Gene therapy for lamin A-related deficiencies

Abstract

Provided herein is a recombinant AAV (rAAV) comprising an AAV capsid and a vector genome packaged therein, the vector genome comprising an AAV 5' inverted terminal repeat (ITR), an expression cassette comprising an open reading frame (ORF) for mature human lamin A (hLamin A) under the control of regulatory sequences directing expression of hLamin A in a target cell, and an AAV 3' ITR. Also provided are pharmaceutical compositions comprising the rAAV described herein in a formulation buffer, as well as methods for treating idiopathic dilated cardiomyopathy (DCM) or diseases associated with mutations in the lamin A (LMNA) gene.

Description

[Technical field]

[0001] Electronic Sequence Listing Reference The electronic sequence listing entitled "UPN-22-9866PCT.xml", which was filed herewith and was created on December 22, 2022, and is 62,136 bytes in size, and the contents of the electronic sequence listing (e.g., the sequences and text therein) are hereby incorporated by reference in their entirety. [Background technology]

[0002] The prevalence of idiopathic dilated cardiomyopathy (DCM) is estimated to be 1:500 (Hershberger, R., Hedges, D. & Morales, A. Dilated cardiomyopathy: the complexity of a diverse genetic architecture. Nat Rev Cardiol 10, 531-547 (2013)). It has been estimated that approximately 40% of the genetic causes of DCM are due to rare variants in fewer than three genes that subsequently affect the function of various proteins, among which the lamin A gene (LMNA) is the most important. The estimated prevalence of lamin A gene (LMNA) mutations in idiopathic DCM is 5.9% (19 / 324) among unrelated DCM probands (USA) (Parks SB, Kushner JD, Nauman D, et al. Lamin A / C mutation analysis in a cohort of 324 unrelated patients with idiopathic or familial dilated cardiomyopathy. Am Heart J 2008;156:161-9) and 6% (35 / 561) among DCM patients (Norway) (Hasselberg et al. European Heart Journal (2018) 39,853-860). The most common loss-of-function mutations cause adult-onset dilated cardiomyopathy with conduction defects. The overall estimated prevalence of LMNA-related cardiomyopathy is 1:8,000 population (40,000 patients in the US). Patients typically present with atrial arrhythmias or atrioventricular (AV) conduction defects in the fourth to fifth decades of life, then progress to complete AV block, dilated cardiomyopathy, ventricular arrhythmias, and end-stage heart failure. By age 60, most patients have had an implantable cardioverter-defibrillator placed, have undergone a heart transplant, or have died.

[0003] Lamin A and C proteins are essential structural components of the nuclear envelope and also play a role in gene expression through their interaction with chromatin. Mutations in the Lamin A / C (LMNA) gene are associated with diverse clinical phenotypes beyond DCM, including neuropathy, muscular dystrophy, progeria, and lipodystrophy (Kang et al. BMB Reports 2018;51:327-37).

[0004] There is no standard treatment or cure for idiopathic DCM and LMNA-related disorders. There is a continuing need in the art for compositions and methods for the effective treatment of idiopathic DCM and LMNA-related disorders. Summary of the Invention

[0005] In one aspect, provided herein is a recombinant adeno-associated virus comprising a capsid and having a vector genome packaged therein, the vector genome comprising an AAV 5' inverted terminal repeat (ITR), an expression cassette, and an AAV 3' ITR, the expression cassette comprising an engineered open reading frame (ORF) for a mature human lamin A (hLaminA) coding sequence encoding a mature hLaminA lacking a preprotein carboxy (C)-terminal tail, the ORF being operably linked to regulatory control sequences directing expression of the mature hLaminA protein in a cell, the regulatory control sequences comprising a promoter, optionally an enhancer, and a polyadenylation (polyA) sequence. In certain embodiments, the ORF has a nucleic acid sequence of SEQ ID NO: 4, or a nucleic acid sequence at least 90% identical to SEQ ID NO: 4, encoding a mature hLaminA lacking a preprotein carboxy (C)-terminal tail. In certain embodiments, the ORF is operably linked to regulatory control sequences comprising a promoter that is a cardiac promoter. In certain embodiments, the promoter is a chicken cardiac troponin T promoter. In certain embodiments, the regulatory control sequence further comprises a CMV IE enhancer, a rabbit globin polyadenylation sequence, and / or optionally a WPRE element.

[0006] In certain embodiments, the expression cassette has the nucleic acid sequence of SEQ ID NO: 2, or a nucleic acid sequence at least 90% identical to SEQ ID NO: 2. In certain embodiments, the vector genome has the nucleic acid sequence of SEQ ID NO: 1 (CMV-IE.chTNTp.LaminA.RBG). In certain embodiments, the capsid is an AAVhu68 capsid, an AAVhu95 capsid, or an AAVhu96 capsid.

[0007] In a further aspect, compositions and pharmaceutical compositions are provided herein that comprise the rAAV or vector described herein and an aqueous suspension medium.In certain embodiments, the rAAV or composition thereof is for use in treating idiopathic dilated cardiomyopathy (DCM) or diseases associated with mutations in the Lamin A (LMNA) gene.In certain embodiments, the diseases associated with mutations in the LMNA gene are selected from muscular dystrophy, neuropathy, lipodystrophy, and partial progeroid.

[0008] In another aspect, provided herein is a method for treating or alleviating or ameliorating one or more symptoms of idiopathic dilated cardiomyopathy (DCM) in a subject in need of such treatment or alleviation or amelioration.In a further aspect, provided herein is a method for treating or alleviating or ameliorating one or more symptoms of disease associated with mutations in Lamin A (LMNA) gene in a subject.In certain embodiments, the idiopathic DCM is early-onset idiopathic DCM.In certain embodiments, the idiopathic DCM is adult-onset idiopathic DCM with conduction defects. In certain embodiments, the disease associated with a loss-of-function mutation in the LMNA gene is selected from muscular dystrophy, neuropathy, lipodystrophy, partial progeroid, and optionally further selected from Emery-Dreifuss muscular dystrophy (EDMD), Malouf syndrome (MLF), congenital muscular dystrophy (MDC), limb-girdle muscular dystrophy type 1B (LGMD1B), Charcot-Marie-Tooth disease type 2B1 (CMT2B1), axonal neuropathy, familial partial lipodystrophy type 2 (FPLD2), mandibuloacral dysplasia lipodystrophy (MAD), mandibuloacral dysplasia type A (MADA), atypical Werner syndrome (AWS), premature aging syndrome (progeria), and Hutchinson-Gilford progeria syndrome (HGPS). In certain embodiments, the disease symptoms include atrioventricular (AV) conduction block, atrial arrhythmias including atrial fibrillation, atrial flutter and atrial tachycardia, ventricular arrhythmias including sustained ventricular tachycardia and ventricular fibrillation (VF). In certain embodiments, the method further includes combination therapy with beta-blockers, angiotensin-converting enzyme (ACE) inhibitors, diuretics, implantable cardioverter defibrillators (ICDs), pacemakers (PMs), and / or cardiac resynchronization therapy (CRT).

[0009] In another aspect, provided herein is a recombinant nucleic acid molecule comprising an expression cassette of SEQ ID NO: 2. In certain embodiments, the nucleic acid molecule is a plasmid. In certain embodiments, a packaging cell is provided that comprises the expression cassette, vector genome, or plasmid.

[0010] These and other aspects of the invention will become apparent from the following detailed description of the invention. [Brief description of the drawings]

[0011] [Figure 1A] Relative levels of gene transfer into NHP hearts for AAVhu68 plotted as fold change in RNA sequencing reads (prevalence of RNA reads in tissue versus administered vector concentration). [Figure 1B] 1 shows the level of transduction in mouse hearts following administration of AAVhu68 or AAVhu95, which contains the gene encoding green fluorescent protein, plotted as percent of GFP-positive area. [Figure 1C] 1 shows the level of transduction in mouse hearts following administration of AAVhu68 or AAVhu95, which contains the gene encoding green fluorescent protein, plotted as number of copies / ng RNA. [Figure 2A] Kaplan-Meier survival plots of lamin knockout (KO) and wild-type (WT) mice neonatally administered either vehicle control or AAVhu68.hLaminA intravenously at a dose of 5 x 10 10 GC (approximately 3 x 10 13 GC / kg). [Figure 2B] Shown are the measured body weights of lamin knockout (KO) and wild-type (WT) mice neonatally administered intravenously with either vehicle control or AAVhu68.hLaminA at a dose of 5×10 10 GC (approximately 3×10 13 GC / kg). [Figure 2C] Representative Western blots confirming expression of Lamin A in the heart and lack of expression of Lamin A in the liver following administration of AAVhu68.hLaminA in knockout mice are shown. [Figure 3A] Representative microscopic images from immunohistochemistry (IHC) analysis of anti-human lamin staining of FRG mouse liver tissue following transplantation of human hepatocytes (human cells are cells that exhibit lamin staining) are shown. [Figure 3B]Representative microscopic images from immunohistochemistry (IHC) analysis of anti-human lamin staining of mouse cardiac tissue following neonatal administration of vehicle control are shown. [Figure 3C] Representative microscopic images from immunohistochemistry (IHC) analysis of anti-human lamin staining of mouse heart tissue following neonatal intravenous administration of AAVhu68.hLaminA at a dose of 5 x 10 10 GC (approximately 3 x 10 13 GC / kg) are shown. [Figure 4A] Representative electrocardiogram analysis showing RR intervals (sec) over time in lamin A KO mice administered AAV (0-23) is shown. [Figure 4B] Representative electrocardiogram analyses showing RR intervals (seconds) over time in lamin A KO mice administered AAV (24–48) are shown. [Figure 4C] Representative electrocardiogram analysis showing the RR interval (sec) over time in WT mice administered vehicle (PBS) is shown (0-12). [Figure 4D] Representative electrocardiogram analyses showing RR intervals (seconds) over time in WT mice administered vehicle (PBS) are shown (13–26). [Figure 5A] Representative electrocardiogram analysis showing RR intervals (seconds) over time in Lamin A KO mice administered vehicle (PBS) is shown (0-18). [Figure 5B] Representative electrocardiogram analysis showing RR intervals (seconds) over time in lamin A KO mice administered vehicle (PBS) is shown (19-38). [Figure 5C] Representative electrocardiogram analysis showing RR intervals (seconds) over time in Lamin A KO mice administered vehicle (PBS) is shown (zoomed in 5A, times 0-10). [Figure 5D] Representative electrocardiogram analysis showing RR intervals (seconds) over time in Lamin A KO mice administered vehicle (PBS) is shown (zoomed in 5B, times 7.00-7.45). [Figure 6A]Shown are echocardiogram results in wild-type (WT) and knockout (KO) mice administered vehicle (PBS) or AAV, plotted as ejection fraction (%) (One-way Anova nonparametric Kruskal-Wallis with Dunn's multiple comparison test: P<0.05, **P<0.01, ***P<0.01, ****P<0.0001). [Figure 6B] Shown are echocardiogram results in wild-type (WT) and knockout (KO) mice administered vehicle (PBS) or AAV, plotting fractional shortening (%) (One-way Anova nonparametric Kruskal-Wallis with Dunn's multiple comparison test: P<0.05, **P<0.01, ***P<0.01, ****P<0.0001). [Figure 6C] Shown are echocardiogram results in wild-type (WT) and knockout (KO) mice administered vehicle (PBS) or AAV, plotted as stroke volume (µL) (One-way Anova nonparametric Kruskal-Wallis with Dunn's multiple comparison test: P<0.05, **P<0.01, ***P<0.01, ****P<0.0001). [Figure 7A] Representative images of histological analysis of cardiac tissue in knockout mice following administration of PBS in knockout mice are shown. [Figure 7B] Representative images of histological analysis of cardiac tissue in knockout mice following administration of AAV-LMNA in knockout mice are shown, confirming expression of Lamin A in ventricular cardiac cells. [Figure 8] Representative Western blot analysis of Lamin A expression in mice administered AAVhu95-LMNA is shown. [Figure 9A] LMNA telemetry study results plotted as percent of WT-PBS of LMNA expression in wild type and heterozygous knockout mice following administration of either PBS (control) or AAV-LMNA. [Figure 9B]LMNA telemetry study results plotted as percent of WT-PBS of LMNC expression in wild type and heterozygous knockout mice following administration of either PBS (control) or AAV-LMNA. [Figure 9C] Representative Western blot analysis of heart samples of lamin A and lamin C expression in AAVhu95-LMNA administered mice (wild type and heterozygous knockout mice) is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Provided herein are sequences, expression cassettes, and vectors expressing human functional mature lamin A (hLAmin A) protein, and compositions containing them.Also provided herein are methods useful for treating and / or alleviating symptoms of LMNA cardiomyopathy (e.g., idiopathic dilated cardiomyopathy (DCM) or diseases associated with mutations in the lamin A (LMNA) gene).In certain embodiments, human lamin A (hLAmin A) protein is delivered via the AAV provided herein.

