mg / kg, about 1.6 mg / kg, about 1.7 mg / kg, about 1.8 mg / kg, about 1.9 mg / kg, about 2.0 mg / kg, about 2.5 mg / kg, or about 3.0 mg / kg; about 0.01 mg / kg to about 0.1 mg / kg, about 0.1 mg / kg to about 0.25 mg / kg, about 0.25 mg / kg to about 0.4 mg / kg; about 0.4 mg / kg to about 0.5 mg / kg, about 0.5 mg / kg to about 0.75 mg / kg, about 0.75 mg / kg to about 1.0 mg / kg, about 1.0 mg / kg to about 1.5 mg / kg, about 1.5 mg / kg to about 2.0 mg / kg, about 2.0 mg / kg to about 2.5 mg / kg, or about 2.5 mg / kg to about 3.0 mg / kg. In specific embodiments, the third dose of the polynucleotide (e.g., mRNA) is between about 0.01-2.0 mg / kg, about 0.1-1.5 mg / kg, about 0.1-0.4 mg / kg, about 0.4-0.7 mg / kg, about 0.7-1.0 mg / kg, about 1.0-1.3 mg / kg, about 1.3 -1.5 mg / kg, about 1.5 -2.0 mg / kg, about 0.1 mg / kg, about 0.2 mg / kg, about 0.3 mg / kg, about 0.4 mg / kg, about 0.5 mg / kg, about 0.6 mg / kg, about 0.7 mg / kg, about 0.75 mg / kg, about 0.8 mg / kg, about 0.9 mg / kg, or about 1.0 mg / kg of the polynucleotide (e.g., mRNA). In specific embodiments, the third dose is about 0.1 mg / kg of the polynucleotide (e.g., mRNA). In specific embodiments, the third dose is about 0.2 mg / kg of the polynucleotide (e.g., mRNA). In specific embodiments, the third dose is about 0.3 mg / kg of the polynucleotide (e.g., mRNA). In specific embodiments, the third dose is about 0.4 mg / kg of the polynucleotide (e.g., mRNA). In specific embodiments, the third dose is about 0.5 mg / kg of the polynucleotide (e.g., mRNA). In

[0772] WBD (US) 4897-9494-7440vl 96 Aty Docket No. P89339 2080WO (01275) specific embodiments, the third dose is about 0.6 mg / kg of the polynucleotide (e.g., mRNA). In specific embodiments, the third dose is about 0.7 mg / kg of the polynucleotide (e.g., mRNA). In specific embodiments, the third dose is about 0.8 mg / kg of the polynucleotide (e.g., mRNA). In specific embodiments, the third dose is about 0.9 mg / kg of the polynucleotide (e.g., mRNA). In specific embodiments, the third dose is about 1.0 mg / kg of the polynucleotide (e.g., mRNA). In specific embodiments, the third dose is about 1.1 mg / kg of the polynucleotide (e.g., mRNA). In specific embodiments, the third dose is about 1.2 mg / kg of the polynucleotide (e.g., mRNA). In specific embodiments, the third dose is about 1.3 mg / kg of the polynucleotide (e.g., mRNA). In specific embodiments, the third dose is about 1.4 mg / kg of the polynucleotide (e.g., mRNA). In specific embodiments, the third dose is about 1.5 mg / kg of the polynucleotide (e.g., mRNA).

[0773] In some embodiments, the first dose is about 0.1 mg / kg of the polynucleotide (e.g., mRNA), the second dose is about 0.1 mg / kg of the polynucleotide (e.g., mRNA), and the third dose is about 0.1 mg / kg of the polynucleotide (e.g., mRNA).

[0774] In some embodiments, the first dose is about 0.2 mg / kg of the polynucleotide (e.g., mRNA), the second dose is about 0.2 mg / kg of the polynucleotide (e.g., mRNA), and the third dose is about 0.2 mg / kg of the polynucleotide (e.g., mRNA).

[0775] In some embodiments, the first dose is about 0.3 mg / kg of the polynucleotide (e.g., mRNA), the second dose is about 0.3 mg / kg of the polynucleotide (e.g., mRNA), and the third dose is about 0.3 mg / kg of the polynucleotide (e.g., mRNA).

[0776] In some embodiments, the first dose is about 0.4 mg / kg of the polynucleotide (e.g., mRNA), the second dose is about 0.4 mg / kg of the polynucleotide (e.g., mRNA), and the third dose is about 0.4 mg / kg of the polynucleotide (e.g., mRNA).

[0777] In some embodiments, the first dose is about 0.5 mg / kg of the polynucleotide (e.g., mRNA), the second dose is about 0.5 mg / kg of the polynucleotide (e.g., mRNA), and the third dose is about 0.5 mg / kg of the polynucleotide (e.g., mRNA).

[0778] In some embodiments, the first dose is about 0.6 mg / kg of the polynucleotide (e.g., mRNA), the second dose is about 0.6 mg / kg of the polynucleotide (e.g., mRNA), and the third dose is about 0.6 mg / kg of the polynucleotide (e.g., mRNA).

[0779] In some embodiments, the first dose is about 0.7 mg / kg of the polynucleotide (e.g., mRNA), the second dose is about 0.7 mg / kg of the polynucleotide (e.g., mRNA), and the third dose is about 0.7 mg / kg of the polynucleotide (e.g., mRNA).

[0780] In some embodiments, the first dose is about 0.8 mg / kg of the polynucleotide (e.g., mRNA), the second dose is about 0.8 mg / kg of the polynucleotide (e.g., mRNA), and the third dose is about 0.8 mg / kg of the polynucleotide (e.g., mRNA).

[0781] WBD (US) 4897-9494-7440vl 97 Atty Docket No. P89339 2080WO (01275) In some embodiments, the first dose is about 0.9 mg / kg of the polynucleotide (e.g., mRNA), the second dose is about 0.9 mg / kg of the polynucleotide (e.g., mRNA), and the third dose is about 0.9 mg / kg of the polynucleotide (e.g., mRNA).

[0782] In some embodiments, the first dose is about 1.0 mg / kg of the polynucleotide (e.g., mRNA), the second dose is about 1.0 mg / kg of the polynucleotide (e.g., mRNA), and the third dose is about 1.0 mg / kg of the polynucleotide (e.g., mRNA).

[0783] In some embodiments, the subject is further administered one or more additional doses of the pharmaceutical composition following administration of the third dose (e.g., a fourth dose, a fifth dose, a sixth dose). In some such embodiments, further doses are each administered about 4 weeks following administration of the previous dose. In some such embodiments, further doses are each administered about 5 weeks following administration of the previous dose. In some such embodiments, further doses are each administered about 6 weeks following administration of the previous dose. In some such embodiments, further doses are each administered about 7 weeks following administration of the previous dose. In some such embodiments, further doses are each administered about 8 weeks following administration of the previous dose. In some such embodiments, further doses are each administered about 9 weeks following administration of the previous dose. In some such embodiments, further doses are each administered about 10 weeks following administration of the previous dose. In some such embodiments, further doses are each administered about 11 weeks following administration of the previous dose. In some such embodiments, further doses are each administered about 12 weeks following administration of the previous dose. In some such embodiments, further doses are each administered up to about 52 weeks following administration of the previous dose.

[0784] In some embodiments, any further doses may be administered at the same interval as the time between the first, second, and third doses. In other embodiments, any further doses may be administered at a different time interval than the time between the first, second, and third doses.

[0785] In some embodiments, the first dose, the second dose, the third dose, and any further doses are equivalent.

[0786] In some embodiments, the further doses are higher than the third dose.

[0787] In some embodiments, the further doses are lower than the third dose.

[0788] For example, a further dose of the polynucleotide (e.g., mRNA) can be about 0.01 mg / kg to about 3 mg / kg, for example, about 0.01 mg / kg, about 0.1 mg / kg, about 0.2 mg / kg, about 0.25 mg / kg, about 0.3 mg / kg, about 0.4 mg / kg, about 0.5 mg / kg, about 0.6 mg / kg, about 0.7 mg / kg, about 0.75 mg / kg, about 0.8 mg / kg, about 0.9 mg / kg, about 1.0 mg / kg, about 1.1 mg / kg, about 1.2 mg / kg, about 1.3 mg / kg, about 1.4 mg / kg, about 1.5 mg / kg, about 1.6 mg / kg, about 1.7 mg / kg, about 1.8 mg / kg, about 1.9 mg / kg, about 2.0 mg / kg, about 2.5 mg / kg, or about 3.0 mg / kg; about WBD (US) 4897-9494-7440vl 98 Aty Docket No. P89339 2080WO (01275) 0.01 mg / kg to about 0.1 mg / kg, about 0.1 mg / kg to about 0.25 mg / kg, about 0.25 mg / kg to about 0.4 mg / kg; about 0.4 mg / kg to about 0.5 mg / kg, about 0.5 mg / kg to about 0.75 mg / kg, about 0.75 mg / kg to about 1.0 mg / kg, about 1.0 mg / kg to about 1.5 mg / kg, about 1.5 mg / kg to about 2.0 mg / kg, about 2.0 mg / kg to about 2.5 mg / kg, or about 2.5 mg / kg to about 3.0 mg / kg.

[0789] In specific embodiments, a further dose of the polynucleotide (e.g., mRNA) is between about 0.01-2.0 mg / kg, about 0.1-1.5 mg / kg, about 0.1-0.4 mg / kg, about 0.4-0.7 mg / kg, about 0.7- 1.0 mg / kg, about 1.0-1.3 mg / kg, about 1.3-1.5 mg / kg, about 1.5-2.0 mg / kg, about 0.1 mg / kg, about 0.2 mg / kg, about 0.3 mg / kg, about 0.4 mg / kg, about 0.5 mg / kg, about 0.6 mg / kg, about 0.7 mg / kg, about 0.75 mg / kg, about 0.8 mg / kg, about 0.9 mg / kg, or about 1.0 mg / kg of the polynucleotide (e.g., mRNA).