[0013] The various nucleic acid sequences provided herein are useful for packaging a functional mature hLamin A coding sequence into a suitable vector (e.g., rAAV) or genetic element (e.g., a plasmid) useful for production.

[0014] The Lamin A / C gene (LMNA) is located at the chromosome 1q21.2 locus and contains alternative splicing sites that encode the Lamin A or Lamin C proteins (Kang S., et al., 2018). The native amino acid sequences of Lamin A and Lamin C are identical over the first 566 amino acids, but Lamin C has a unique carboxy (C-) terminal sequence of six amino acids "VSGSRR" (SEQ ID NO: 21). In contrast, the mature Lamin A protein has at its C-terminus "GSHCSSSGDPAEYNLRSRTVLCGTCGQPADKASASGSGAQVGGPISSGSSASSVTVTRSYRSVGGSGGGSFGDNLVTRSY" (SEQ ID NO: 22).

[0015] SEQ ID NO:19 provides 664 amino acids of full length lamin A preprotein, i.e. mature lamin A with the carboxy terminus as above, as well as an 18 amino acid carboxy tail containing a CaaX motif (i.e. CSIM) and a farnesylation motif at the carboxy (C) terminus of the amino acid sequence. Prelamin A is matured by protease-mediated cleavage of the last 18 amino acids to yield mature lamin A. SEQ ID NO:5 refers to the mature lamin A protein lacking the preprotein C-terminal tail motif (LLGNSSPRTQSPQNCSIM; SEQ ID NO:20).

[0016] As used herein, the terms "functional lamin A" and / or "functional mature human lamin A" refer to a protein having the amino acid sequence of a mature lamin A protein having the sequence of SEQ ID NO: 5, or a sequence about 95% to about 100% identical thereto, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, 99.9% identical thereto, and values ​​therebetween (as determined over a contiguous amino acid sequence that provides at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, or more than 100% of the same and / or greater biological activity or function as wild-type mature human lamin A). This biological activity or function may be determined by any suitable means, such as in vitro assays, animal models, or monitoring of a patient following treatment for correction of symptoms of a condition associated with dysfunctional or non-functional lamin A. In certain embodiments, the mutant mature human lamin A may have one or more conservative amino acid substitutions, e.g., 1 to 30 amino acid changes, compared to the amino acid sequence of SEQ ID NO: 5. In certain embodiments, the mutant mature human lamin A protein may be about 95% to about 100% identical to SEQ ID NO: 5 and may include one or more conservative, non-conservative amino acid substitutions, as well as insertions and / or deletions. In certain embodiments, the substitution resulting in a mutant human lamin A having SEQ ID NO: 21 ("VSGSRR") in the region of amino acids 567 to 572 (referenced to SEQ ID NO: 5) is excluded. In certain embodiments, about 10% to about 100% of wild-type mature human lamin. In certain embodiments, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, or at least 95% of normal wild-type mature human lamin A activity and / or function is achieved. In certain embodiments, greater than 100% of normal wild-type mature human lamin A activity and / or function is achieved, for example, about 105%, about 110%, about 115%, about 120%, about 125%, about 130% or more.

[0017] As described herein, any variant or mutant of mature human lamin A expressed from the nucleic acid sequences provided herein or variations thereof restores the desired function, alleviates symptoms, or ameliorates symptoms associated with DCM or diseases associated with mutations in the LMNA gene. The amino acid substitutions are selected to avoid any changes that render mature human lamin A dysfunctional or non-functional, for example, by introducing substitutions associated with diseases as described herein.

[0018] As used herein, "conservative amino acid replacement" or "conservative amino acid substitution" refers to the modification, replacement, or substitution of an amino acid with a different amino acid having similar biochemical properties (e.g., charge, hydrophobicity, and size) known by those skilled in the art. Also see, for example, FRENCH et al. What is a conservative substitution? Journal of Molecular Evolution, March 1983, Volume 19, Issue 2, pp 171-175 and YAMPOLSKY et al. The Exchangeability of Amino Acids in Proteins, Genetics. 2005 Aug; 170(4): 1459-1472, each of which is incorporated herein by reference in its entirety. Without wishing to be bound by theory, conservative amino acid replacement excludes amino acid substitutions in the mature Lamin A protein that are associated with disease, as described herein.

[0019] The following are examples of amino acid substitutions that may render mature human lamin A dysfunctional or non-functional: For example, amino acid substitutions in lamin A associated with DCM may include one or more of Q6X, E203K, R25G, R25P, E203G, R25W, L215P, L59R, R225X, R60G, Y267C, E82K, E317K, L85R, A347K, R89L, R349L, K97E, Q355X, S143P, R399C, E161K, R435C, R190W, R541C, D192G, R541S, N195K, S573L, S573L, R133P, E358K, L530P, R584H, T623S, and R644C (with reference to the amino acid sequence of the lamin A preprotein; SEQ ID NO: 19). The amino acid substitutions in lamin A protein associated with muscular dystrophies are Q6X, G232E, G449D, A57P, N39S, R25G, R25P, L248P, R453W, L59R, R50P, Y259X, R25G, R249Q, L454P, R249W, E358K, E33G, R2 49W, N456I, L302P, R377H, L35V, F260L, N456K, E358K, R377L, N39S, Y267C, D461Y , L380S, R399C, A43T, S268P, W467R, R453P, Y481H, Y45C, L271P, I469T, R455P, R5 0S, Q294P, W520S, N456D, I63S, S295P, R527P, I63N, S303P, T528K, E65G, R336Q, T 528R, R89C, R343Q, L529P, R133P, E358K, L530P, L140P, E361K, R541H, T150P, M37 The amino acid substitutions in the Lamin A protein associated with neuropathy may include one or more of the following: 1K, R541S, R189P, R377L, R541P, R190Q, R386K, G602S, R196S, R401C, R624H, H222P, V442A, H222Y, D446V (with reference to the amino acid sequence of the Lamin A preprotein; SEQ ID NO: 19). The amino acid substitutions in the Lamin A protein associated with neuropathy may include R298C (with reference to the amino acid sequence of the Lamin A preprotein; SEQ ID NO: 19).Amino acid substitutions in Lamin A protein associated with lipodystrophy may include one or more of R25W, V440M, R60G, R471C, R62G, R527C, ΔK208, R527H, D230N, A529V, G456D, R482W, R482Q, R482L, P485R, K486N, S573L, R582H, R584H (with reference to the amino acid sequence of Lamin A preprotein; SEQ ID NO: 19). Amino acid substitutions in Lamin A proteins associated with partial progeroids may include one or more of A57P, T10I, R133L, S143E, L140R, S143F, D300N, E145K, Q656Q, R471C, R527C, T528M, M540T, K542N, E578V, V607V, G608S, G608G, T623S (with reference to the amino acid sequence of Lamin A preprotein; SEQ ID NO: 19). See also Kang, S., et al., Laminopathies; Mutations on single gene and various human genetic diseases, BMB Reports 2018, 51(7):327-337; Rankin, J., et al., The laminopathies: a clinical review, Clin. Genet., 2006, 70:261-274; Vigouroux C, Bonne G. Laminopathies: One Gene, Two Proteins, Five Diseases. In: Madame Curie Bioscience Database [Internet]. Austin (TX): Landes Bioscience; 2000-2013, ncbi.nlm.nih.gov / books / NBK6151 / (all of which are incorporated by reference in their entireties).

[0020] In one embodiment, functional mature hLamin A has an amino acid sequence of SEQ ID NO:5, or an amino acid sequence at least about 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or 99.9%) identical thereto.

[0021] In certain embodiments, functional mature hLamin A protein ameliorates symptoms or delays progression of LMNA cardiomyopathy (e.g., idiopathic dilated cardiomyopathy (DCM)) or disease associated with mutations in the Lamin A (LMNA) gene in animal models. One exemplified animal model is the LMNA knockout (LMNA-ko) mouse. Other suitable models may be used. LMNA cardiomyopathy symptoms or progression may be assessed using a variety of assays / methods, including, but not limited to, survival plots (e.g., Kaplan-Meier survival plots), weight monitoring, echocardiograms (echo), and electrocardiograms (EKG or ECG). In certain embodiments, administration or expression of a functional mature hLamin A protein in an animal model results in an alleviation of symptoms of LMNA cardiomyopathy or a delay in the progression of LMNA cardiomyopathy as indicated by an assay result that is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, or more than 100% of that obtained in a corresponding wild-type animal. In certain embodiments, administration or expression of a functional mature hLamin A protein in an animal model of LMNA cardiomyopathy results in an alleviation of symptoms of LMNA cardiomyopathy or a delay in the progression of LMNA cardiomyopathy as indicated by an improved assay result that is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, or more than 100% of that obtained in a corresponding untreated animal of LMNA cardiomyopathy.

[0022] Lamin A open reading frame and nucleic acid molecule In one aspect, provided herein is an hLamin A coding sequence that is an engineered hLamin A coding sequence. In one embodiment, the engineered sequence is useful for improving production, transcription, expression, or safety in a subject. In another embodiment, the engineered sequence is useful for increasing the efficacy of the resulting therapeutic composition or treatment. In a further embodiment, the engineered sequence is useful for increasing the efficacy of the expressed functional native hLamin A protein, but may also allow for lower doses of therapeutic reagents that deliver the functional protein, increasing safety.

[0023] In one aspect, provided herein is a recombinant nucleic acid molecule comprising an engineered hLamin A coding sequence that encodes a functional mature human lamin A (hLamin A). In certain embodiments, the engineered hLamin A coding sequence comprises the nucleic acid sequence of SEQ ID NO: 4, or a sequence that is about 90%, at least 95% identical, at least 97% identical, at least 98% identical, or 99%-100% identical to SEQ ID NO: 4 and expresses a functional mature hLamin A protein.

[0024] In certain embodiments, the engineered hLaminA coding sequence is a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:5, which is at least 90% identical thereto (e.g., 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 at least 99.9%) identical thereto.

[0025] A "nucleic acid" as described herein may be RNA, DNA, or modifications thereof, may be single-stranded or double-stranded, and may be selected from the group including, for example, nucleic acids encoding proteins of interest, oligonucleotides, nucleic acid analogs, such as peptide-nucleic acids (PNAs), pseudocomplementary PNAs (pc-PNAs), locked nucleic acids (LNAs), etc. Such nucleic acid sequences include, for example, protein-encoding nucleic acid sequences that act as, for example, but are not limited to, transcriptional repressors, antisense molecules, ribozymes, hypo-inhibitory nucleic acid sequences, such as, but are not limited to, RNAi, shRNAi, siRNA, microRNAi (mRNAi), antisense oligonucleotides, etc.

[0026] The terms "percent identity (%)", "sequence identity", "percent sequence identity", or "percent identical" in the context of nucleic acid sequences refer to residues in two sequences that are the same when aligned for correspondence. The length of sequence identity comparison can be, and is preferred, over the entire length of a genome, the entire length of a gene coding sequence, or a fragment of at least about 500-5000 nucleotides. However, identity between smaller fragments, e.g., of at least about 9 nucleotides, usually at least about 20-24 nucleotides, at least about 28-32 nucleotides, at least about 36 or more nucleotides, can also be desired.

[0027] Percent identity can be readily determined for amino acid sequences spanning the entire length of a protein, polypeptide, about 32 amino acids, about 330 amino acids, or peptide fragments thereof, or the corresponding nucleic acid sequence encoding the sequence. Suitable amino acid fragments can be at least about 8 amino acids in length and can be up to about 700 amino acids in length. Generally, when referring to "identity", "homology", or "similarity" between two different sequences, the "identity", "homology", or "similarity" is determined with reference to "aligned" sequences. An "aligned" sequence or "alignment" refers to multiple nucleic acid or protein (amino acid) sequences, often including corrections for missing or additional bases or amino acids, as compared to a reference sequence.

[0028] The alignment is performed using any of a variety of publicly or commercially available multiple sequence alignment programs. Sequence alignment programs are available for amino acid sequences, including, for example, "Clustal X", "Clustal Omega", "MAP", "PIMA", "MSA", "BLOCKMAKER", "MEME", and "Match-Box" programs. Generally, any of these programs are used with default settings, but those skilled in the art can change these settings as needed. Alternatively, those skilled in the art can utilize another algorithm or computer program that provides at least the same level of identity or alignment as that provided by the referenced algorithm and program. See, for example, JD Thomson et al, Nucl. Acids. Res., "A comprehensive comparison of multiple sequence alignments", 27(13):2682-2690 (1999).

[0029] Multiple sequence alignment programs are also available for nucleic acid sequences. Examples of such programs include "Clustal W", "Clustal Omega", "CAP Sequence Assembly", "BLAST", "MAP" and "MEME", which are accessible through web servers on the Internet. Other sources of such programs are known to those skilled in the art. Alternatively, the Vector NTI utility is also used. There are also numerous algorithms known in the art that can be used to measure nucleotide sequence identity, including those included in the programs mentioned above. As another example, polynucleotide sequences can be compared using Fasta™, a program in GCG version 6.1. Fasta™ provides alignment and percent sequence identity of the best overlapping regions between the query and search sequences. For example, percent sequence identity between nucleic acid sequences can be determined using Fasta™ using its default parameters (word size 6 and NOPAM factor for the scoring matrix) provided in GCG version 6.1, which is incorporated herein by reference.

[0030] The nucleic acid sequences described herein can be cloned using routine molecular biology techniques or generated de novo by DNA synthesis, which can be carried out using routine procedures by service companies operating in the field of DNA synthesis and / or molecular cloning (e.g., GeneArt, GenScript, Life Technologies, Eurofins). The nucleic acid sequences encoding the miRNAs or modified snRNAs described herein can be assembled and placed in any suitable genetic element, e.g., naked DNA, phage, transposon, cosmid, episome, etc., for example, to generate a non-viral delivery system (e.g., RNA-based systems, naked DNA, etc.) or to generate a viral vector in a packaging host cell and / or to deliver to a host cell of interest, and introduce the sequences carried therein into the host cell. In one embodiment, the genetic element is a vector. In one embodiment, the genetic element is a plasmid. The methods used to generate such engineered constructs are known to those skilled in the art of nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012).

[0031] It is to be understood that the hLamin A coding sequences described herein are intended to apply to other compositions, regimens, aspects, embodiments, and methods described throughout the specification.

[0032] Expression cassettes and vector genomes Provided herein are nucleic acid sequences, also referred to as expression cassettes, that include an engineered hLamin A coding sequence under the control of regulatory sequences that direct expression of functional mature hLamin A in a target cell. In certain embodiments, the expression cassette includes an open reading frame (ORF) for a functional mature hLamin A coding sequence that encodes a functional mature hLamin A lacking the preprotein carboxy (C)-terminal tail, the ORF operably linked to regulatory control sequences that direct expression of the mature functional hLamin A protein in the cell, the regulatory control sequences including a promoter, a hybrid promoter, optionally an enhancer, and a polyadenylation (polyA) sequence.

[0033] As used herein, an "expression cassette" includes a biologically useful nucleic acid sequence (e.g., a gene cDNA encoding a protein, an enzyme, or other useful gene product, mRNA, etc.) and a regulatory sequence operably linked thereto that directs or regulates the transcription, translation, and / or expression of the nucleic acid sequence and its gene product. As used herein, an "operably linked" sequence includes both regulatory sequences that are contiguous or non-contiguous with the nucleic acid sequence and regulatory sequences that act in trans or cis with the nucleic acid sequence. Such regulatory sequences typically include, for example, one or more of a promoter, enhancer, intron, Kozak sequence, polyadenylation sequence, and TATA signal. An expression cassette may contain, among other elements, regulatory sequences upstream (5') of the gene sequence, e.g., one or more of a promoter, enhancer, intron, etc., and enhancer, or regulatory sequences downstream (3') of the gene sequence, e.g., one or more of a 3' untranslated region (3'UTR) that includes a polyadenylation site. In certain embodiments, the regulatory sequence is operably linked to the nucleic acid sequence of the gene product, and the regulatory sequence is separated from the nucleic acid sequence of the gene product by an intervening nucleic acid sequence, i.e., the 5' untranslated region (5'UTR). In certain embodiments, the expression cassette comprises one or more nucleic acid sequences of the gene product. In some embodiments, the expression cassette can be a monocistronic or bicistronic expression cassette. In other embodiments, the term "transgene" refers to one or more DNA sequences from an exogenous source that are inserted into a target cell.

[0034] Typically, such expression cassettes can be used to generate viral vectors and contain coding sequences for the gene products described herein flanked by packaging signals of the viral genome and other expression control sequences such as those described herein, In certain embodiments, a vector genome may contain two or more expression cassettes.

[0035] The term "exogenous" as used to describe a nucleic acid sequence or protein means that the nucleic acid or protein does not naturally occur in the chromosome or at the location present in the host cell. An exogenous nucleic acid sequence also refers to a sequence that is derived from and inserted into the same host cell or subject, but exists in a non-native state, e.g., in a different copy number or under the control of different regulatory elements.

[0036] An expression cassette may contain, among other elements, regulatory sequences upstream (5') of the gene sequence, such as one or more of a promoter, a hybrid promoter, an enhancer, an intron, etc., and regulatory sequences downstream (3') of the gene sequence, such as one or more enhancers, or a 3' untranslated region (3'UTR) containing a polyadenylation (polyA) site.

[0037] In certain embodiments, the regulatory sequence includes one or more of the following: a promoter, an enhancer, an intron, a transcription factor, a transcription terminator, an efficient RNA processing signal such as a splicing and polyadenylation site signal (polyA), a sequence that stabilizes cytoplasmic mRNA (e.g., Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element (WPRE)), and a sequence that improves translation efficiency (i.e., Kozak consensus sequence). In certain embodiments, the selected promoter is a constitutive promoter. In certain embodiments, the promoter is a ubiquitous promoter. For example, such promoters include chicken beta-actin (CB) promoter, a hybrid of the cytomegalovirus immediate early enhancer and chicken beta-actin promoter (CB7 promoter), human cytomegalovirus (CMV) promoter, ubiquitin C promoter (UbC), early and late promoters of simian virus 40 (SV40), U6 promoter, metallothionein promoter, EFlα promoter, ubiquitin promoter, hypoxanthine phosphoribosyltransferase (HPRT) promoter, dihydrofolate reductase (DHFR) promoter (Scharfmann et al., Proc. Natl. Acad. Sci. USA 88:4626-4630 (1991), adenosine deaminase promoter, phosphoglycerol kinase (PGK) promoter, pyruvate kinase promoter, phosphoglycerol mutase promoter, beta-actin promoter (Lai et al., Proc. Natl. Acad. Sci. USA 86:10006-10010 (1989)), the long terminal repeats (LTRs) of Moloney leukemia virus and other retroviruses, the thymidine kinase promoter of herpes simplex virus, as well as other constitutive promoters known to those skilled in the art.

[0038] In certain embodiments, the promoter is a tissue or cell specific promoter. In certain embodiments, the promoter is a cardiac specific promoter, such as cardiac troponin T (cTNT), desmin (DES), alpha-myosin heavy chain (α-MHC), myosin light chain 2 (MLC-2) promoter. See also Pacak, CA, et al., Tissue specific promoters improve specificity of AAV9 mediated transgene expression following intra-vascular gene delivery in neonatal mice, Genetic Vaccines and Therapy 2008, 6:13. In certain embodiments, the expression cassette comprises a promoter that is chicken cardiac troponin T promoter (also referred to as chicken TnT or chTnT). In certain embodiments, the chTnT promoter comprises the nucleic acid sequence of SEQ ID NO: 7. In certain embodiments, the promoter is a hybrid promoter. In certain embodiments, the promoter is a hybrid cardiac promoter. As used herein, the term "hybrid promoter" refers to a regulatory control sequence that comprises a hybrid between an enhancer, a spacer sequence, and a promoter sequence. In certain embodiments, the hybrid cardiac promoter comprises a CMV IE enhancer sequence, a spacer sequence, and a chicken cardiac troponin T promoter. In certain embodiments, the spacer sequence is less than 100% identical to the sequences in the examples herein. In certain embodiments, the spacer sequence comprises at least 2 to at least 10 nucleotides. In certain embodiments, the spacer sequence is at least 9 nucleotides. In certain embodiments, the spacer comprises the nucleic acid sequence "CAATAGCTT". In certain embodiments, the spacer sequence comprises the nucleic acid sequence "CA". In certain embodiments, the spacer sequence is selected so that it does not encode any protein, peptide, or vector genome element.Also see U.S. Provisional Patent Application No. 63 / 293,678, filed December 24, 2021, which is incorporated by reference in its entirety into this specification.

[0039] In certain embodiments, the hybrid cardiac promoter comprises the nucleic acid sequence of SEQ ID NO: 23. In certain embodiments, the hybrid cardiac promoter comprises a nucleic acid sequence that is at least 99% identical to SEQ ID NO: 23. Variations in the nucleic acid sequence of the hybrid cardiac promoter include substitutions of nucleotides in the spacer sequence, and optionally insertions and deletions of nucleotides in the spacer sequence.

[0040] In one embodiment, the expression of the gene product is controlled by a regulable promoter that provides tight control over the transcription of the sequence encoding the gene product, for example, a pharmacological agent, or a transcription factor that is activated by a pharmacological agent or, in an alternative embodiment, a physiological stimulus. A promoter system that is leak-free and can be tightly controlled is preferred. Examples of regulable promoters that are ligand-dependent transcription factor complexes include, but are not limited to, members of the nuclear receptor superfamily that are activated by their respective ligands (e.g., glucocorticoids, estrogens, progestins, retinoids, ecdysone, and analogs and mimetics thereof), and rTTA that is activated by tetracycline. In one aspect, the gene switch is an EcR-based gene switch. Examples of such systems include, but are not limited to, those described in U.S. Pat. Nos. 6,258,603, 7,045,315, U.S. Published Patent Application Nos. 2006 / 0014711, 2007 / 0161086, and International Published Application No. WO01 / 70816. Examples of chimeric ecdysone receptor systems are described in U.S. Pat. No. 7,091,038, U.S. Published Patent Application Nos. 2002 / 0110861, 2004 / 0033600, 2004 / 0096942, 2005 / 0266457, and 2006 / 0100416, and International Published Application Nos. WO01 / 70816, WO02 / 066612, WO02 / 066613, WO02 / 066614, WO02 / 066615, WO02 / 29075, and WO2005 / 108617, each of which is incorporated by reference in its entirety. An example of a non-steroidal ecdysone agonist regulatory system is the RheoSwitch® Mammalian Inducible Expression System (New England Biolabs, Ipswich, Mass.).

[0041] Still other promoter systems include, but are not limited to, tetracycline (tet) response elements (e.g., those described by Gossen & Bujard (1992, Proc. Natl. Acad. Sci. USA 89:5547-551); or those described by Lee et al. (1981, Nature 294:228-232); Hynes et al. (1981, Proc. Natl. Acad. Sci. USA 78:2038-2042); Klock et al. (1987, Nature 329:734-736); and Israel & Kaufman (1989, Nucl. Acids 329:734-736). Res. 17:2589-2604), as well as other inducible promoters known in the art. These response elements may include hypoxia response elements (HREs) that bind HIF-Iα and β, metal ion response elements, such as those described by Mayo et al. (1982, Cell 29:99-108); Brinster et al. (1982, Nature 296:39-42), and Searle et al. (1985, Mol. Cell. Biol. 5:1480-1489); or heat shock elements, such as those described by Nouer et al. (in: Heat Shock Response, ed. Nouer, L., CRC, Boca Raton, Fla., ppI67-220, 1991).