[0790] In specific embodiments, a further dose is about 0.1 mg / kg of the polynucleotide (e.g., mRNA). In specific embodiments, a further dose is about 0.2 mg / kg of the polynucleotide (e.g., mRNA). In specific embodiments, a further dose is about 0.3 mg / kg of the polynucleotide (e.g., mRNA). In specific embodiments, a further dose is about 0.4 mg / kg of the polynucleotide (e.g., mRNA). In specific embodiments, a further dose is about 0.5 mg / kg of the polynucleotide (e.g., mRNA). In specific embodiments, a further dose is about 0.6 mg / kg of the polynucleotide (e.g., mRNA). In specific embodiments, a further dose is about 0.7 mg / kg of the polynucleotide (e.g., mRNA). In specific embodiments, a further dose is about 0.8 mg / kg of the polynucleotide (e.g., mRNA). In specific embodiments, a further dose is about 0.9 mg / kg of the polynucleotide (e.g., mRNA). In specific embodiments, a further dose is about 1.0 mg / kg of the polynucleotide (e.g., mRNA). In specific embodiments, a further dose is about 1.1 mg / kg of the polynucleotide (e.g., mRNA). In specific embodiments, a further dose is about 1.2 mg / kg of the polynucleotide (e.g., mRNA). In specific embodiments, a further dose is about 1.3 mg / kg of the polynucleotide (e.g., mRNA). In specific embodiments, a further dose is about 1.4 mg / kg of the polynucleotide (e.g., mRNA). In specific embodiments, a further dose is about 1.5 mg / kg of the polynucleotide (e.g., mRNA).

[0791] In some embodiments, the first dose is about 0.1 mg / kg of the polynucleotide (e.g., mRNA), the second dose is about 0.1 mg / kg of the polynucleotide (e.g., mRNA), the third dose is about 0.1 mg / kg of the polynucleotide (e.g., mRNA), and the further dose is about 0.1 mg / kg of the polynucleotide (e.g., mRNA).

[0792] In some embodiments, the first dose is about 0.2 mg / kg of the polynucleotide (e.g., mRNA), the second dose is about 0.2 mg / kg of the polynucleotide (e.g., mRNA), the third dose is about 0.2 mg / kg of the polynucleotide (e.g., mRNA), and the further dose is about 0.2 mg / kg of the polynucleotide (e.g., mRNA).

[0793] WBD (US) 4897-9494-7440vl 99 Atty Docket No. P89339 2080WO (01275) In some embodiments, the first dose is about 0.3 mg / kg of the polynucleotide (e.g., mRNA), the second dose is about 0.3 mg / kg of the polynucleotide (e.g., mRNA), the third dose is about 0.3 mg / kg of the polynucleotide (e.g., mRNA), and the further dose is about 0.3 mg / kg of the polynucleotide (e.g., mRNA).

[0794] In some embodiments, the first dose is about 0.4 mg / kg of the polynucleotide (e.g., mRNA), the second dose is about 0.4 mg / kg of the polynucleotide (e.g., mRNA), the third dose is about 0.4 mg / kg of the polynucleotide (e.g., mRNA), and the further dose is about 0.4 mg / kg of the polynucleotide (e.g., mRNA).

[0795] In some embodiments, the first dose is about 0.5 mg / kg of the polynucleotide (e.g., mRNA), the second dose is about 0.5 mg / kg of the polynucleotide (e.g., mRNA), the third dose is about 0.5 mg / kg of the polynucleotide (e.g., mRNA), and the further dose is about 0.5 mg / kg of the polynucleotide (e.g., mRNA).

[0796] In some embodiments, the first dose is about 0.6 mg / kg of the polynucleotide (e.g., mRNA), the second dose is about 0.6 mg / kg of the polynucleotide (e.g., mRNA), the third dose is about 0.6 mg / kg of the polynucleotide (e.g., mRNA), and the further dose is about 0.6 mg / kg of the polynucleotide (e.g., mRNA).

[0797] In some embodiments, the first dose is about 0.7 mg / kg of the polynucleotide (e.g., mRNA), the second dose is about 0.7 mg / kg of the polynucleotide (e.g., mRNA), the third dose is about 0.7 mg / kg of the polynucleotide (e.g., mRNA), and the further dose is about 0.7 mg / kg of the polynucleotide (e.g., mRNA).

[0798] In some embodiments, the first dose is about 0.8 mg / kg of the polynucleotide (e.g., mRNA), the second dose is about 0.8 mg / kg of the polynucleotide (e.g., mRNA), the third dose is about 0.8 mg / kg of the polynucleotide (e.g., mRNA), and the further dose is about 0.8 mg / kg of the polynucleotide (e.g., mRNA).

[0799] In some embodiments, the first dose is about 0.9 mg / kg of the polynucleotide (e.g., mRNA), the second dose is about 0.9 mg / kg of the polynucleotide (e.g., mRNA), the third dose is about 0.9 mg / kg of the polynucleotide (e.g., mRNA), and the further dose is about 0.9 mg / kg of the polynucleotide (e.g., mRNA).

[0800] In some embodiments, the first dose is about 1.0 mg / kg of the polynucleotide (e.g., mRNA), the second dose is about 1.0 mg / kg of the polynucleotide (e.g., mRNA), the third dose is about 1.0 mg / kg of the polynucleotide (e.g., mRNA), and the further dose is about 1.0 mg / kg of the polynucleotide (e.g., mRNA).

[0801] Example initial dosing and anticipated dose escalation plan are set forth in Tables 6 and 7, respectively. Example determination algorithm for second dose and third dose, are set forth in Figures 16-19.

[0802] WBD (US) 4897-9494-7440vl 100 Aty Docket No. P89339 2080WO (01275) As appropriate, the dosage or dosing frequency of the engineered meganuclease, or the polynucleotide encoding the same, may be adjusted over the course of the treatment, based on the judgment of the administering physician. Appropriate doses will depend, among other factors, on the specifics of any lipid nanoparticle chosen, on the route of administration, on the subject being treated (z.e., age, weight, sex, and general condition of the subject), and the mode of administration. Thus, the appropriate dosage may vary from patient to patient. An appropriate effective amount can be readily determined by one of skill in the art or treating physician. Dosage treatment may be a single dose schedule or, if multiple doses are required, a multiple dose schedule. Moreover, the subject may be administered as many doses as appropriate. One of skill in the art can readily determine an appropriate number of doses. The dosage may need to be adjusted to take into consideration an alternative route of administration or balance the therapeutic benefit against any side effects.

[0803] Administration of Medications to Prevent or Treat Adverse Effects

[0804] The administration of the therapeutically effective dose of a pharmaceutical composition comprising a plurality of LNPs provided herein may cause side effects, such as an infusion-related reaction (IRR) or cytokine release syndrome (CRS). An IRR is an adverse event that can occur after a drug infusion, usually within minutes to hours after a drug infusion, and can cause various symptoms including rash, flushing, urticaria, pruritus, wheezing, dyspnea, bronchospasm, hypotension, lightheadedness, dizziness, weakness, abdominal pain, nausea, vomiting, cramps, diarrhea, and back pain. A CRS is an acute systemic inflammatory syndrome characterized by fever and multiple organ dysfunction. The following medications may be used to prevent or treat adverse effects (e.g., an IRR or CRS) related to administration of the pharmaceutical composition provided herein: acetaminophen, histamine (Hl / 2) blockers, non-steroidal anti-inflammatory drugs (NSAIDs), epinephrine, and / or corticosteroids. Supplemental oxygen and / or IV fluids may also be used as clinically indicated.

[0805] In some embodiments, the subject is administered a steroid. A steroid can be administered between about 4-24 hours prior to administration of the pharmaceutical composition, such as about 4-6, 6-10, 10-14, 14-18, or 18-24 hours prior, or about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours prior to administration of the pharmaceutical composition. In some embodiments, the subject is administered a steroid between about 6-18 hours prior to administration of the pharmaceutical composition. In some embodiments, the subject is administered a steroid between about 10-14 hours prior to administration of the pharmaceutical composition. In some embodiments, the subject is administered a steroid about 12 hours prior to administration of the pharmaceutical composition. In some embodiments, the steroid is

[0806] WBD (US) 4897-9494-7440vl 101 Atty Docket No. P89339 2080WO (01275) administered orally. In some embodiments, the steroid is dexamethasone. In some embodiments, the dexamethasone is administered at a dose of about 8 mg.

[0807] In some embodiments, the subject is administered a steroid between about 1-3 hours prior to administration of the pharmaceutical composition. In some embodiments, the subject is administered a steroid between about 1-2 hours prior to administration of the pharmaceutical composition. In some embodiments, the steroid is administered intravenously. In some embodiments, the steroid is dexamethasone. In some embodiments, the dexamethasone is administered at a dose of about 10 mg.

[0808] In some embodiments, the subject is administered a histamine receptor 1 (Hl) blocker between about 1-3 hours prior to administration of the pharmaceutical composition. In some embodiments, the subject is administered an Hl blocker. The subject can be administered an Hl blocker between about 1-2 hours prior to administration of the pharmaceutical composition. In some embodiments, the Hl blocker is administered intravenously. In some embodiments, the Hl blocker is diphenhydramine. In some embodiments, the diphenhydramine is administered at a dose of about 50 mg. In some embodiments, the Hl blocker is administered orally. In some embodiments, the Hl blocker is cetirizine. In some embodiments, the cetirizine is administered at a dose of about 10 mg.

[0809] In some embodiments, the subject is administered a histamine receptor 2 (H2) blocker. The subject can be administered an H2 blocker between about 1-3 hours prior to administration of the pharmaceutical composition. In some embodiments, the subject is administered an H2 blocker between about 1-2 hours prior to administration of the pharmaceutical composition. In some embodiments, the H2 blocker is administered orally or intravenously. In some embodiments, the H2 blocker is famotidine. In some embodiments, the famotidine is administered at a dose of about 20 mg.