[0042] Using such promoters, expression of the transgene can be regulated, for example, by the Tet-on / off system (Gossen et al., 1995, Science 268:1766-9; Gossen et al., 1992, Proc. Natl. Acad. Sci. USA., 89(12):5547-51); the TetR-KRAB system (Urrutia R., 2003, Genome Biol., 4(10):231; Deuschle U et al., 1995, Mol Cell Biol.(4):1907-14); the mifepristone (RU486)-regulated system (Geneswitch; Wang Y et al., 1994, Proc. Natl. Acad. Sci. USA., 91(17):8180-4; Schillinger et al., 2003, Proc. Natl. Acad. Sci. USA., 91(17):8180-4); al., 2005, Proc. Natl. Acad. Sci. USA. 102(39):13789-94); and a humanized tamoxifen-dependent regulatory system (Roscilli et al., 2002, Mol. Ther. 6(5):653-63).

[0043] In another embodiment, the gene switch is based on heterodimerization of FK506-binding protein (FKBP) with the FKBP rapamycin-related protein (FRAP) and is regulated through rapamycin or its non-immunosuppressant analogues.Examples of such systems include, but are not limited to, ARGENT™ transcription technology (ARIAD Pharmaceuticals, Cambridge, Mass.), and the systems described in U.S. Pat. Nos. 6,015,709, 6,117,680, 6,479,653, 6,187,757, and 6,649,595, U.S. Publication No. 2002 / 0173474, U.S. Publication No. 2009 / 10100535, U.S. Pat. No. 5,834,266, U.S. Pat. No. 7,109,317, U.S. Pat. No. 7,485,441, U.S. Pat. No. 5,830,462, U.S. Pat. No. 5,830,462, U.S. Pat. No. 5,771,226, and U.S. Pat. ,869,337, U.S. Patent No. 5,871,753, U.S. Patent No. 6,011,018, U.S. Patent No. 6,043,082, U.S. Patent No. 6,046,047, U.S. Patent No. 6,063,625, U.S. Patent No. 6,140,120, U.S. Patent No. 6,165,787, U.S. Patent No. 6,972,193, U.S. Patent No. 6,326,166, U.S. Patent No. 7,008,780, U.S. Patent No. 6,133,456, U.S. Patent No. 6,150,527, U.S. Patent No. 6,506,379 No. 6,258,823, U.S. Patent No. 6,693,189, U.S. Patent No. 6,127,521, U.S. Patent No. 6,150,137, U.S. Patent No. 6,464,974, U.S. Patent No. 6,509,152, U.S. Patent No. 6,015,709, U.S. Patent No. 6,117,680, U.S. Patent No. 6,479,653, U.S. Patent No. 6,187,757, U.S. Patent No. 6,649,595, U.S. Patent No. 6,984,635, U.S. Patent No. 7,067,526, U.S. Patent No. Nos. 7,196,192, 6,476,200, 6,492,106, WO94 / 18347, WO96 / 20951, WO96 / 06097, WO97 / 31898, WO96 / 41865, WO98 / 02441, WO95 / 33052, WO99 / 110508, WO99 / 110510, WO99 / 36553, WO99 / 41258, WO01 / 114387, the ARGENT™ Regulated Transcription Plasmid Kit, Takara Bio iDimerize Regulated Transcription Kit, or equivalent kits from QuantiTect, Sensiscript, and the like, each of which is incorporated herein by reference in its entirety.These systems are designed to be induced by rapamycin or one of its analogs, called "rapalogs". Examples of suitable rapamycins are provided in the references listed above in connection with the description of the ARGENT™ system. In one embodiment, the molecule is rapamycin (e.g., marketed as Rapamune™ by Pfizer). In another embodiment, the rapalog known as AP21967 [ARIAD] is used. Examples of these dimerizer molecules include, but are not limited to, rapamycin, FK506, FK1012 (a homodimer of FK506), rapamycin analogs ("rapalogs") that are readily prepared by chemical modification of natural products to add "bumps" that reduce or eliminate affinity for endogenous FKBP and / or FRAP. Examples of rapalogs include, but are not limited to, AP26113 (Ariad), AP1510 (Amara, JF, et al., 1997, Proc Natl Acad Sci USA, 94(20):10618-23), AP22660, AP22594, AP21370, AP22594, AP23054, AP1855, AP1856, AP1701, AP1861, AP1692, and AP1889, which have "bumps" designed to minimize interaction with endogenous FKBP. Still other rapalogs may be selected, such as AP23573 [Merck]. In certain embodiments, rapamycin or a suitable analog may be delivered locally or systemically to AAV-transfected cells.

[0044] In certain embodiments, the expression cassette comprises one or more expression enhancers. In one embodiment, the expression cassette contains two or more expression enhancers. These enhancers can be the same or different from each other. In certain embodiments, the enhancer is a cytomegalovirus immediate early enhancer (CMV IE enhancer). In certain embodiments, the CMV IE enhancer comprises the nucleic acid of SEQ ID NO: 8. In certain embodiments, the enhancer is a cardiac enhancer. In certain embodiments, the cardiac enhancer is a chicken troponin T enhancer. In certain embodiments, the enhancer is a rat alpha-myosin heavy enhancer. This / these enhancers may be in two copies located adjacent to each other. Alternatively, the double copies of the enhancer may be separated by one or more sequences. In further embodiments, the enhancer(s) are selected from one or more of the APB enhancer, ABPS enhancer, alpha mic / bik enhancer, TTR enhancer, en34 enhancer, ApoE enhancer, CMV enhancer, or RSV enhancer. In yet another embodiment, the regulatory element comprises an intron. In further embodiments, the intron is selected from chicken beta actin intron (CBA), human beta globin, IVS2, SV40 (Promega), bGH, alpha-globulin, beta-globulin, collagen, ovalbumin, or p53. See also WO2011 / 126808. In one embodiment, the regulatory element comprises polyA. In further embodiments, the polyA is synthetic or from bovine growth hormone (bGH), human growth hormone (hGH), SV40, rabbit β-globin (RBG), or modified RBG (mRBG). Optionally, one or more sequences may be selected to stabilize the mRNA. One example of such a sequence is a modified WPRE sequence, which may be engineered upstream of the polyA sequence and downstream of the coding sequence [see, e.g., MA Zanta-Boussif, et al, Gene Therapy (2009) 16:605-619.

[0045] In certain embodiments, the expression cassette may include one or more expression enhancers, such as post-transcriptional regulatory elements from woodchuck (WPRE), human (HPRE), ground squirrel (GPRE), or arctic ground squirrel (AGSPRE) hepatitis virus, or synthetic post-transcriptional regulatory elements. These expression enhancing elements are particularly advantageous when placed in the 3'UTR and may significantly increase mRNA stability and / or protein yield. In certain embodiments, the provided expression cassette includes a regulatory sequence that is a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) or a variant thereof. Suitable WPRE sequences are provided in the vector genomes described herein and are known in the art (e.g., as described in U.S. Patent Nos. 6,136,597, 6,287,814, and 7,419,829, which are incorporated by reference). In certain embodiments, the WPRE is a variant that has been mutated to eliminate expression of the Woodchuck Hepatitis B Virus X (WHX) protein, including, for example, a mutation in the start codon of the WHX gene. See also Kingsman SM, Mitrophanous K., & Olsen JC (2005), Potential Oncogene Activity of the Woodchuck Hepatitis Post-Transcriptional Regulatory Element (Wpre). "Gene Ther. 12(1):3-4; and Zanta-Boussif MA, Charrier S., Brice-Ouzet A., Martin S., Opolon P., Thrasher AJ, Hope TJ, & Galy A. (2009), Validation of a Mutated Pre Sequence Allowing High and Sustained Transgene Expression While Abrogating Whv-X Protein Synthesis: Application to the Gene Therapy of Was, Gene Ther. 16(5):605-19, both of which are incorporated by reference in their entireties.In other embodiments, the enhancer is selected from a non-viral source. In certain embodiments, the WPRE sequence is absent.

[0046] In certain embodiments, the expression cassette comprises a regulatory control sequence comprising a cardiac promoter. In certain embodiments, the expression cassette comprises a regulatory control sequence comprising a cardiac troponin T (cTnT) promoter. In certain embodiments, the expression cassette comprises a regulatory control sequence comprising a hybrid cardiac promoter comprising a CMV IE enhancer, a spacer sequence, and a chicken cardiac troponin T promoter (chTnT). In certain embodiments, the expression cassette comprises a chTnT comprising the nucleic acid sequence of SEQ ID NO:7 with a CMV IE enhancer comprising the nucleic acid sequence of SEQ ID NO:8. In certain embodiments, the expression cassette comprises a hybrid cardiac promoter comprising the nucleic acid sequence of SEQ ID NO:23, or a sequence at least 99% identical to SEQ ID NO:23. In certain embodiments, the regulatory control sequence comprises a polyA sequence that is a rabbit beta globin polyA sequence. In certain embodiments, the expression cassette comprises a rabbit beta globin polyA sequence comprising the nucleic acid sequence of SEQ ID NO:9. In certain embodiments, the expression cassette comprises a chTnT promoter, optionally with a CMV IE enhancer-hLamin A coding sequence-rabbit beta-globin polyA. In certain embodiments, the expression cassette comprises, 5' to 3', a CMV IE enhancer, a chTnT promoter, an hLamin A coding sequence, and a rabbit beta-globin polyA. In certain embodiments, the expression cassette comprises a nucleic acid sequence of SEQ ID NO:2, or a sequence 90% identical to SEQ ID NO:2. In certain embodiments, the expression cassette comprises a nucleic acid sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99 to at least 100% identical to SEQ ID NO:2.

[0047] In one particular embodiment, the expression cassette is CMVe.chTNTp.Lamin.RBG comprising the nucleic acid sequence of SEQ ID NO:2.

[0048] In certain embodiments, an expression cassette containing the hLamin A coding sequence may contain other regulatory sequences therefor. The necessary regulatory sequences are operably linked to the hLamin A coding sequence in a manner that allows its transcription, translation, and / or expression in the target cell.

[0049] In a further aspect, provided herein is a vector genome comprising an AAV 5' inverted terminal repeat (ITR), an expression cassette, and an AAV 3' ITR, wherein the expression cassette comprises a nucleic acid sequence encoding a functional mature hLamin A gene operably linked to expression control sequences that direct its expression in a cell containing a selected gene.

[0050] In certain embodiments, the vector genome comprises an engineered nucleic acid sequence that includes an open reading frame (ORF) for a functional mature hLamin A coding sequence that encodes a functional mature hLamin A lacking the preprotein carboxy (C)-terminal tail, the ORF being operably linked to regulatory control sequences that direct expression of the mature, functional hLamin A protein in a cell, the regulatory control sequences including a promoter, optionally an enhancer, and a polyadenylation (polyA) sequence.

[0051] In certain embodiments, the vector genome comprises an expression cassette having a nucleic acid sequence of SEQ ID NO:2 or a sequence at least about 90% identical to SEQ ID NO:2. In certain embodiments, the vector genome comprises a nucleic acid molecule comprising, 5' to 3', AAV5'ITR-hybrid cardiac promoter-engineered hLamin A coding sequence-rabbit beta globin polyA-AAV3'ITR. In certain embodiments, the vector genome comprises a nucleic acid molecule comprising, 5' to 3', AAV5'ITR-optionally CMV IE promoter-optionally spacer sequence-chTnT promoter-engineered hLamin A coding sequence-rabbit beta globin polyA-AAV3'ITR. In certain embodiments, the vector genome comprises a nucleic acid molecule comprising, 5' to 3', AAV5'ITR-CMV IE promoter-space ... sequence of SEQ ID NO:1. In certain embodiments, the vector genome comprises a nucleic acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99 to at least 100% identical to SEQ ID NO:1.

[0052] In certain embodiments, the vector genome is 5'ITR.CMVe.chTNTp.Lamin.RBG.3'ITR comprising the nucleic acid sequence of SEQ ID NO:1, where "CMVe.chTNTp" refers to a hybrid cardiac promoter comprising a CMV IE enhancer, a spacer sequence, and a chicken cardiac troponin T promoter.