[0810] In some embodiments, the subject is administered a steroid between about 2-6 hours following administration of the pharmaceutical composition. In some embodiments, the subject is administered a steroid about 4 hours following administration of the pharmaceutical composition. In some embodiments, the steroid is administered orally. In some embodiments, the steroid is dexamethasone. In some embodiments, the dexamethasone is administered at a dose of about 4 mg.

[0811] In some embodiments, the subject is administered: a) a steroid (e.g., dexamethasone orally at a dose of about 10 mg) about 12 hours prior to administration of the pharmaceutical composition; b) a steroid (e.g., dexamethasone intravenously at a dose of about 10 mg) about 1-2 hours prior to administration of the pharmaceutical composition; c) an Hl blocker (e.g., diphenhydramine intravenously at a dose of about 50 mg; or cetirizine orally at a dose of about 10 mg) about 1-2 hours prior to administration of the pharmaceutical composition; d) a H2 blocker (e.g., famotidine WBD (US) 4897-9494-7440vl 102 Aty Docket No. P89339 2080WO (01275) intravenously or orally at a dose of about 20 mg) about 1-2 hours prior to administration of the pharmaceutical composition; and e) a steroid (e.g., dexamethasone orally at a dose of about 4 mg) about 4 hours following administration of the pharmaceutical composition.

[0812] As appropriate, the dosage or dosing frequency of the medications for adverse effect, or the polynucleotide encoding the same, may be adjusted over the course of the treatment, based on the judgment of the administering physician.

[0813] Nucleos(t)ide Analogue Therapy

[0814] In some embodiments, the subject has been administered a nucleos(t)ide analogue therapy immediately prior to administration of the pharmaceutical composition and achieved an adequate virologic response. For example, the subject may have been administered a nucleos(t)ide analogue therapy for at least 6 months immediately prior to administration of the pharmaceutical composition and achieved an adequate virologic response.

[0815] In some embodiments, the subject continues to be administered the nucleos(t)ide analogue therapy following administration of the pharmaceutical composition. In some embodiments, the subject continues to be administered the nucleos(t)ide analogue therapy for at least 8 weeks following the last administration of the pharmaceutical composition.

[0816] In some embodiments, the nucleos(t)ide analogue is discontinued therapy if: a) serum HBsAg concentrations are less than 0.05 lU / mL, serum HBV DNA concentrations are less than 10 HJ / mL, and serum alanine transaminase (ALT) concentrations are less than 1.5 x upper limit of normal (ULN); or b) serum HBsAg concentrations are greater than 0.05 HJ / mL and less than 50 lU / mL, serum HBV DNA concentrations are less than 10 HJ / mL, and ALT concentrations are less than 1.5 x upper limit of normal (ULN), serum HBV RNA concentrations are undetectable, and HBcrAg concentrations are undetectable, wherein the conditions of a) or b) must be met on two occasions at least 4 weeks apart, and no earlier than 8 weeks since the last dose of the pharmaceutical composition.

[0817] In some embodiments, the subject is monitored for about 48 weeks following discontinuation of nucleos(t)ide analogue therapy for achievement of a functional cure or a partial cure. In some embodiments, a functional cure comprises sustained serum HBsAg concentrations below 0.05 lU / mL with or without seroconversion of antibody to HBsAg for at least 24 weeks following the last dose of the pharmaceutical composition. In some embodiments, a partial cure comprises sustained reductions of serum HBsAg concentrations to less than 50 lU / mL but greater than 0.05 lU / mL, serum HBV DNA concentrations less than 10 lU / mL, and serum ALT concentrations less than 1.5 x ULN for at least 24 weeks following the last dose of the pharmaceutical composition.

[0818] WBD (US) 4897-9494-7440vl 103 Atty Docket No. P89339 2080WO (01275) Subject Population

[0819] Any subject (or patient) population in need of treatment with the pharmaceutical composition provided herein can be administered the pharmaceutical composition. For example, the subject may have hepatitis B infection, and may suffer from CHB. The subject may have chronic hepatitis B with compensated liver disease. In some embodiments, the subject has received a nucleos(t)ide analogue therapy immediately prior to administration of the pharmaceutical composition and achieved an adequate virologic response. In some embodiments, the subject has received a nucleos(t)ide analogue therapy for at least 6 months immediately prior to administration of the pharmaceutical composition. In some embodiments, the subject meets the following conditions:

[0820] • chronically infected with HBeAg-negative HBV;

[0821] • being treated with a nucleos(t)ide analog;

[0822] • have evidence of ongoing infection (as evidenced by the presence of HBsAg) therefore putting them as risk of disease progression; and

[0823] • have normally functioning liver, and have no signs of liver cirrhosis or hepatocellular carcinoma.

[0824] Therapeutic Effects; Endpoints

[0825] According to the methods provided herein, therapeutically effective amount of a pharmaceutical composition comprising a plurality of LNPs comprising a polynucleotide encoding an engineered meganuclease described herein (“meganuclease pharmaceutical composition”) can be administered to a subject in need thereof for the treatment of HBV infection or a disease associated with HBV infection, such as CHB with compensated liver disease. Such administration can result in decrease in levels of markers associated with HBV infection or a disease associated with HBV infection (e g., HBsAg, HBV DNA, HBV RNA, HBcrAg, HBV cccDNA) or decrease in one or more signs or symptoms associated with the HBV infection or a disease associated with HBV infection.

[0826] For example, following administration of the pharmaceutical composition provided herein, HBsAg may be no longer detectable in the serum or plasma of the subject, and administration of the pharmaceutical composition may be discontinued. Following administration of the pharmaceutical composition provided herein, HBsAg may be no longer detectable in the serum or plasma of the subject in the absence of antiviral treatment. In some embodiments, after one or more administrations of the pharmaceutical composition as described herein, the subject does not exhibit symptoms of the disease associated with HBV infection in the absence of antiviral treatment.

[0827] WBD (US) 4897-9494-7440vl 104 Atty Docket No. P89339 2080WO (01275) HBsAg

[0828] The serum HBsAg concentration can be reduced in the subject following administration of the meganuclease pharmaceutical composition compared to the serum HBsAg concentration prior to the administration. As compared to “prior to administration” as used herein refers to as compared to an appropriate control, such as i) a sample from the subject prior to administration or ii) a sample from control subjects not having administered the meganuclease pharmaceutical composition provided herein. Without wishing to be bound by theory, the serum HBsAg concentrations in control subjects without administration of the meganuclease pharmaceutical composition provided herein can be about 200-10000 lU / ml. In certain embodiments, the control subjects meet the following criteria:

[0829] • chronically infected with HBeAg-negative HBV;

[0830] • being treated with a nucleos(t)ide analog;

[0831] • have not been administered the pharmaceutical composition provided herein;

[0832] • have evidence of ongoing infection (as evidenced by the presence of HBsAg) therefore putting them as risk of disease progression; and

[0833] • have normally functioning liver, and have no signs of liver cirrhosis or hepatocellular carcinoma.

[0834] The reduction in the serum HBsAg concentration can be expressed by % reduction from that in a baseline / control sample taken prior to the administration of the meganuclease pharmaceutical composition. For example, the serum HBsAg concentration is reduced by at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%; about 50%-100%, 55%-100%, 60%-100%, 65%-100%, 70%-100%, 75%-100%, 80%-100%, 85%-100%, 90%-100%, 95%-100%, 99%-100%, 50%-55%, 55%-60%, 60%-65%, 65%-70%, 70%-75%, 75%-80%, 80%- 85%, 85%-90%, 90%-95%, 95%-99%; or about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% in the subject following administration of the meganuclease pharmaceutical composition compared to the serum HBsAg concentration prior to the administration. In specific embodiments, the serum HBsAg concentration is reduced by about 50%- 100%, about 90%-100%, about 95%-100%, or about 99%-100% in the subject following administration of the meganuclease pharmaceutical composition compared to the serum HBsAg concentration prior to the administration. In some embodiments, the serum HBsAg concentration is reduced by 100% in the subject following administration of the meganuclease pharmaceutical composition compared to the serum HBsAg concentration prior to the administration.

[0835] The reduction in the serum HBsAg concentration can be expressed as logio changes (Alogio) from that in a baseline / control sample taken prior to administration of the meganuclease

[0836] WBD (US) 4897-9494-7440vl 105 Atty Docket No. P89339 2080WO (01275) pharmaceutical composition. For example, the logio change in the serum HBsAg concentration is at least about -0.1, -0.2. -0.3, -0.4, -0.5, -0.6, -0.7, -0.8, -0.9, -1.0, -1.1, -1.2, -1.3, -1.4, -1.5, -1.6, -1.7, -1.8, -1.9, -2.0, -2.1, -2.2, -2.3, -2.4, -2.5, -2.6, -2.7, -2.8, -2.9, -3.0, -3.1, -3.2, -3.3, -3.4, -3.5, -3.6, - 3.7, -3.8, -3.9, or -4.0; about -0.1 to -4.0, -0.1 to -2.5, -0.1 to -2.0, -0.1 to -1.5, -0.1 to -1.0, -0.1 to - 0.5, -0.5 to -1.0, -1.0 to -1.5, -1.5 to -2.0, -2.0 to -2.5, -2.5 to -3.0, -3.0 to -3.5, -3.5 to -4.0; or about - 0.1, -0.2. -0.3, -0.4, -0.5, -0.6, -0.7, -0.8, -0.9, -1.0, -1.1, -1.2, -1.3, -1.4, -1.5, -1.6, -1.7, -1.8, -1.9, -2.0, -2.1, -2.2, -2.3, -2.4, -2.5, -2.6, -2.7, -2.8, -2.9, -3.0, -3.1, -3.2, -3.3, -3.4, -3.5, -3.6, -3.7, -3.8, -3.9, or -4.0 in the subject following administration of the meganuclease pharmaceutical composition compared to the serum HBsAg concentration prior to the administration. In specific embodiments, the logio change in the serum HBsAg concentration is about -0.1 to -4.0, about -1.0 to -4.0, about -2.0 to -4.0, about -2.0 to -3.0, or about -3.0 to -4.0 in the subject following administration of the meganuclease pharmaceutical composition compared to the serum HBsAg concentration prior to the administration. The HBsAg concentration in a sample has “no changes” when the absolute values of the logio changes are no greater than (i.e., less than or equal to) 0.2 from the baseline. A “reduction” in the HBsAg concentration in a sample refers to a reduction with the absolute values of the logio changes greater than 0.2 (i.e., the fold change values less than 10'°2) from the baseline, whether or not the post reduction HBsAg concentration in the sample is above, at, or below the lower limit of quantification (LLQ) (i.e., 0.05 lU / ml).

[0837] In some embodiments, the serum HBsAg concentration in the subject is less than 50 HJ / mL following administration of the meganuclease pharmaceutical composition. For example, the serum HBsAg concentration in the subject can be less than 45, 40, 35, 30, 25, 20, 15, 10, 5, 1, 0.5, 0.1, or 0.05 HJ / mL following administration of the meganuclease pharmaceutical composition. Without wishing to be bound by theory, the serum HBsAg concentrations in CHB subjects without administration of the meganuclease pharmaceutical composition provided herein are about 200- 10000 lU / ml. The serum HBsAg concentration in the subject can be undetectable following administration of the meganuclease pharmaceutical composition. “Undetectable” in the context of HBsAg in a sample as used herein refers to the HBsAg concentration in the sample being below the LLQ, i.e., 0.05 lU / ml. The HBsAg concentration in a sample has “no changes” when the changes are no greater than (i.e., less than or equal to) 10°2lU / ml from the baseline. A “reduction” in the HBsAg concentration in a sample refers to reduction in the HBsAg concentration in the sample that are greater than 10°2lU / ml from the baseline, whether or not the post reduction HBsAg concentration in the sample is above, at, or below the LLQ (i.e., 0.05 lU / ml).

[0838] HBsAg concentrations in a sample can be measured by standard methods for measuring antigen concentrations in a sample, for example by using a serological assay, such as an enzyme- linked immunoassay (ELISA) or radioimmunoassay.

[0839] WBD (US) 4897-9494-7440vl 106 Aty Docket No. P89339 2080WO (01275) HBV DNA

[0840] The plasma HBV DNA concentration can be reduced in the subject following administration of the pharmaceutical composition compared to the plasma HBV DNA concentration prior to the administration. As compared to “prior to administration” as used herein refers to as compared to an appropriate control, such as i) a sample from the subject prior to administration or ii) a sample from a control subject (or control subjects) not having administered the meganuclease pharmaceutical composition provided herein. Without wishing to be bound by theory, the plasma HBV DNA concentrations in control subjects without administration of the meganuclease pharmaceutical composition provided herein can be less than about 20 lU / ml. In certain embodiments, the control subjects meet the following criteria:

[0841] • chronically infected with HBeAg-negative HBV;

[0842] • being treated with a nucleos(t)ide analog;

[0843] • have not been administered the meganuclease pharmaceutical composition provided herein;

[0844] • have evidence of ongoing infection (as evidenced by the presence of HBsAg) therefore putting them as risk of disease progression; and

[0845] • have normally functioning liver, and have no signs of liver cirrhosis or hepatocellular carcinoma.

[0846] For example, the plasma HBV DNA concentration is reduced by at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%; about 50%-100%, 55%-100%, 60%- 100%, 65%-100%, 70%-100%, 75%-100%, 80%-100%, 85%-100%, 90%-100%, 95%-100%, 99%- 100%, 50%-55%, 55%-60%, 60%-65%, 65%-70%, 70%-75%, 75%-80%, 80%-85%, 85%-90%, 90%-95%, 95%-99%; or about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% in the subject following administration of the meganuclease pharmaceutical composition compared to the plasma HBV DNA concentration prior to the administration. In specific embodiments, the plasma HBV DNA concentration is reduced by about 50%-100%, about 90%- 100%, about 95%-100%, or about 99%-100% in the subject following administration of the meganuclease pharmaceutical composition compared to the plasma HBV DNA concentration prior to the administration. In some embodiments, the plasma HBV DNA concentration is reduced by 100% in the subject following administration of the meganuclease pharmaceutical composition compared to the serum HBV DNA concentration prior to the administration.

[0847] In some embodiments, the plasma HBV DNA concentration is less than 10 lU / ml following administration of the meganuclease pharmaceutical composition. For example, the plasma HBV DNA concentration can be less than 9, 8, 7, 6, 5, 4, or 3 lU / ml following administration of the meganuclease pharmaceutical composition. In some embodiments, the plasma HBV DNA

[0848] WBD (US) 4897-9494-7440vl 107 Atty Docket No. P89339 2080WO (01275) concentration is undetectable following administration of the meganuclease pharmaceutical composition. “Undetectable” as used herein refers to the plasma HBV DNA concentration being below the lower limit of quantification (i.e., 2.7 lU / ml).

[0849] HBV DNA concentrations in a sample can be measured by standard methods for measuring DNA concentrations in a sample, for example by PCR and quantitative real-time PCR.

[0850] HBV RNA

[0851] The serum HBV RNA concentration can be reduced in the subject following administration of the meganuclease pharmaceutical composition compared to the serum HBV RNA concentration prior to the administration. As compared to “prior to administration” as used herein refers to as compared to an appropriate control, such as i) a sample from the subject prior to administration or ii) a sample from a control subject (or control subjects) not having administered the meganuclease pharmaceutical composition provided herein. In certain embodiments, the control subjects meet the following criteria:

[0852] • chronically infected with HBeAg-negative HBV;

[0853] • being treated with a nucleos(t)ide analog;

[0854] • have not been administered the meganuclease pharmaceutical composition provided herein;

[0855] • have evidence of ongoing infection (as evidenced by the presence of HBsAg) therefore putting them as risk of disease progression; and

[0856] • have normally functioning liver, and have no signs of liver cirrhosis or hepatocellular carcinoma.

[0857] For example, the serum HBV RNA concentration is reduced by at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%; about 50%-100%, 55%-100%, 60%- 100%, 65%-100%, 70%-100%, 75%-100%, 80%-100%, 85%-100%, 90%-100%, 95%-100%, 99%- 100%, 50%-55%, 55%-60%, 60%-65%, 65%-70%, 70%-75%, 75%-80%, 80%-85%, 85%-90%, 90%-95%, 95%-99%; or about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% in the subject following administration of the meganuclease pharmaceutical composition compared to the serum HBV RNA concentration prior to the administration. In specific embodiments, the serum HBV RNA concentration is reduced by about 50%-100%, about 90%- 100%, about 95%-100%, or about 99%-100% in the subject following administration of the meganuclease pharmaceutical composition compared to the serum HBV RNA concentration prior to the administration. In some embodiments, the serum HBV RNA concentration is reduced by 100% in the subject following administration of the meganuclease pharmaceutical composition compared to the serum HBV RNA concentration prior to the administration. In some embodiments, the plasma HBV RNA concentration is undetectable following administration of the meganuclease WBD (US) 4897-9494-7440vl 108 Aty Docket No. P89339 2080WO (01275) pharmaceutical composition. “Undetectable” as used herein refers to the serum HBV RNA concentration being below the lower limit of quantification (i.e., 10 copies / mL).

[0858] HBV RNA concentrations in a sample can be measured by standard methods for measuring RNA concentrations in a sample, for example by quantitative real time PCR or RT-PCR.

[0859] HBcrAg

[0860] The serum HBcrAg concentration can be reduced in the subject following administration of the meganuclease pharmaceutical composition compared to the serum HBcrAg concentration prior to the administration. As compared to “prior to administration” as used herein refers to as compared to an appropriate control, such as i) a sample from the subject prior to administration or ii) a sample from a control subject (or control subjects) not having administered the meganuclease pharmaceutical composition provided herein. In certain embodiments, the control subjects meet the following criteria:

[0861] • chronically infected with HBeAg-negative HBV;

[0862] • being treated with a nucleos(t)ide analog;

[0863] • have not been administered the meganuclease pharmaceutical composition provided herein;

[0864] • have evidence of ongoing infection (as evidenced by the presence of HBsAg) therefore putting them as risk of disease progression; and

[0865] • have normally functioning liver, and have no signs of liver cirrhosis or hepatocellular carcinoma.

[0866] For example, the serum HBcrAg concentration is reduced by at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%; about 50%-100%, 55%-100%, 60%-100%, 65%-100%, 70%-100%, 75%-100%, 80%-100%, 85%-100%, 90%-100%, 95%-100%, 99%-100%, 50%-55%, 55%-60%, 60%-65%, 65%-70%, 70%-75%, 75%-80%, 80%-85%, 85%-90%, 90%- 95%, 95%-99%; or about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% in the subject following administration of the meganuclease pharmaceutical composition compared to the serum HBcrAg concentration prior to the administration. In specific embodiments, the serum HBcrAg concentration is reduced by about 50%-100%, about 90%-100%, about 95%-100%, or about 99%-100% in the subject following administration of the meganuclease pharmaceutical composition compared to the serum HBcrAg concentration prior to the administration. In some embodiments, the serum HBcrAg concentration is reduced by 100% in the subject following administration of the meganuclease pharmaceutical composition compared to the serum HBcrAg concentration prior to the administration. In some embodiments, the serum HBcrAg concentration is undetectable following administration of the meganuclease pharmaceutical composition.

[0867] WBD (US) 4897-9494-7440vl 109 Atty Docket No. P89339 2080WO (01275) “Undetectable” as used herein refers to the serum HBcrAg concentration being below the lower limit of quantification (i.e., less than 1 kU / mL).

[0868] HBcrAg concentrations in a sample can be measured by standard methods for measuring antigen concentrations in a sample, for example by a serological assay, such as a chemiluminescent enzyme immunoassay (CLEIA), a chemiluminescent immunoassay (CLIA), or radioimmunoassay.