[0053] In certain embodiments, the target cell is a cardiac tissue cell. In certain embodiments, the target cell is a cardiac cell. In certain embodiments, the target cell is any other cell that expresses functional mature Lamin A protein in a subject without idiopathic DCM or a disease associated with a mutation in the LMNA gene.

[0054] As used herein, "vector genome" refers to a nucleic acid sequence packaged inside a parvovirus (e.g., rAAV) capsid that forms a viral particle. Such a nucleic acid sequence contains AAV inverted terminal repeats (ITRs). In the examples herein, the vector genome contains, at a minimum, from 5' to 3', an AAV 5'ITR (also referred to as 5'ITR), a coding sequence(s) (i.e., transgene(s)), and an AAV 3'ITR (also referred to as 3'ITR). ITRs from AAV2, a source AAV different from the capsid, or ITRs other than the full-length ITRs may be selected. In certain embodiments, the ITRs are from the same AAV source as the AAV that provides the rep function or trans-complementing AAV during production. Additionally, other ITRs may be used, such as self-complementary (scAAV) ITRs. Both single-stranded and self-complementary (sc) AAVs are included in rAAV. A transgene is a nucleic acid coding sequence heterologous to the vector sequence that encodes a polypeptide, protein, functional RNA molecule (e.g., miRNA, miRNA inhibitor), or other gene product of interest. The nucleic acid coding sequence is operably linked to regulatory components in a manner that allows transcription, translation, and / or expression of the transgene in cells of the target tissue. Suitable components of a vector genome are discussed in more detail herein. In one example, a "vector genome" contains, at a minimum, a nucleic acid sequence encoding hLamin A operably linked 5' to 3' to vector-specific sequences, regulatory control sequences that direct its expression in the target cells, and the vector-specific sequences can be terminal repeat sequences that specifically package the vector genome into a viral vector capsid or envelope protein. For example, AAV inverted terminal repeats are utilized for packaging into AAV and certain other parvovirus capsids. In certain embodiments, the vector genome is an expression cassette having at its extreme 5' and 3' ends inverted terminal repeat (ITR) sequences necessary for packaging the vector genome into an AAV capsid, and containing therebetween the hLamin A gene described herein operably linked to sequences that direct its expression.In certain embodiments, the vector genome may, at a minimum, comprise, 5' to 3', an AAV 5' ITR, a coding sequence(s), and an AAV 3' ITR. In certain embodiments, the ITRs may be selected from an AAV source other than AAV2, a different AAV source than the capsid, or a full-length ITR. In certain embodiments, the ITRs are from the same AAV source as the AAV that provides the rep function or trans-complementing AAV during production. Additionally, other ITRs may be used.

[0055] The AAV sequences of the vector typically contain cis-acting 5' and 3' inverted terminal repeat sequences (see, e.g., BJ Carter, in "Handbook of Parvoviruses", ed., P. Tijsser, CRC Press, pp. 155 168 (1990)). The ITR sequences are approximately 145 bp in length. Preferably, substantially complete sequences encoding the ITRs are used in the molecule, although some minimal modification of these sequences is tolerated. The ability to modify these ITR sequences is within the skill of the art. (See, e.g., texts such as Sambrook et al, "Molecular Cloning. A Laboratory Manual", 2d ed., Cold Spring Harbor Laboratory, New York (1989); and K. Fisher et al., J. Virol., 70:520 532 (1996)). One example of such a molecule used is a "cis-acting" plasmid containing a transgene, where the selected transgene sequence and associated regulatory elements are flanked by 5' and 3' AAV ITR sequences. In one embodiment, the ITRs are from a different AAV than the one supplying the capsid. In one embodiment, the ITR sequences are from AAV2. However, ITRs from other AAV sources may be selected. A shortened version of the 5' ITR (termed ΔITR) has been described, in which the D sequence and terminal separation sites (trs) are deleted. In one particular embodiment, the vector genome comprises a 130 base pair shortened AAV2 ITR, in which the external A elements are deleted. Without wishing to be bound by theory, it is believed that the shortened ITRs are restored to the wild-type length of 145 base pairs during vector DNA amplification, using the internal (A') elements as templates. In other embodiments, full-length AAV 5' and 3' ITRs are used. When the source of the ITRs is from AAV2 and the AAV capsid is from another AAV source, the resulting vector may be referred to as pseudotyped, however, other configurations of these elements may also be suitable.

[0056] It is to be understood that the compositions in the expression cassettes and vector genomes described herein are intended to apply to the other compositions, regimens, aspects, embodiments, and methods described throughout this specification.

[0057] vector In one aspect, provided herein is a vector comprising an engineered open reading frame (ORF) for mature human lamin A (hLaminA), wherein the ORF has a mature hLaminA coding sequence that is a nucleic acid sequence encoding a functional mature human lamin A (hLaminA) lacking the preprotein carboxy (C)-terminal tail, the ORF is operably linked to regulatory control sequences that direct expression of mature hLaminA in a cell, the hLaminA coding sequence comprising a nucleic acid sequence of SEQ ID NO:4, or a nucleic acid sequence that is at least 90% identical to SEQ ID NO:4 and encodes the amino acid sequence of SEQ ID NO:5, and the regulatory control sequences comprise a cardiac-specific promoter.

[0058] In certain embodiments, the vector comprises an hLamin A coding sequence comprising the nucleic acid sequence of SEQ ID NO:4, or a sequence that is at least 90%, at least 95% identical, at least 97% identical, at least 98% identical, or 99%-100% identical to SEQ ID NO:4 and expresses a functional mature hLamin A protein. In certain embodiments, the vector comprises an hLamin A coding sequence comprising the nucleic acid sequence of SEQ ID NO:4, or a nucleic acid sequence that is at least 90% (e.g., 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 at least 99.9%) identical thereto and encodes the amino acid sequence of SEQ ID NO:5.

[0059] A "vector" as used herein is a biological or chemical moiety that contains a nucleic acid sequence and can be introduced into a suitable target cell for replication or expression of the nucleic acid sequence. Examples of vectors include, but are not limited to, recombinant viruses, plasmids, lipoplexes, polymersomes, polyplexes, dendrimers, cell penetrating peptide (CPP) conjugates, magnetic particles, or nanoparticles. In one embodiment, a vector is a nucleic acid molecule into which an exogenous or heterologous nucleic acid or engineered nucleic acid encoding a functional SGSH can be inserted and then introduced into a suitable target cell. Such vectors preferably have one or more origins of replication and one or more sites into which recombinant DNA can be inserted. Vectors often have a means by which cells containing the vector can be selected from cells that do not, for example, the vector encodes a drug resistance gene. Common vectors include plasmids, viral genomes, and "artificial chromosomes". Conventional methods for generating, producing, characterizing, or quantifying vectors are available to those skilled in the art.

[0060] In one embodiment, the vector is a non-viral plasmid and includes the described expression cassettes, e.g., "naked DNA," "naked plasmid DNA," naked RNA, and mRNA, associated with various compositions and nanoparticles, including micelles, liposomes, cationic lipid-nucleic acid compositions, polyglycan compositions, and other polymer, lipid, and / or cholesterol-based-nucleic acid conjugates, and other constructs such as those described herein. See, e.g., X. Su, et al, Mol. Pharmaceutics, 2011, 8(3), pp774-787; web publication: March 21, 2011; WO2013 / 182683, WO2010 / 053572, and WO2012 / 170930 (all of which are incorporated herein by reference).

[0061] In certain embodiments, the vectors described herein are "replication-defective viruses" or "viral vectors," which refer to synthetic or artificial viral particles in which an expression cassette containing a nucleic acid sequence encoding hLamin A is packaged into a viral capsid or envelope, and any viral genomic sequences packaged within the viral capsid or envelope are replication-defective (i.e., retain the ability to infect target cells but cannot generate progeny virions). In one embodiment, the genome of the viral vector does not contain genes encoding enzymes required for replication (the genome can be engineered to be "gutless," containing only nucleic acid sequences encoding hLamin A flanked by signals required for amplification and packaging of the artificial genome), but these genes can be supplied during production. It is therefore considered safe for use in gene therapy, since replication and infection by progeny virions cannot occur except in the presence of viral enzymes required for replication.

[0062] As used herein, a recombinant viral vector is an adeno-associated virus (AAV), adenovirus, bocavirus, hybrid AAV / bocavirus, herpes simplex virus, or lentivirus.

[0063] As used herein, the term "host cell" may refer to a packaging cell line in which a vector (e.g., recombinant AAV) is produced. A host cell may be a prokaryotic or eukaryotic cell (e.g., human, insect, or yeast) that contains exogenous or heterologous DNA that has been introduced into the cell by any means (e.g., electroporation, calcium phosphate precipitation, microinjection, transformation, viral infection, transfection, liposome delivery, membrane fusion techniques, high-speed DNA-coated pellets, viral infection, and protoplast fusion). Examples of host cells may include, but are not limited to, isolated cells, cell cultures, Escherichia coli cells, yeast cells, human cells, non-human cells, mammalian cells, non-mammalian cells, insect cells, HEK-293 cells, liver cells, kidney cells, cells of the central nervous system, cardiac cells, or stem cells.

[0064] It is to be understood that the vector compositions described herein are intended to apply to the other compositions, regimens, aspects, embodiments, and methods described throughout this specification.

[0065] Recombinant adeno-associated virus (rAAV) Provided herein is a recombinant adeno-associated virus (rAAV) useful for treating idiopathic dilated cardiomyopathy or diseases associated with dysfunctional LMNA gene, such as diseases caused by complete or partial loss-of-function mutations. The rAAV comprises (a) an AAV capsid and (b) a vector genome packaged in the AAV capsid of (a). Advantageously, the selected AAV capsid targets the cells to be treated. In certain embodiments, the capsid is from clade F. However, in certain embodiments, another AAV capsid source, i.e., clade A, may be selected. In certain embodiments, the AAV capsid is an AAVhu68 capsid. In certain embodiments, the AAV capsid is an AAVhu95 capsid. In certain embodiments, the AAV capsid is an AAVhu96 capsid. The vector genome comprises an AAV 5' inverted terminal repeat (ITR), an engineered nucleic acid sequence encoding a functional mature hLamin A as described herein, regulatory sequences that direct expression of hLamin A in target cells, and the AAV 3' ITR.

[0066] In one aspect, rAAV.hLaminA is for use in the treatment of idiopathic DCM. In certain embodiments, rAAV.hLaminA is for use in the treatment of early-onset idiopathic DCM. In certain embodiments, rAAV.hLaminA is for use in the treatment of adult-onset idiopathic DCM. In certain embodiments, rAAV comprises a vector genome comprising a 5'AAV ITR, an expression cassette, and a 3'AAV ITR, the expression cassette comprising an open reading frame (ORF) for a functional mature hLamin A coding sequence encoding a functional mature hLamin A lacking a preprotein carboxy (C)-terminal tail, the ORF being operably linked to regulatory control sequences directing expression of the mature functional hLamin A protein in a cell, the regulatory control sequences comprising a promoter, optionally an enhancer, and a polyadenylation sequence (rAAV.hLaminA). In certain embodiments, the rAAV comprises a vector genome comprising an expression cassette having a nucleic acid sequence of SEQ ID NO:2, or a sequence at least about 90% identical to SEQ ID NO:2. In certain embodiments, the rAAV comprises a vector genome comprising a nucleic acid molecule comprising, 5' to 3', AAV5' ITR-hybrid cardiac promoter-engineered hLamin A coding sequence-rabbit beta globin polyA-AAV3' ITR. In certain embodiments, the rAAV comprises a vector genome comprising a nucleic acid molecule comprising, 5' to 3', AAV5' ITR-optionally CMV IE promoter-optionally spacer sequence-chTnT promoter-engineered hLamin A coding sequence-rabbit beta globin polyA-AAV3' ITR. In certain embodiments, the rAAV comprises a vector genome comprising a nucleic acid molecule comprising, 5' to 3', AAV5' ITR-CMV IE promoter-spacer sequence-chTnT promoter-engineered hLamin A coding sequence-rabbit beta globin polyA-AAV3' ITR.In certain embodiments, the rAAV comprises a vector genome comprising the nucleic acid sequence of SEQ ID NO:1, or a sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% to at least 100% identical to SEQ ID NO:1 (rAAV.CMV-IE.chTNTp.LaminA.RBG).