[0869] HBV cccDNA

[0870] The HBV cccDNA concentration in the target cells can be reduced in the subject following administration of the meganuclease pharmaceutical composition compared to an appropriate control. An “appropriate control” can be the HBV cccDNA concentration in samples (e.g., cells) from control subjects. In certain embodiments, the control subjects meet the following criteria:

[0871] • chronically infected with HBeAg-negative HBV;

[0872] • being treated with a nucleos(t)ide analog;

[0873] • have not been administered the meganuclease pharmaceutical composition provided herein;

[0874] • have evidence of ongoing infection (as evidenced by the presence of HBsAg) therefore putting them as risk of disease progression; and

[0875] • have normally functioning liver, and have no signs of liver cirrhosis or hepatocellular carcinoma.

[0876] Additionally or alternatively, an “appropriate control” can be a sample (e.g., cell) taken from the subject prior to administration of the meganuclease pharmaceutical composition. One skilled in the art is able to select an appropriate control.

[0877] For example, the HBV cccDNA concentration in target cells is reduced by at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%; about 50%-100%, 55%- 100%, 60%-100%, 65%-100%, 70%-100%, 75%-100%, 80%-100%, 85%-100%, 90%-100%, 95%- 100%, 99%-100%, 50%-55%, 55%-60%, 60%-65%, 65%-70%, 70%-75%, 75%-80%, 80%-85%, 85%-90%, 90%-95%, 95%-99%; or about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% in the subject following administration of the meganuclease pharmaceutical composition compared to an appropriate control (e.g., HBV cccDNA concentration in target cells in control subjects, HBV cccDNA concentration in target cells prior to the administration of the meganuclease pharmaceutical composition). In specific embodiments, the HBV cccDNA concentration in target cells is reduced by about 50%-100%, about 90%-100%, about 95%-100%, or about 99%-100% in the subject following administration of the meganuclease pharmaceutical composition compared to an appropriate control. In some embodiments, the HBV cccDNA concentration in target cells is reduced by 100% in the subject following administration of the meganuclease pharmaceutical composition compared to an appropriate control. In some WBD (US) 4897-9494-7440vl 110 Aty Docket No. P89339 2080WO (01275) embodiments, the HBV cccDNA concentration in target cells in the subject is undetectable following administration of the meganuclease pharmaceutical composition. “Undetectable” as used herein refers to the HBV cccDNA concentration in target cells being below the lower limit of quantification.

[0878] HBV cccDNA concentrations in target cells can be measured by standard methods for measuring DNA concentrations in a sample, for example PCR and quantitative real-time PCR.

[0879] Indel in HB V cccDNA or HB V genome

[0880] At least about 50% of HBV cccDNA present in the target cells can comprise an indel at the recognition sequence after administration of the meganuclease pharmaceutical composition. For example, at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%; about 50%-100%, 55%-100%, 60%-100%, 65%-100%, 70%-100%, 75%-100%, 80%-100%, 85%- 100%, 90%-100%, 95%-100%, 99%-100%, 50%-55%, 55%-60%, 60%-65%, 65%-70%, 70%- 75%, 75%-80%, 80%-85%, 85%-90%, 90%-95%, 95%-99%; or about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of HBV cccDNA present in the target cells can comprise an indel at the recognition sequence after administration of the meganuclease pharmaceutical composition. In some embodiments, between about 50%-100%, about 80%-100%, about 90-100%, or about 95%-100% of HBV cccDNA present in the target cells comprises an indel at the recognition sequence after administration of the meganuclease pharmaceutical composition.

[0881] In some embodiments, the indel inactivates the pol gene. In some embodiments, the indel inactivates an HbsAg gene.

[0882] Additionally or alternatively, at least about 50% of integrated HBV genomes or genome fragments present in the genome of the target cells can comprise an indel at the recognition sequence after administration of the meganuclease pharmaceutical composition. For example, at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%; about 50%- 100%, 55%-100%, 60%-100%, 65%-100%, 70%-100%, 75%-100%, 80%-100%, 85%-100%, 90%- 100%, 95%-100%, 99%-100%, 50%-55%, 55%-60%, 60%-65%, 65%-70%, 70%-75%, 75%-80%, 80%-85%, 85%-90%, 90%-95%, 95%-99%; or about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of integrated HBV genomes or genome fragments present in the genome of the target cells can comprise an indel at the recognition sequence after administration of the meganuclease pharmaceutical composition. In some embodiments, between about 50%-100%, about 80%-100%, about 90-100%, or about 95%-100% of integrated HBV genomes or genome fragments present in the genome of the target cells comprises an indel at the recognition sequence after administration of the meganuclease pharmaceutical composition.

[0883] WBD (US) 4897-9494-7440vl 111 Aty Docket No. P89339 2080WO (01275) In some embodiments, the indel inactivates the pol gene. In some embodiments, the indel inactivates an HbsAg gene.

[0884] The presence and amount of indels in the cccDNA or the integrated HB V genomes or genome fragments in the target cells can be assessed by standard methods for assessing indels in DNA, including PCR, real-time quantitative PCR, and sequencing.

[0885] EXAMPLES

[0886] The embodiments of the disclosure are further illustrated by the following examples, which should not be construed as limiting. Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific substances and procedures described herein. Such equivalents are intended to be encompassed in the scope of the claims that follow the examples below.

[0887] EXAMPLE 1

[0888] GLP Acute Toxicity, Toxicokinetics, and Biodistribution Study in Non-human Primates

[0889] A GLP study was conducted in NHP to determine the potential acute toxicity, toxicokinetic (TK) characteristics, and biodistribution (BD) of PBGENE-HBV.

[0890] In these studies, the PBGENE-HBV drug substance is an mRNA sequence encoding an engineered meganuclease referred to as HBV 11-12L.1090 E80 (SEQ ID NO: 5) that targets a specific 22-base pair (bp) location in hepatitis B virus (HBV) covalently closed circular (ccc) DNA and integrated HBV DNA. This specific 22-bp sequence (SEQ ID NO: 3) in the HBV genome is denoted as HBV target site 11-12 (HBV 11-12). In HBV-infected patients, cleavage of the HBV 11- 12 recognition sequence in HBV cccDNA can result in degradation of the cccDNA or the formation of an insertion / deletion (indel) in the pol gene that results in gene inactivation. The HBV 11-12 sequence can also be present in HBV genomes or genome fragments that are integrated into the nuclear genome, in which case cleavage of the HBV 11-12 sequence can result in the formation of an inactivating indel.

[0891] The mRNA sequence encoding the HBV 11-12L.1090 E80 meganuclease includes a Cap-1 structure at the 5’ terminus (m7G(5’)ppp(5’)(2’OMeA)pG), a 5’ untranslated region (5’ UTR) from the albumin gene (ALB) (SEQ ID NO: 13), an open reading frame (ORF) containing the coding sequence of the meganuclease (SEQ ID NO: 7), sequences encoding simian virus 40 nuclear localization signals (SV40 NLS) at the 5’ and 3’ extremes of the ORF (SEQ ID NOs: 11 and 12 at the 5’ and 3’ ends, respectively), a 3’ UTR from the small nuclear ribonucleoprotein polypeptides B and Bl genes (SNRPB) (SEQ ID NO: 15), and finally a poly- A tail (SEQ ID NO: 19), as shown in WBD (US) 4897-9494-7440vl 112 Aty Docket No. P89339 2080WO (01275) Figure 2. The HBV 11-12L.1090 E80 nuclease comprises a polypeptide linker designed to covalently connect two engineered, I-Crel-derived subunits and develop electrostatic contacts within the backbone of the nuclease. The codon sequence of the meganuclease has been optimized to improve mRNA translation and stability (referred to as a “MAX construct”). Certain bases in the mRNA sequence are substituted with modified nucleotides. The full length of the mRNA is set forth in SEQ ID NO: 20.

[0892] The PBGENE-HBV drug product is an LNP encapsulating the PBGENE-HBV mRNA drug substance, formulated in Dulbecco’s phosphate-buffered saline (pH 7.4) with 300 mM sucrose. The composition of the PBGENE-HBV LNP is provided in Table 3.

[0893] Table 3: Composition of PBGENE-HBV LNP

[0894] WBD (US) 4897-9494-7440vl 113 Aty Docket No. P89339 2080WO (01275) BP = British Pharmacopoeia; Ch.P = Chinese Pharmacopoeia; DS = drug substance; JP = Japanese Pharmacopoeia; NF = National Formulary; Ph.. Eur. = European Pharmacopoeia; QS = quantum satis; USP = United States Pharmacopeia; WFI = water for injection

[0895] 1Fi rst GMP DP Batch used USP compendial materials only, DP GMP batch 2 uses multi-compendial material. 2Meets USP / Ph. Eur compendial requirements

[0896] The molar ratio of Bis(2 -butyloctyl) 10-(N-(3-(pyrrolidin-l- yl)propyl)nonanamido)nonadecanedioate (“Lipid 1”) : 2- [2-(co -methoxy (polyethyleneglycol2000) ethoxy]-N,N-ditetradecylacetamide (“Lipid 2”) : DSPC:cholesterol in said LNP is targeted to be about 47.5:2.5: 10:40, the LNP particle size is between about 55-75 nm, the N:P ratio is targeted to be about 6: 1, the pH of the drug product is targeted between 6.7-7.6, the poly dispersity index is equal to, or less than, 0.20, the osmolality of the formulation is between about 495-605 mOsm / kg, and the encapsulation percentage is targeted to exceed 90%. The PBGENE-HBV drug substance is diluted into normal saline to the desired concentration prior to administration by intravenous infusion.

[0897] PBGENE-HBV was administered by a 60-minute IV (Restrained Peripheral Infusion- Intermittent) infusion using a sing...