[0067] In certain embodiments, the AAV capsid for the compositions and methods described herein is selected based on the target cell. In certain embodiments, the AAV capsid transduces cardiac cells. In certain embodiments, other AAV capsids may be selected.

[0068] In certain embodiments, the clade F AAV capsid is an AAVhu68 capsid [see, e.g., US2020 / 0056159; PCT / US21 / 55436; SEQ ID NOs: 10 and 11 for nucleic acid sequences, and SEQ ID NO: 12 for amino acid sequence], an AAVhu95 capsid [see, e.g., U.S. Provisional Application No. 63 / 251,599, filed October 2, 201; SEQ ID NOs: 13 and 14 (hu95 nucleic acid sequences) and SEQ ID NO: 15 (hu95 amino acid sequence)], or an AAVhu95 capsid [see, e.g., U.S. Provisional Application No. 63 / 251,599, filed October 2, 201; SEQ ID NOs: 13 and 14 (hu95 nucleic acid sequences) and SEQ ID NO: 15 (hu95 amino acid sequence)]. , AAVhu96 capsid [see, e.g., U.S. Provisional Application No. 63 / 251,599, filed October 2, 201; SEQ ID NOs: 16 and 17 (hu96 nucleic acid sequence), and SEQ ID NO: 18 (hu96 amino acid sequence), or AAV9 [see, e.g., U.S. Pat. No. 7,906,111], or engineered mutants and variants thereof [see, e.g., WO2020 / 200499; WO2003 / 054197]. See also, International Patent Application No. PCT / US2022 / 077315, filed September 30, 2022, which is incorporated by reference in its entirety.

[0069] In certain embodiments, the AAV capsid is a non-clade F capsid, e.g., a clade A, B, C, D, or E capsid. In certain embodiments, the non-clade F capsid is AAV1 or a variant thereof. In certain embodiments, the AAV capsid transduces a target cell other than a cardiac cell. In certain embodiments, the AAV capsid is a clade A capsid (e.g., AAV1, AAV6, AAVrh91), a clade B capsid (e.g., AAV2), a clade C capsid (e.g., hu53), a clade D capsid (e.g., AAV7), or a clade E capsid (e.g., rh10).

[0070] As used herein, the term "clade" in relation to a group of AAVs refers to a group of AAVs that are phylogenetically related to each other as determined based on an alignment of AAV vp1 amino acid sequences using a neighbor-joining algorithm with a bootstrap value of at least 75% (out of at least 1000 replicates) and a Poisson-corrected distance measure of 0.05 or less. Neighbor-joining algorithms have been described in the literature. See, e.g., M. Nei and S. Kumar, Molecular Evolution and Phylogenetics, Oxford University Press, New York (2000). Computer programs are available that can be used to implement this algorithm. For example, the MEGA v2.1 program implements a modified Nei-Gojobori method. Using these techniques and computer programs, and the sequence of the AAV vp1 capsid protein, one of skill in the art can readily determine whether a selected AAV is contained within one of the clades identified herein, another clade, or outside these clades. See, e.g., G Gao, et al, J Virol, 2004 Jun;78(10):6381-6388, which identifies clades A, B, C, D, E, and F, and provides the nucleic acid sequences of novel AAVs, GenBank Accession Nos. AY530553-AY530629. See also WO2005 / 033321.

[0071] rAAV is composed of an AAV capsid and a vector genome. The AAV capsid is a collection of a heterogeneous population of vp1 proteins, a heterogeneous population of vp2 proteins, and a heterogeneous population of vp3 proteins. As used herein, the term "heterogeneous" or any grammatical variation thereof, when used to refer to vp capsid proteins, refers to a collection of non-identical members, for example, having vp1, vp2, or vp3 monomers (proteins) with different modified amino acid sequences.

[0072] As used herein, the term "heterologous" or any grammatical variation thereof when used to refer to vp capsid proteins refers to a population of non-identical members, e.g., having vp1, vp2, or vp3 monomers (proteins) with different modified amino acid sequences. The term "heterologous population" used in reference to vp1, vp2, and vp3 proteins (alternatively referred to as isoforms) refers to differences in the amino acid sequences of vp1, vp2, and vp3 proteins within a capsid. AAV capsids contain subpopulations within vp1, vp2, and vp3 proteins that have modifications from predicted amino acid residues. These subpopulations contain, at a minimum, certain deamidated asparagine (N or Asn) residues. For example, certain subpopulations contain at least one, two, three, or four highly deamidated asparagine (N) positions in asparagine-glycine pairs, and optionally further contain other deamidated amino acids, where the deamidation results in an amino acid change and other optional modifications.

[0073] In certain embodiments, AAV capsids are provided that have a heterogeneous population of AAV capsid isoforms (i.e., VP1, VP2, VP3) that contain multiple highly deamidated "NG" positions. In certain embodiments, the highly deamidated positions are at the positions identified below with reference to the predicted full-length VP1 amino acid sequence. In other embodiments, the capsid gene is modified such that the referenced "NG" is removed and a mutant "NG" is engineered into another position.

[0074] As used herein, the terms "target cell" and "target tissue" may refer to any cell or tissue intended to be transduced by the subject AAV vector or in which expression of hLamin A is desired. The terms may refer to any one or more of muscle, liver, lung, airway epithelium, central nervous system, neurons, eye (visual cells), or heart. In certain embodiments, the term "target cell" is intended to refer to a cell of a subject undergoing treatment for a disease associated with a mutation in the idiopathic Lamin A or LMNA gene. In certain embodiments, the vector is delivered to the target cell ex vivo. In certain embodiments, the vector is delivered to the target cell in vivo.

[0075] Additionally, provided herein is an rAAV production system useful for producing the rAAV described herein. The production system comprises a cell culture comprising (a) a nucleic acid sequence encoding an AAV capsid protein, (b) a vector genome, and (c) sufficient AAV rep and helper functions to enable packaging of the vector genome into an AAV capsid. In certain embodiments, the vector genome is SEQ ID NO: 1. In certain embodiments, the cell culture is a human embryonic kidney 293 cell culture. In certain embodiments, the AAV rep is from a different AAV. In certain embodiments, the AAV rep is from AAV2. In certain embodiments, the AAV rep coding sequence and the cap gene are on the same nucleic acid molecule, and optionally, there is a spacer between the rep sequence and the cap gene.

[0076] For use in producing AAV viral vectors (e.g., recombinant (r)AAV), the vector genome can be carried on any suitable vector, e.g., a plasmid, that is delivered to a packaging host cell. Plasmids useful in the present invention can be engineered to be suitable for replication and packaging in prokaryotic, insect, or mammalian cells in vitro, among others. Suitable transfection techniques and packaging host cells are known and / or can be readily designed by one of skill in the art.

[0077] In certain embodiments, a plasmid useful for the production of rAAV particles is provided, the plasmid comprising a vector genome comprising an AAV 5'ITR, an expression cassette, and an AAV 3'ITR, the expression cassette comprising an engineered nucleic acid sequence comprising an open reading frame (ORF) for a functional mature hLamin A coding sequence encoding a functional mature hLamin A lacking a preprotein carboxy (C)-terminal tail, the ORF being operably linked to regulatory control sequences that direct expression of the mature functional hLamin A protein in a cell, the regulatory control sequences comprising a promoter, optionally an enhancer, and a polyadenylation (polyA) sequence. In certain embodiments, a nucleic acid (e.g., a plasmid) useful for rAAV production comprises a vector genome comprising an AAV 5'ITR, a hybrid cardiac promoter (comprising a CMV IE enhancer, a spacer sequence, and a chTnT promoter), a functional mature hLamin A coding sequence, a rabbit beta globin polyA sequence, and an AAV 3'ITR. In certain embodiments, a nucleic acid (e.g., a plasmid) useful for rAAV production comprises a vector genome comprising the nucleic acid sequence of SEQ ID NO:1.

[0078] Methods for generating and isolating AAV suitable for use as a vector are known in the art. See generally, for example, Grieger & Samulski, 2005, Adeno-associated virus as a gene therapy vector: Vector development, production and clinical applications, Adv. Biochem. Engin / Biotechnol. 99:119-145; Buning et al., 2008, Recent developments in adeno-associated virus vector technology, J. Gene Med. 10:717-733; and the references cited below, each of which is incorporated herein by reference in its entirety. As used herein, gene therapy vector refers to the rAAV described herein that is suitable for use in treating patients. The ITRs are the only AAV components required in cis in the same construct as the nucleic acid molecule containing the gene in order to package the gene into virions. The cap and rep genes can be supplied in trans.

[0079] In one embodiment, the selected genetic element can be delivered to the AAV packaging cell by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion techniques, high-speed DNA-coated pellets, viral infection, and protoplast fusion. Stable AAV packaging cells can also be produced. The methods used to produce such constructs are known to those skilled in the art of nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, for example, Molecular Cloning: A Laboratory Manual, ed. Green and Sambrook, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012).

[0080] The term "AAV intermediate" or "AAV vector intermediate" refers to assembled rAAV capsids that lack the desired genomic sequence packaged therein. These may also be referred to as "empty" capsids. Such capsids may not contain detectable genomic sequences of an expression cassette, or may contain only partially packaged genomic sequences that are insufficient to achieve expression of a gene product. These empty capsids are non-functional for introducing a gene of interest into a host cell.

[0081] The recombinant adeno-associated virus (AAV) described herein can be produced using known techniques. See, for example, WO2003 / 042397; WO2005 / 033321, WO2006 / 110689; US7588772B2. Such methods involve culturing a host cell that contains a nucleic acid sequence encoding an AAV capsid protein; a functional rep gene; an expression cassette consisting of, at a minimum, an AAV inverted terminal repeat (ITR) and a transgene; and sufficient helper functions to allow the expression cassette to be packaged into an AAV capsid protein. Methods for producing capsids, coding sequences therefor, and methods for the production of rAAV viral vectors have been described. See, for example, Gao, et al, Proc. Natl. Acad. Sci. USA100(10), 6081-6086(2003) and US2013 / 0045186A1.

[0082] In one embodiment, a producer cell culture is provided that is useful for producing a recombinant AAV having a capsid selected from AAVhu68, AAVhu95, or AAVhu96. Such a cell culture contains a nucleic acid that expresses an AAVhu68 capsid protein in a host cell (e.g., SEQ ID NO:10 or SEQ ID NO:11; a vector genome containing a non-AAV nucleic acid sequence encoding a nucleic acid molecule suitable for packaging into an AAVhu68 capsid, e.g., a gene operably linked to AAV ITRs and a regulatory sequence that directs expression of the gene in a host cell; and sufficient AAV rep and adenovirus helper functions to allow packaging of the vector genome into a recombinant AAVhu68, or AAVhu95 capsid (e.g., SEQ ID NO:13 or SEQ ID NO:14), AAVhu96 capsid (e.g., SEQ ID NO:16 or SEQ ID NO:17). In one embodiment, the cell culture is a mammalian cell (e.g., human embryonic kidney 293 cells, among others) or an insect cell (e.g., Spodoptera frugiperda (Sf9) cells). In certain embodiments, the baculovirus provides the helper functions necessary for packaging the vector genome into recombinant AAVhu68, AAVhu95, or AAVhu96 capsids.

[0083] Optionally, the rep function is provided by an AAV other than AAV2 selected to complement the source of the ITRs.

[0084] In one embodiment, the cells are produced in a suitable cell culture (e.g., HEK293 or Sf9) or suspension. Methods for producing gene therapy vectors described herein include methods well known in the art, such as the generation of plasmid DNA used in the production of gene therapy vectors, the generation of vectors, and the purification of vectors. In some embodiments, the gene therapy vector is an AAV vector, and the generated plasmids are AAV cis-plasmids encoding the AAV vector genome and the gene of interest, AAV trans-plasmids containing the AAV rep and cap genes, and adenovirus helper plasmids. The vector production process may include method steps such as initiation of cell culture, passaging of cells, seeding of cells, transfection of cells with plasmid DNA, medium exchange with serum-free medium after transfection, and harvesting of vector-containing cells and culture medium. The harvested vector-containing cells and culture medium are referred to herein as crude cell harvest. In yet another system, the gene therapy vector is introduced into insect cells by infection with a baculovirus-based vector. For a review of these production systems, see generally, e.g., Zhang et al., 2009, Adenovirus-adeno-associated virus hybrid for large-scale recombinant adeno-associated virus production, Human Gene Therapy 20:922-929, the contents of each of which are incorporated herein by reference in their entireties. Methods of making and using these and other AAV production systems are also described in the following U.S. patents, the contents of each of which are incorporated herein by reference in their entirety: U.S. Patent Nos. 5,139,941; 5,741,683; 6,057,152; 6,204,059; 6,268,213; 6,491,907; 6,660,514; 6,951,753; 7,094,604; 7,172,893; 7,201,898; 7,229,823; and 7,439,065.

[0085] The crude cell harvest may then be subjected to process steps such as concentration of the vector harvest, diafiltration of the vector harvest, microfluidization of the vector harvest, nuclease digestion of the vector harvest, filtration of the microfluidized intermediate, crude purification by chromatography, crude purification by ultracentrifugation, buffer exchange by tangential flow filtration, and / or formulation and filtration to prepare bulk vector. Affinity chromatography purification followed by anion exchange resin chromatography is used to purify the vector drug product and remove empty capsids. These methods are described in further detail in WO2017 / 160360, entitled "Scalable Purification Method for AAV9," filed December 9, 2016, which is incorporated herein by reference. A method for purifying AAV8, WO2017 / 100676, filed December 9, 2016, and a method for purifying rh10, WO2017 / 100704, filed December 9, 2016, entitled "Scalable Purification Method for AAVrh10" (also filed December 11, 2015), and a method for purifying AAV1, WO2017 / 100674, filed December 9, 2016 for "Scalable Purification Method for AAV1", filed December 11, 2015, are all incorporated herein by reference. However, other suitable methods may be selected.

[0086] To calculate the content of empty and full particles, the VP3 band volume for a selected sample (e.g., in the examples herein, a preparation purified by iodixanol gradient, number of genome copies (GC) = number of particles) is plotted against the GC particles loaded. The resulting linear equation (y = mx + c) is used to calculate the number of particles in the band volume of the peak of the test sample. The number of particles (pt) per 20 μL loaded is then multiplied by 50 to obtain particles (pt) / mL. Dividing Pt / mL by GC / mL gives the ratio of particles to genome copies (pt / GC). Pt / mL-GC / mL gives empty pt / mL. Dividing empty pt / mL by pt / mL and multiplying by 100 gives the percentage of empty particles.

[0087] Generally, methods for assaying AAV vector particles containing empty capsids and packaged genomes are known in the art. See, for example, Grimm et al., Gene Therapy (1999) 6:1322-1330; Sommer et al., Molec. Ther. (2003) 7:122-128. To test for denatured capsids, the method involves subjecting the processed AAV stock to SDS-polyacrylamide gel electrophoresis, consisting of any gel capable of separating the three capsid proteins, for example, a gradient gel containing 3-8% Tris-acetate in a buffer, then running the gel until the sample material is separated, and blotting the gel onto a nylon or nitrocellulose membrane, preferably nylon. The anti-AAV capsid antibody is then used as the primary antibody that binds to the denatured capsid protein, preferably an anti-AAV capsid monoclonal antibody, most preferably a B1 anti-AAV-2 monoclonal antibody (Wobus et al., J. Virol. (2000) 74:9281-9293). A secondary antibody is then used that binds to the primary antibody and includes a means for detecting binding with the primary antibody, more preferably an anti-IgG antibody that contains a detection molecule covalently bound to the antibody, most preferably a sheep anti-mouse IgG antibody covalently bound to horseradish peroxidase. To semi-quantitatively determine the binding between the primary and secondary antibodies, a method for detecting binding is used, preferably a detection method capable of detecting radioisotope radiation, electromagnetic radiation, or colorimetric changes, most preferably a chemiluminescence detection kit. For example, for SDS-PAGE, samples from column fractions can be taken and heated in SDS-PAGE loading buffer containing a reducing agent (e.g., DTT), and capsid proteins resolved in precast gradient polyacrylamide gels (e.g., Novex). Silver staining may be performed using SilverXpress (Invitrogen, CA) according to the manufacturer's instructions, or other suitable staining methods, i.e., SYPRO Ruby or Coomassie dye.In one embodiment, the concentration of AAV vector genome (vg) in the column fractions can be measured by quantitative real-time PCR (Q-PCR). The samples are diluted and digested with DNaseI (or another suitable nuclease) to remove exogenous DNA. After inactivation of the nuclease, the samples are further diluted and amplified using a TaqMan™ fluorogenic probe specific for the primers and the DNA sequence between the primers. The number of cycles required to reach a defined level of fluorescence (threshold cycle, Ct) is measured for each sample on an Applied Biosystems Prism 7700 sequence detection system. Plasmid DNA containing the same sequence as contained in the AAV vector is used to generate a standard curve in the Q-PCR reaction. The cycle threshold (Ct) value obtained from the sample is used to determine the vector genome titer by normalizing it to the Ct value of the plasmid standard curve. An endpoint assay based on digital PCR can also be used.

[0088] In one embodiment, an optimized q-PCR method utilizing a broad-spectrum serum protease, such as Proteinase K (e.g., commercially available from Qiagen), is used. More specifically, the optimized qPCR genomic titer assay is similar to the standard assay, except that after DNase I digestion, the sample is diluted with Proteinase K buffer and treated with Proteinase K, followed by heat inactivation. Suitably, the sample is diluted with an amount of Proteinase K buffer equal to the sample size. Proteinase K buffer may be concentrated 2-fold or more. Typically, the Proteinase K treatment is about 0.2 mg / mL, but may vary from 0.1 mg / mL to about 1 mg / mL. The treatment step is generally performed at about 55° C. for about 15 minutes, but may be performed at lower temperatures (e.g., about 37° C. to about 50° C.) for longer periods (e.g., about 20 minutes to about 30 minutes) or at higher temperatures (e.g., up to about 60° C.) for shorter periods (e.g., about 5 to 10 minutes). Similarly, heat inactivation is generally at about 95°C for about 15 minutes, although the temperature may be lowered (e.g., about 70 to about 90°C) and the time may be extended (e.g., about 20 to about 30 minutes). The sample is then diluted (e.g., 1000-fold) and subjected to TaqMan analysis as described for standard assays.

[0089] Additionally or alternatively, droplet digital PCR (ddPCR) may be used. For example, a method for determining single-stranded and self-complementary AAV vector genome titer by ddPCR has been described. See, for example, M. Lock et al, Hu Gene Therapy Methods, Hum Gene Ther Methods. 2014 Apr; 25 (2): 115-25. doi: 10.1089 / hgtb.2013.131. Epub 2014 Feb 14.

[0090] In certain embodiments, the rAAV manufacturing process described herein involves methods described in U.S. Provisional Patent Application No. 63 / 371,597, filed August 16, 2022, and U.S. Provisional Patent Application No. 63 / 371,592, filed August 16, 2022, which are incorporated by reference in their entireties herein.

[0091] Briefly, a method for separating rAAVhu68 (or AAVhu95 or AAVhu96) particles having packaged genome sequences from genome-defective AAVhu68 (or AAVhu95 or AAVhu96) intermediates involves subjecting a suspension containing recombinant AAVhu68 (or AAVhu95 or AAVhu96) viral particles and AAVhu68 (or AVhu95 or AAVhu96) capsid intermediates to high performance liquid chromatography, where the AAVhu68 (or AAVhu95 or AAVhu96) viral particles and AAVhu68 (or AAVhu95 or AAVhu96) intermediates are bound to a strong anion exchange resin equilibrated at a pH of about 10.2 and subjected to a salt gradient while monitoring the eluate for ultraviolet absorbance at about 260 nanometers (nm) and about 280 nm. The pH can range from about 10 to 10.4, although it is less optimal for rAAVhu68 (or AAVhu95 or AAVhu96). In this method, AAV full capsids are collected from fractions that are eluted when the A260 / A280 ratio reaches an inflection point. In one example, for an affinity chromatography step, the diafiltered product may be applied to an affinity resin (Life Technologies) that efficiently captures AAV2 serotypes. Under these ionic conditions, a significant percentage of residual cellular DNA and proteins flow through the column, and AAV particles are efficiently captured.

[0092] rAAV.hLamin A (e.g., rAAV.CMVe.chTNTp.Lamin.RBG) is suspended in a suitable physiologically compatible composition (e.g., buffered saline). This composition can be frozen for storage, later thawed, and optionally diluted with a suitable diluent. Alternatively, the vector can be prepared as a composition suitable for delivery to a patient without undergoing a freezing and thawing step.

[0093] As used herein, the term "NAb titer" is a measure of how many neutralizing antibodies (e.g., anti-AAV Nabs) are produced that neutralize the physiological effects of the targeted epitope (e.g., AAV). Anti-AAV NAb titers can be measured, for example, as described in Calcedo, R., et al., Worldwide Epidemiology of Neutralizing Antibodies to Adeno-Associated Viruses. Journal of Infectious Diseases, 2009.199(3):p.381-390 (incorporated herein by reference).

[0094] The abbreviation "sc" refers to self-complementary. "Self-complementary AAV" refers to constructs in which the coding region carried by the recombinant AAV nucleic acid sequence is designed to form an intramolecular double-stranded DNA template. Upon infection, rather than waiting for cell-mediated synthesis of the second strand, the two complementary halves of scAAV will associate to form one double-stranded DNA (dsDNA) unit ready for immediate replication and transcription. See, for example, DM McCarty et al, "Self-complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis", Gene Therapy, (August 2001), Vol 8, Number 16, Pages 1248-1254. Self-complementary AAVs are described, for example, in U.S. Patent Nos. 6,596,535, 7,125,717, and 7,456,683, each of which is incorporated by reference herein in its entirety.

[0095] "Replication-defective virus" or "viral vector" refers to a synthetic or artificial viral particle in which an expression cassette containing a gene of interest is packaged into a viral capsid or envelope, and any viral genomic sequences packaged within the viral capsid or envelope are replication-defective, i.e., they are unable to produce progeny virions, but can retain the ability to infect target cells. In one embodiment, the genome of the viral vector does not contain genes encoding enzymes required for replication (the genome can be engineered to be "gutless", containing only the gene of interest flanked by signals required for amplification and packaging of the artificial genome), but these genes can be supplied during production. It is therefore considered safe for use in gene therapy, since replication and infection by progeny virions cannot occur except in the presence of viral enzymes required for replication.

[0096] As used herein, the terms "rAAV" and "artificial AAV", which are used interchangeably, refer to, but are not limited to, an AAV that includes a capsid protein and a vector genome packaged therein, the vector genome including a nucleic acid heterologous to the AAV. In one embodiment, the capsid protein is a non-naturally occurring capsid. Such an artificial capsid can be produced by any suitable technique using a selected AAV sequence (e.g., a fragment of vp1 capsid protein) in combination with a heterologous sequence that can be obtained from a different selected AAV that is a non-contiguous portion of the same AAV, from a non-AAV viral source, or from a non-viral source. The artificial AAV can be, but is not limited to, a pseudotyped AAV capsid, a chimeric AAV capsid, a recombinant AAV capsid, or a "humanized" AAV capsid. A pseudotyped vector in which the capsid of one AAV is replaced with a heterologous capsid protein is useful in the present invention. In one embodiment, AAV2 / 5 and AAV2 / 8 are exemplary pseudotype vectors. The selected genetic elements can be delivered by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion techniques, high-speed DNA-coated pellets, viral infection, and protoplast fusion. The methods used to create such constructs are known to those skilled in the art of nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012).

[0097] Often, rAAV particles are referred to as DNase-resistant. However, in addition to this endonuclease (DNase), other endo- and exo-nucleases can be used in the purification steps described herein to remove contaminating nucleic acids. Such nucleases can be selected to degrade single-stranded and / or double-stranded DNA, as well as RNA. Such steps can include a single nuclease, or a mixture of nucleases directed to different targets, which can be endonucleases or exonucleases.