Claims

1. CLAIMS1. A lipid nanoparticle (LNP) comprising a polynucleotide, wherein said polynucleotide comprises a nucleic acid sequence encoding an engineered meganuclease that binds and cleaves a recognition sequence comprising SEQ ID NO: 3 in a polymerase (pol) gene of a hepatitis B virus (HBV) genome or HBV genome fragment, and wherein said LNP comprises: a) Bis(2 -butyloctyl) 10-(N-(3 -(pyrrolidin- 1 -yl)propyl)nonanamido)nonadecanedioate; b) 2-[2-(o -methoxy (polyethyleneglycol2000) ethoxy]-N,N-ditetradecylacetamide; c) l,2-distearoyl-sn-glycero-3 -phosphocholine (DSPC); and d) cholesterol.

2. The LNP of claim 1, wherein the molar concentration of Bis(2 -butyloctyl) 10-(N-(3- (pyrrolidin-l-yl)propyl)nonanamido)nonadecanedioate in said LNP is between about 45% to about 50% of the total molar lipid concentration.

3. The LNP of claim 1 or claim 2, wherein the molar concentration of Bis(2 -butyloctyl) 10-(N-(3 -(pyrrolidin- l-yl)propyl)nonanamido)nonadecanedioate in said LNP is about 47.5% of the total molar lipid concentration.

4. The LNP of any one of claims 1-3, wherein the molar concentration of 2-[2-(o- methoxy (polyethyleneglycol2000) ethoxy]-N,N-ditetradecylacetamide in said LNP is between about 1.5% to about 3.5% of the total molar lipid concentration.

5. The LNP of any one of claims 1-4, wherein the molar concentration of 2-[2-(o- methoxy (polyethyleneglycol2000) ethoxy]-N,N-ditetradecylacetamide in said LNP is about 2.5% of the total molar lipid concentration.

6. The LNP of any one of claims 1-5, wherein the molar concentration of DSPC in said LNP is between about 7.5% to about 12.5% of the total molar lipid concentration.

7. The LNP of any one of claims 1-6, wherein the molar concentration of DSPC in said LNP is about 10% of the total molar lipid concentration.WBD (US) 4897-9494-7440vl 167 Aty Docket No. P89339 2080WO (01275)8. The LNP of any one of claims 1-7, wherein the molar concentration of cholesterol in said LNP is between about 37.5% to about 42.5% of the total molar lipid concentration.

9. The LNP of any one of claims 1-8, wherein the molar concentration of cholesterol in said LNP is about 40% of the total molar lipid concentration.

10. The LNP of any one of claims 1-9, wherein the molar ratio of Bis(2 -butyloctyl) 10- (N-(3-(pyrrolidin-l-yl)propyl)nonanamido)nonadecanedioate:2-[2-(o -methoxy (polyethyleneglycol2000) ethoxy]-N,N-ditetradecylacetamide:DSPC:cholesterol in said LNP is about 47.5:2.5: 10:40.

11. The LNP of any one of claims 1-10, wherein said LNP has a particle size of between about 55-75 nm.

12. The LNP of any one of claims 1-11, wherein said LNP has a particle size of between about 62-73 nm.

13. The LNP of any one of claims 1-12, wherein said LNP has a particle size of about 65 nm.

14. The LNP of any one of claims 1-13, wherein said engineered meganuclease comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 5 or SEQ ID NO: 6.

15. The LNP of any one of claims 1-14, wherein said engineered meganuclease comprises an amino acid sequence set forth in SEQ ID NO: 5 or SEQ ID NO: 6.

16. The LNP of any one of claims 1-15, wherein said nucleic acid sequence encoding said engineered meganuclease comprises a sequence having at least 95% sequence identity to SEQ ID NO: 7 or SEQ ID NO: 8.

17. The LNP of any one of claims 1-16, wherein said nucleic acid sequence encoding said engineered meganuclease comprises a sequence set forth in SEQ ID NO: 7 or SEQ ID NO: 8.WBD (US) 4897-9494-7440vl 168 Atty Docket No. P89339 2080WO (01275)18. The LNP of any one of claims 1-17, wherein said engineered meganuclease comprises a 5’ nuclear localization sequence (NLS) at its N-terminus.

19. The LNP of claim 18, wherein said 5’ NLS comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 11.

20. The LNP of claim 18 or claim 19, wherein said 5’ NLS comprises an amino acid sequence set forth in SEQ ID NO: 11.

21. The LNP of any one of claims 1-20, wherein said engineered meganuclease comprises a 3’ nuclear localization sequence (NLS) at its C-terminus.

22. The LNP of any one of claims 21, wherein said 3’ NLS comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 12.

23. The LNP of any one of claims 21 or claim 22, wherein said 3’ NLS comprises an amino acid sequence set forth in SEQ ID NO: 12.

24. The LNP of any one of claims 1-23, wherein said engineered meganuclease comprises a 5’ NLS at its N-terminus comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 11, and a 3’ NLS at its C-terminus comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 12.

25. The LNP of any one of claims 1-24, wherein said engineered meganuclease comprises a 5’ NLS at its N-terminus comprising an amino acid sequence set forth in SEQ ID NO: 11, and a 3’ NLS at its C-terminus comprising an amino acid sequence set forth in SEQ ID NO: 12.

26. The LNP of any one of claims 1-25, wherein said polynucleotide comprises a 5’ untranslated region (UTR) sequence.

27. The LNP of claim 26, wherein said 5’ UTR sequence comprises a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 13.WBD (US) 4897-9494-7440vl 169 Atty Docket No. P89339 2080WO (01275)28. The LNP of claim 26 or claim 27, wherein said 5’ UTR sequence comprises a nucleic acid sequence set forth in SEQ ID NO: 13.

29. The LNP of any one of claims 1-28, wherein said polynucleotide comprises a 3’ untranslated region (UTR) sequence.

30. The LNP of claim 29, wherein said 3’ UTR sequence comprises a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 15.

31. The LNP of claim 29 or claim 30, wherein said 3’ UTR sequence comprises a nucleic acid sequence set forth in SEQ ID NO: 15.

32. The LNP of any one of claims 1-31, wherein said polynucleotide comprises a 5’ UTR sequence that comprises a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 13, and a 3’ UTR sequence that comprises a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 15.

33. The LNP of any one of claims 1-32, wherein said polynucleotide comprises a 5’ UTR sequence that comprises a nucleic acid sequence set forth in SEQ ID NO: 13, and a 3’ UTR sequence that comprises a nucleic acid sequence set forth in SEQ ID NO: 15.

34. The LNP of any one of claims 1-33, wherein said polynucleotide comprises a Kozak sequence comprising a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 17.

35. The LNP of any one of claims 1-34, wherein said polynucleotide comprises a Kozak sequence comprising a nucleic acid sequence set forth in SEQ ID NO: 17.

36. The LNP of any one of claims 1-35, wherein said polynucleotide comprises a polyA sequence.

37. The LNP of claim 36, wherein said polyA sequence comprises a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 19.WBD (US) 4897-9494-7440vl 170 Atty Docket No. P89339 2080WO (01275)38. The LNP of claim 36 or claim 37, wherein said polyA sequence comprises a nucleic acid sequence set forth in SEQ ID NO: 19.

39. The LNP of any one of claims 1-38, wherein said polynucleotide comprises a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 20 or SEQ ID NO: 21.

40. The LNP of any one of claims 1-39, wherein said polynucleotide comprises a nucleic acid sequence set forth SEQ ID NO: 20 or SEQ ID NO: 21.

41. The LNP of any one of claims 1-40, wherein said polynucleotide is a messenger RNA (mRNA).

42. The LNP of claim 41, wherein said mRNA comprises a 5’ cap structure.

43. The LNP of claim 42, wherein said 5’ cap structure comprises (m7G(5’)ppp(5’)(2’OMeA)pG).

44. The LNP of any one of claims 41-43, wherein uridine bases in said mRNA are modified to pseudouridine, Nl-methyl-pseudouridine, 5-methoxyuridine, or 2-thiouridine.

45. The LNP of any one of claims 41-44, wherein said LNP has an N:P ratio of between about 4: 1-8: 1.

46. The LNP of any one of claims 41-45, wherein said LNP has an N:P ratio of about 5: 1-7: 1.

47. The LNP of any one of claims 41-46, wherein said LNP has an N:P ratio of about 6: 1.

48. The LNP of any one of claims 41-47, wherein said mRNA comprises a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 20 or SEQ ID NO: 21.

49. The LNP of any one of claims 41-48, wherein said mRNA comprises a nucleic acid sequence set forth in SEQ ID NO: 20 or SEQ ID NO: 21.WBD (US) 4897-9494-7440vl 171 Atty Docket No. P89339 2080WO (01275)50. An LNP comprising an mRNA, wherein said mRNA comprises a nucleic acid sequence encoding an engineered meganuclease that binds and cleaves a recognition sequence comprising SEQ ID NO: 3 in a pol gene of an HBV genome or HBV genome fragment, wherein said LNP comprises:(a) Bis(2-butyloctyl) 10-(N-(3-(pyrrolidin-l-yl)propyl)nonanamido)nonadecanedioate at a molar concentration of between about 45% to about 50% of the total molar lipid concentration;(b) 2-[2-(o -methoxy (polyethyleneglycol2000) ethoxy]-N,N-ditetradecylacetamide at a molar concentration of between about 1.5% to about 3.5% of the total molar lipid concentration;(c) DSPC at a molar concentration of between about 7.5% to about 12.5% of the total molar lipid concentration; and(d) cholesterol at a molar concentration of between about 37.5% to about 42.5% of the total molar lipid concentration; wherein said LNP has a particle size of between about 55-75 nm, wherein said mRNA comprises the nucleic acid sequence set forth in SEQ ID NO: 20 or SEQ ID NO: 21, wherein said mRNA comprises a 5’ cap structure comprising (m7G(5’)ppp(5’)(2’OMeA)pG), and wherein said LNP has an N:P ratio of between about 4: 1-8: 1.

51. The LNP of claim E, wherein the molar ratio of Bis(2 -butyloctyl) 10-(N-(3- (pyrrolidin-l-yl)propyl)nonanamido)nonadecanedioate:2-[2-(o -methoxy (polyethyleneglycol2000) ethoxy]-N,N-ditetradecylacetamide:DSPC:cholesterol in said LNP is about 47.5:2.5: 10:40.

52. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a plurality of said LNP of any one of claims 1-51.

53. The pharmaceutical composition of claim 52, wherein said pharmaceutical composition comprises between about 15.7-19.2 mg / mL of Bis(2 -butyloctyl) 10-(N-(3-(pyrrolidin- l-yl)propyl)nonanamido)nonadecanedioate.

54. The pharmaceutical composition of claim 52 or claim 53, wherein said pharmaceutical composition comprises between about 17.4-18.5 mg / mL of Bis(2 -butyloctyl) 10- (N-(3-(pyrrolidin-l-yl)propyl)nonanamido)nonadecanedioate.WBD (US) 4897-9494-7440vl 172 Atty Docket No. P89339 2080WO (01275)55. The pharmaceutical composition of any one of claims 52-54, wherein said pharmaceutical composition comprises between about 2.2-3.0 mg / mL of 2- [2-(o -methoxy (polyethyleneglycol2000) ethoxy]-N,N-ditetradecylacetamide.

56. The pharmaceutical composition of any one of claims 52-55, wherein said pharmaceutical composition comprises between about 2.6-2.8 mg / mL of 2- [2-(o -methoxy (polyethyleneglycol2000) ethoxy]-N,N-ditetradecylacetamide.

57. The pharmaceutical composition of any one of claims 52-56, wherein said pharmaceutical composition comprises between about 2.5-3.4 mg / mL of DSPC.

58. The pharmaceutical composition of any one of claims 52-57, wherein said pharmaceutical composition comprises between about 3.

3. -3.4 mg / mL of DSPC.

59. The pharmaceutical composition of any one of claims 52-58, wherein said pharmaceutical composition comprises between about 5.5-7.0 mg / mL of cholesterol.

60. The pharmaceutical composition of any one of claims 52-59, wherein said pharmaceutical composition comprises between about 6.4-6.6 mg / mL of cholesterol.

61. The pharmaceutical composition of any one of claims 52-60, wherein said pharmaceutical composition comprises between about 0.8-1.2 mg / mL of said polynucleotide.

62. The pharmaceutical composition of any one of claims 52-61, wherein said pharmaceutical composition comprises between about 0.95-1.1 mg / mL of said polynucleotide.

63. The pharmaceutical composition of any one of claims 52-62, wherein said pharmaceutical composition comprises about 1.0 mg / mL of said polynucleotide.

64. The pharmaceutical composition of any one of claims 52-63, wherein said plurality of LNPs are formulated in phosphate-buffered saline and sucrose.

65. The pharmaceutical composition of claim 64, wherein said sucrose is at a concentration of about 300 mM.WBD (US) 4897-9494-7440vl 173 Atty Docket No. P89339 2080WO (01275)66. The pharmaceutical composition of any one of claims 52-65, wherein said pharmaceutical composition has a pH between about 6.7-7.6.

67. The pharmaceutical composition of any one of claims 52-66, wherein said pharmaceutical composition has a pH of about 6.92-7.42.

68. The pharmaceutical composition of any one of claims 52-66, wherein said pharmaceutical composition has a pH of about 7.4-7.5.

69. The pharmaceutical composition of any one of claims 52-68, wherein said pharmaceutical composition has a poly dispersity index equal to, or less than, 0.20.

70. The pharmaceutical composition of any one of claims 52-69, wherein the osmolality of said pharmaceutical composition is between about 495-605 mOsm / kg.

71. The pharmaceutical composition of any one of claims 52-70, wherein the osmolality of said pharmaceutical composition is about 564-603 mOsm / kg.

72. The pharmaceutical composition of any one of claims 52-71, wherein about 85% or more of said plurality of LNPs comprise said polynucleotide.

73. A method for treating a disease associated with hepatitis B virus infection, said method comprising administering to an HBV-infected subject a therapeutically effective dose of a pharmaceutical composition comprising a plurality of said LNP of any one of claims 1-49, wherein said plurality of said LNP are delivered to target cells in said subject that comprise an HBV genome or HBV genome fragment, wherein said engineered meganuclease is expressed in said target cells and produces a cleavage site at a recognition sequence comprising SEQ ID NO: 3 in a pol gene in said HBV genome or said HBV genome fragment.

74. The method of claim 73, wherein said polynucleotide is an mRNA.

75. The method of claim 74, wherein said LNP is said LNP of any one of claims 41-49.WBD (US) 4897-9494-7440vl 174 Atty Docket No. P89339 2080WO (01275)76. The method of any one of claims 73-75, wherein said HBV genome or said HBV genome fragment is comprised by cccDNA.

77. The method of claim 76, wherein said cccDNA is eliminated following generation of said cleavage site.

78. The method of claim 76, wherein said pol gene is inactivated in said cccDNA by introduction of said indel at said cleavage site.

79. The method of claim 77 or claim 78, wherein said inactivated pol gene does not encode an active and / or full-length HBV polymerase protein.

80. The method of any one of claims 73-75, wherein said HBV genome or said HBV genome fragment is comprised in the genome of said target cells.

81. The method of claim 80, wherein said pol gene is inactivated in said HBV genome or said HBV genome fragment by introduction of an indel at said cleavage site.

82. The method of claim 80 or claim 81, wherein said genome is the nuclear genome.

83. The method of claim 80 or claim 81, wherein said genome is the mitochondrial genome.

84. The method of any one of claims 80-83, wherein said inactivated pol gene does not encode an active and / or full-length HBV polymerase protein.

85. The method of any one of claims 73-84, wherein said method inactivates an HBsAg gene in said HBV genome or said HBV genome fragment.

86. The method of any one of claims 73-85, wherein said target cells are liver cells.

87. The method of claim 86, wherein said liver cells are hepatocytes.

88. The method of any one of claims 73-87, wherein said pharmaceutical composition is said pharmaceutical composition of any one of claims 52-72.WBD (US) 4897-9494-7440vl 175 Aty Docket No. P89339 2080WO (01275)89. The method of any one of claims 73-88, wherein said pharmaceutical composition is administered to said subject by intravenous (IV) administration.

90. The method of any one of claims 73-89, wherein said pharmaceutical composition is administered by IV administration over about 2 hours.

91. The method of any one of claims 73-90, wherein said pharmaceutical composition is administered at a dose of between about 0.01-2.0 mg / kg of said polynucleotide.

92. The method of any one of claims 73-91, wherein said pharmaceutical composition is administered at a dose of between about 0.1-1.5 mg / kg of said polynucleotide.

93. The method of any one of claims 73-92, wherein said pharmaceutical composition is administered at a dose of about 0.1 mg / kg of said polynucleotide.

94. The method of any one of claims 73-92, wherein said pharmaceutical composition is administered at a dose of about 0.2 mg / kg of said polynucleotide.

95. The method of any one of claims 73-92, wherein said pharmaceutical composition is administered at a dose of about 0.3 mg / kg of said polynucleotide.

96. The method of any one of claims 73-92, wherein said pharmaceutical composition is administered at a dose of about 0.4 mg / kg of said polynucleotide.

97. The method of any one of claims 73-92, wherein said pharmaceutical composition is administered at a dose of about 0.5 mg / kg of said polynucleotide.

98. The method of any one of claims 73-92, wherein said pharmaceutical composition is administered at a dose of about 0.8 mg / kg of said polynucleotide.

99. The method of any one of claims 73-92, wherein said pharmaceutical composition is administered at a dose of about 1.0 mg / kg of said polynucleotide.WBD (US) 4897-9494-7440vl 176 Atty Docket No. P89339 2080WO (01275)100. The method of any one of claims 73-99, wherein said subject is administered a single dose of said pharmaceutical composition on day 0.

101. The method of claim 100, wherein said subject is further administered a second dose of said pharmaceutical composition following administration of said first dose.

102. The method of claim 100 or claim 101, wherein said subject is further administered a second dose of said pharmaceutical composition about 8 weeks following administration of said first dose.

103. The method of claim 101 or claim 102, wherein said first dose and said second dose are equivalent.

104. The method of claim 101 or claim 102, wherein said second dose is higher than said first dose.

105. The method of claim 101 or claim 102, wherein said second dose is lower than said first dose.

106. The method of any one of claims 101-103, wherein said first dose is about 0.1 mg / kg of said polynucleotide and said second dose is about 0.1 mg / kg of said polynucleotide.

107. The method of any one of claims 101-103, wherein said first dose is about 0.2 mg / kg of said polynucleotide and said second dose is about 0.2 mg / kg of said polynucleotide.

108. The method of any one of claims 101-103, wherein said first dose is about 0.3 mg / kg of said polynucleotide and said second dose is about 0.3 mg / kg of said polynucleotide.

109. The method of any one of claims 101-103, wherein said first dose is about 0.4 mg / kg of said polynucleotide and said second dose is about 0.4 mg / kg of said polynucleotide.

110. The method of any one of claims 101-103, wherein said first dose is about 0.5 mg / kg of said polynucleotide and said second dose is about 0.5 mg / kg of said polynucleotide.WBD (US) 4897-9494-7440vl 177 Atty Docket No. P89339 2080WO (01275)111. The method of any one of claims 101-103, wherein said first dose is about0.8 mg / kg of said polynucleotide and said second dose is about 0.8 mg / kg of said polynucleotide.

112. The method of any one of claims 101-103, wherein said first dose is about 1.0 mg / kg of said polynucleotide and said second dose is about 1.0 mg / kg of said polynucleotide.

113. The method of any one of claims 101-112, wherein said subject is further administered a third dose of said pharmaceutical composition following administration of said second dose.

114. The method of any one of claims 101-113, wherein said subject is further administered a third dose of said pharmaceutical composition about 8 weeks following administration of said second dose.

115. The method of claim 113 or claim 114, wherein said first dose, said second dose, and said third dose are equivalent.

116. The method of claim 113 or claim 114, wherein said third dose is higher than said second dose.

117. The method of claim 113 or claim 114, wherein said third dose is lower than said second dose.

118. The method of any one of claims 113-115, wherein said first dose is about 0.1 mg / kg of said polynucleotide, said second dose is about 0.1 mg / kg of said polynucleotide, and said third dose is about 0.1 mg / kg of said polynucleotide.

119. The method of any one of claims 113-115, wherein said first dose is about 0.2 mg / kg of said polynucleotide, said second dose is about 0.2 mg / kg of said polynucleotide, and said third dose is about 0.2 mg / kg of said polynucleotide.

120. The method of any one of claims 113-115, wherein said first dose is about 0.3 mg / kg of said polynucleotide, said second dose is about 0.3 mg / kg of said polynucleotide, and said third dose is about 0.3 mg / kg of said polynucleotide.WBD (US) 4897-9494-7440vl 178 Atty Docket No. P89339 2080WO (01275)121. The method of any one of claims 113-115, wherein said first dose is about 0.4 mg / kg of said polynucleotide, said second dose is about 0.4 mg / kg of said polynucleotide, and said third dose is about 0.4 mg / kg of said polynucleotide.

122. The method of any one of claims 113-115, wherein said first dose is about 0.5 mg / kg of said polynucleotide, said second dose is about 0.5 mg / kg of said polynucleotide, and said third dose is about 0.5 mg / kg of said polynucleotide.

123. The method of any one of claims 113-115, wherein said first dose is about 0.8 mg / kg of said polynucleotide, said second dose is about 0.8 mg / kg of said polynucleotide, and said third dose is about 0.8 mg / kg of said polynucleotide.

124. The method of any one of claims 113-115, wherein said first dose is about 1.0 mg / kg of said polynucleotide, said second dose is about 1.0 mg / kg of said polynucleotide, and said third dose is about 1.0 mg / kg of said polynucleotide.

125. The method of any one of claims 73-124, wherein said subject is administered a steroid about 12 hours prior to administration of said pharmaceutical composition.

126. The method of any one of claims 73-125, wherein said subject is administered a steroid between about 1-2 hours prior to administration of said pharmaceutical composition.

127. The method of any one of claims 73-126, wherein said subject is administered an Hl blocker between about 1-2 hours prior to administration of said pharmaceutical composition.

128. The method of any one of claims 73-127, wherein said subject is administered an H2 blocker between about 1-2 hours prior to administration of said pharmaceutical composition.

129. The method of any one of claims 73-128, wherein said subject is administered a steroid about 4 hours following administration of said pharmaceutical composition.

130. The method of any one of claims 73-129, wherein said subject has been administered a nucleos(t)ide analogue therapy for at least 6 months immediately prior to administration of said pharmaceutical composition and achieved an adequate virologic response.WBD (US) 4897-9494-7440vl 179 Atty Docket No. P89339 2080WO (01275)131. The method of claim 130, wherein said subject continues to be administered said nucleos(t)ide analogue therapy for at least 8 weeks following the last administration of said pharmaceutical composition.

132. The method of claim 131, wherein said nucleos(t)ide analogue therapy is discontinued if: serum HBsAg concentrations are less than 0.05 lU / mL, serum HBV DNA concentrations are less than 10 lU / mL, and serum alanine transaminase (ALT) concentrations are less than 1.5 x upper limit of normal (ULN); or serum HBsAg concentrations are greater than 0.05 lU / mL and less than 50 lU / mL, serum HBV DNA concentrations are less than 10 lU / mL, and ALT concentrations are less than 1.5 x upper limit of normal (ULN), serum HBV RNA concentrations are undetectable, and HBcrAg concentrations are undetectable, wherein the conditions of a) or b) are met on two occasions at least 4 weeks apart, and no earlier than 8 weeks since the last dose of said pharmaceutical composition, and wherein said subject is monitored for about 48 weeks following discontinuation of nucleos(t)ide analogue therapy for achievement of a functional cure or a partial cure.

133. The method of claim 132, wherein said functional cure comprises sustained serum HBsAg concentrations below 0.05 lU / mL with or without seroconversion of antibody to HBsAg for at least 24 weeks following administration of the last dose of said pharmaceutical composition.

134. The method of claim 132, wherein said partial cure comprises sustained reductions of serum HBsAg concentrations to less than 50 lU / mL but greater than 0.05 lU / mL, serum HBV DNA concentrations less than 10 lU / mL, and serum ALT concentrations less than 1.5 x ULN for at least 24 weeks following administration of the last dose of said pharmaceutical composition.

135. The method of any one of claims 73-134, wherein said subject has chronic hepatitis B with compensated liver disease.

136. The method of any one of claims 73-135, wherein said subject has received a nucleos(t)ide analogue therapy immediately prior to administration of said pharmaceutical composition and achieved an adequate virologic response.WBD (US) 4897-9494-7440vl 180 Atty Docket No. P89339 2080WO (01275)137. The method of any one of claims 73-136, wherein said subject has received a nucleos(t)ide analogue therapy for at least 6 months immediately prior to administration of said pharmaceutical composition.

138. The method of any one of claims 73-137, wherein the serum HBsAg concentration is reduced in said subject following administration of said pharmaceutical composition compared to the serum HBsAg concentration prior to said administration.

139. The method of any one of claims 73-138, wherein the serum HBsAg concentration is reduced by about 90%-100% in said subject following administration of said pharmaceutical composition compared to the serum HBsAg concentration prior to said administration.

140. The method of any one of claims 73-139, wherein the serum HBsAg concentration is reduced by 100% in said subject following administration of said pharmaceutical composition compared to the serum HBsAg concentration prior to said administration.

141. The method of any one of claims 73-140, wherein the serum HBsAg concentration in said subject is less than 50 lU / mL following administration of said pharmaceutical composition.

142. The method of any one of claims 73-141, wherein the serum HBsAg concentration in said subject is less than 0.05 lU / mL following administration of said pharmaceutical composition.

143. The method of any one of claims 73-142, wherein the plasma HBV DNA concentration is reduced in said subject following administration of said pharmaceutical composition compared to the plasma HBV DNA concentration prior to said administration.

144. The method of any one of claims 73-143, wherein the plasma HBV DNA concentration is reduced by about 90%-100% in said subject following administration of said pharmaceutical composition compared to the plasma HBV DNA concentration prior to said administration.

145. The method of any one of claims 73-144, wherein the plasma HBV DNA concentration is reduced by 100% in said subject following administration of said pharmaceutical composition compared to the plasma HBV DNA concentration prior to said administration.WBD (US) 4897-9494-7440vl 181 Aty Docket No. P89339 2080WO (01275)146. The method of any one of claims 73-145, wherein the serum HBV RNA concentration is reduced in said subject following administration of said pharmaceutical composition compared to the serum HBV RNA concentration prior to said administration.

147. The method of any one of claims 73-146, wherein the serum HBV RNA concentration is reduced by about 90%-100% in said subject following administration of said pharmaceutical composition compared to the serum HBV RNA concentration prior to said administration.

148. The method of any one of claims 73-147, wherein the serum HBV RNA concentration is reduced by 100% in said subject following administration of said pharmaceutical composition compared to the serum HBV RNA concentration prior to said administration.

149. The method of any one of claims 73-148, wherein the serum HBcrAg concentration is reduced in said subject following administration of said pharmaceutical composition compared to the serum HBcrAg concentration prior to said administration.

150. The method of any one of claims 73-149, wherein the serum HBcrAg concentration is reduced by about 90%-100% in said subject following administration of said pharmaceutical composition compared to the serum HBcrAg concentration prior to said administration.

151. The method of any one of claims 73-150, wherein the serum HBcrAg concentration is reduced by 100% in said subject following administration of said pharmaceutical composition compared to the serum HBcrAg concentration prior to said administration.

152. The method of any one of claims 73-151, wherein the HBV cccDNA concentration in said target cells is reduced in said subject following administration of said pharmaceutical composition compared to an appropriate control.

153. The method of any one of claims 73-152, wherein the HBV cccDNA concentration in target cells in said subject is reduced by about 90%-100% following administration of said pharmaceutical composition compared to an appropriate control.WBD (US) 4897-9494-7440vl 182 Atty Docket No. P89339 2080WO (01275)154. The method of any one of claims 73-153, wherein the HBV cccDNA concentration in target cells in said subject is reduced by 100% following administration of said pharmaceutical composition compared to an appropriate control.

155. The method of any one of claims 73-154, wherein between about 50%-100% of HBV cccDNA present in said target cells comprises an indel at said recognition sequence after administration of said pharmaceutical composition.

156. The method of any one of claims 73-155, wherein between about 80%-100% of HBV cccDNA present in said target cells comprises an indel at said recognition sequence after administration of said pharmaceutical composition.

157. The method of any one of claims 73-156, wherein between about 90%-100% of HBV cccDNA present in said target cells comprises an indel at said recognition sequence after administration of said pharmaceutical composition.

158. The method of any one of claims 73-157, wherein between about 95%-100% of HBV cccDNA present in said target cells comprises an indel at said recognition sequence after administration of said pharmaceutical composition.

159. The method of any one of claims 73-158, wherein between about 50%-100% of integrated HBV genomes or genome fragments present in the genome of said target cells comprises an indel at said recognition sequence after administration of said pharmaceutical composition.

160. The method of any one of claims 73-159, wherein between about 80%-100% of integrated HBV genomes present in the genome of said target cells comprises an indel at said recognition sequence after administration of said pharmaceutical composition.

161. The method of any one of claims 73-160, wherein between about 90%-100% of integrated HBV genomes present in the genome of said target cells comprises an indel at said recognition sequence after administration of said pharmaceutical composition.

162. The method of any one of claims 73-161, wherein between about 95%-100% of integrated HBV genomes present in the genome of said target cells comprises an indel at said recognition sequence after administration of said pharmaceutical composition.WBD (US) 4897-9494-7440vl 183 Aty Docket No. P89339 2080WO (01275)

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