[0098] The term "nuclease resistant" indicates that the AAV capsid is fully assembled around an expression cassette designed to deliver genes into a host cell, and protects these packaged genomic sequences from degradation (digestion) during a nuclease incubation step designed to remove contaminating nucleic acids that may be present from the production process.

[0099] As used herein, a "subpopulation" of vp proteins refers to a group of vp proteins that have at least one defined common characteristic and that consists of at least one group member and fewer than all members of the reference group, unless otherwise specified. For example, a "subpopulation" of vp1 proteins, unless otherwise specified, is at least one vp1 protein and fewer than all vp1 proteins in an assembled AAV capsid. A "subpopulation" of vp3, unless otherwise specified, can be one vp3 protein and fewer than all vp3 proteins in an assembled AAV capsid. For example, vp1 protein can be a subpopulation of vp proteins in an assembled AAV capsid, vp2 protein can be another subpopulation of vp proteins, and vp3 is yet another subpopulation of vp proteins. In another example, vp1, vp2, and vp3 proteins can contain subpopulations having, e.g., at least one, two, three, or four highly deamidated asparagines, e.g., different modifications at asparagine-glycine pairs.

[0100] Pharmaceutical Compositions In one aspect, provided herein is a pharmaceutical composition comprising a vector as described herein in a formulation buffer. In one embodiment, provided herein is a pharmaceutical composition comprising a rAAV as described herein in a formulation buffer. In one embodiment, the rAAV is at about 1×10 9 Genome copies (GC) / mL ~ approx. 1 x 10 14 In a further embodiment, the rAAV is formulated at about 3×10 9 GC / mL ~ approx. 3×10 13 In yet a further embodiment, the rAAV is formulated at about 1×10 GC / mL. 9 GC / mL ~ approx. 1×10 13 In one embodiment, the rAAV is formulated at a concentration of at least about 1×10 11 It is formulated in GC / mL.

[0101] Also provided herein is a composition comprising the rAAV or vector described herein and an aqueous suspension medium.In certain embodiments, the suspension is formulated for intravenous delivery, intrathecal administration, or intracerebroventricular administration.In one aspect, the composition contains at least one rAAV stock and optional carriers, excipients, and / or preservatives.

[0102] As used herein, a "stock" of rAAV refers to a population of rAAV. Despite the heterogeneity of capsid proteins due to deamidation, rAAV within a stock are expected to share the same vector genome. A stock can include, for example, rAAV with capsids having selected AAV capsid proteins and heterogeneous deamidation patterns characteristic of a selected production system. A stock can be produced from a single production system or pooled from multiple runs of a production system. A variety of production systems can be selected, including but not limited to those described herein.

[0103] As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharma- ceutical active substances is well known in the art. Supplementary active ingredients can also be incorporated into the composition. The phrase "pharmacologically acceptable" refers to molecular entities and compositions that do not produce allergic or similar adverse reactions when administered to a host. Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like can be used to introduce the compositions of the present invention into suitable host cells. In particular, the rAAV vector delivery vector genome can be formulated for delivery either encapsulated in lipid particles, liposomes, vesicles, nanospheres, nanoparticles, or the like.

[0104] In one embodiment, the composition comprises a final formulation suitable for delivery to a subject, for example, an aqueous liquid suspension buffered to a physiologically compatible pH and salt concentration.Optionally, one or more surfactants are present in the formulation.In another embodiment, the composition can be shipped as a concentrate that is diluted for administration to a subject.In other embodiments, the composition can be lyophilized and reconstituted at the time of administration.

[0105] Suitable surfactants or combinations of surfactants may be selected from non-toxic non-ionic surfactants. In one embodiment, a bifunctional block copolymer surfactant terminated in a primary hydroxyl group is selected, such as Pluronic® F68 [BASF], also known as Poloxamer 188, with a neutral pH and an average molecular weight of 8400. Other surfactants and other poloxamers may be selected, i.e., non-ionic triblock copolymers composed of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)), SOLUTOL HS 15 (macrogol-15 hydroxystearate), LABRASOL (polyoxycaprylic acid glyceride), polyoxy 10 oleyl ether, TWEEN (polyoxyethylene sorbitan fatty acid ester), ethanol, and polyethylene glycol. In one embodiment, the formulation contains a poloxamer. These copolymers are generally named with the letter "P" (for poloxamer) followed by three digits, the first two digits x 100 giving the approximate molecular mass of the polyoxypropylene core, and the last digit x 10 giving the percentage of polyoxyethylene content. In one embodiment, poloxamer 188 is selected. In one embodiment, the surfactant may be present in an amount of up to about 0.0005% to about 0.001% (w / w%, weight to weight basis) of the suspension. In another embodiment, the surfactant may be present in an amount of up to about 0.0005% to about 0.001% (v / v%, volume to volume basis) of the suspension. In yet another embodiment, the surfactant is present in an amount of up to about 0.0005% to about 0.001% of the suspension, where n% indicates n grams per 100 mL of suspension.

[0106] In another embodiment, the composition comprises a carrier, diluent, excipient, and / or adjuvant. A suitable carrier can be easily selected by a person skilled in the art in view of the indication for which the introduced virus is intended. For example, one suitable carrier includes saline, which can be formulated with various buffer solutions (e.g., phosphate buffered saline). Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water. The buffer / carrier should contain a component that prevents rAAV from sticking to the injection tube but does not interfere with rAAV binding activity in vivo. A suitable surfactant, or combination of surfactants, can be selected from among non-toxic non-ionic surfactants. In one embodiment, a bifunctional block copolymer surfactant terminated in a primary hydroxyl group is selected, such as Poloxamer 188 (also known under the trade names Pluronic® F68 [BASF], Lutrol® F68, Synperonic® F68, Kolliphor® P188), having a neutral pH and an average molecular weight of 8400. Other surfactants and other poloxamers may be selected, i.e. non-ionic triblock copolymers composed of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)), SOLUTOL HS 15 (macrogol-15 hydroxystearate), LABRASOL (polyoxycapric acid glyceride), polyoxy-oleyl ether, TWEEN (polyoxyethylene sorbitan fatty acid ester), ethanol, and polyethylene glycol. In one embodiment, the formulation contains a poloxamer. These copolymers are generally named with the letter "P" (for poloxamer) followed by three digits, the first two digits x 100 giving the approximate molecular mass of the polyoxypropylene core and the last digit x 10 giving the percentage of polyoxyethylene content. In one embodiment, poloxamer 188 is selected. The surfactant may be present in an amount up to about 0.0005% to about 0.001% of the suspension.

[0107] In certain embodiments, compositions containing rAAV.hLamin A are delivered at a pH ranging from 6.8 to 8, or 7.2 to 7.8, or 7.5 to 8. In certain embodiments, compositions containing rAAV.hLamin A are delivered intravenously at a pH of about 6.5 to about 7.5, which may be desirable. In certain embodiments, compositions containing rAAV.hLamin A are delivered intravenously at a pH of about 6.8 to about 7.2, which may be desirable. However, other pH ranges within the broad range, and subranges of these, may be selected for other delivery routes.

[0108] In certain embodiments, the formulation may contain a buffered saline solution that does not contain sodium bicarbonate. Such formulation may contain a buffered saline solution that contains one or more of sodium phosphate, sodium chloride, potassium chloride, calcium chloride, magnesium chloride, and mixtures thereof in water, such as Harvard buffer. In one embodiment, the buffered solution is PBS.

[0109] Optionally, the compositions of the present invention may contain other conventional pharmaceutical ingredients, such as preservatives or chemical stabilizers, in addition to the rAAV and carrier(s).Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol.Suitable chemical stabilizers include gelatin and albumin.

[0110] The compositions according to the invention may comprise a pharma- ceutically acceptable carrier as defined above. Suitably, the compositions described herein comprise an effective amount of one or more AAVs suspended in a pharma- ceutically suitable carrier and / or admixed with suitable excipients designed for delivery to a subject via injection or by another route and / or device.

[0111] In one embodiment, a therapeutically effective amount of the vector is included in the pharmaceutical composition. The choice of carrier is not a limitation of the present invention. As used herein, "therapeutically effective amount" refers to the amount of a composition containing a nucleic acid sequence encoding hLamin A (or rAAV or a vector thereof) that delivers and expresses an amount of protein sufficient to achieve efficacy in a target cell. In one embodiment, the dosage of the vector is about 1 x 10, including all integers or decimals and endpoints within the range. 9 GC / kg mass ~ approx. 1×10 14 The dosage is adjusted to balance the therapeutic effect against any side effects, and such dosage may vary depending on the therapeutic application for which the recombinant vector is used. The level of expression of the transgene product can be monitored to determine the frequency of dosage that results in the viral vector, preferably the AAV vector containing a minigene. Optionally, a similar dosage regimen as that described for therapeutic purposes can be utilized for immunization using the composition of the present invention.

[0112] The phrase "pharmacologically acceptable" refers to molecular entities and compositions that do not produce allergic or similar adverse reactions when administered to a host.

[0113] As used herein, the term "dose" or "amount" can refer to the total dose or amount delivered to a subject over the course of treatment, or the dose or amount delivered in a single unit (or multiple unit or divided dose) administration.

[0114] Additionally, the replication-deficient virus composition may be administered to a human patient in a dose range of about 1.0×10 (treating an average subject weighing 70 kg), including all integers or fractions within the range. 9 GC~approx. 1.0×10 16 GC range, preferably 1.0 x 10 12 GC~1.0×10 14 The composition can be formulated in dosage units containing an amount of replication-deficient virus in the range of GC. In one embodiment, the composition contains at least 1 x 10 per dose, including all integers or decimals within the range. 9 , 2×10 9, 3×10 9 , 4×10 9 , 5×10 9 , 6×10 9 , 7×10 9 , 8×10 9 , or 9 x 10 9 In another embodiment, the composition is formulated to contain at least 1 x 10 GC per dose, including all integers or decimals within the range. 10 , 2×10 10 , 3×10 10 , 4×10 10 , 5×10 10 , 6×10 10 , 7×10 10 , 8×10 10 , or 9 x 10 10 In another embodiment, the composition is formulated to contain at least 1 x 10 GC per dose, including all integers or decimals within the range. 11 , 2×10 11 , 3×10 11 , 4×10 11 , 5×10 11 , 6×10 11 , 7×10 11 , 8×10 11 , or 9 x 10 11 In another embodiment, the composition is formulated to contain at least 1 x 10 GC per dose, including all integers or decimals within the range. 12 , 2×10 12 , 3×10 12 , 4×10 12 , 5×10 12 , 6×10 12 , 7×10 12 , 8×10 12 , or 9 x 10 12 In another embodiment, the composition is formulated to contain at least 1 x 10 GC per dose, including all integers or decimals within the range. 13 , 2×10 13 , 3×10 13 , 4×10 13 , 5×10 13 , 6×10 13 , 7×10 13 , 8×10 13, or 9 x 10 13 In another embodiment, the composition is formulated to contain at least 1 x 10 GC per dose, including all integers or decimals within the range. 14 , 2×10 14 , 3×10 14 , 4×10 14 , 5×10 14 , 6×10 14 , 7×10 14 , 8×10 14 , or 9 x 10 14 In another embodiment, the composition is formulated to contain at least 1 x 10 GC per dose, including all integers or decimals within the range. 15 , 2×10 15 , 3×10 15 , 4×10 15 , 5×10 15 , 6×10 15 , 7×10 15 , 8×10 15 , or 9 x 10 15 In one embodiment, for human applications, the dose is 1×10 per dose, including all integers or fractions within the range. 10 ~Approx. 1×10 12 It may be in the GC range.

[0115] It is to be understood that the compositions in the pharmaceutical compositions described herein are intended to apply to the other compositions, regimens, aspects, embodiments, and methods described throughout this specification.

[0116] Methods and Uses In one aspect, provided herein is a method of treating a human subject diagnosed with idiopathic dilated cardiomyopathy (DCM) or a disease associated with a mutation in the LMNA gene. Further provided herein is the use of rAAV in the manufacture (preparation) of a medicament for the treatment of a human subject diagnosed with idiopathic dilated cardiomyopathy (DCM) or a disease associated with a mutation in the LMNA gene.

[0117] The method includes administering to a subject a suspension of a vector or rAAV described herein. In one embodiment, the method comprises administering to a subject a suspension of about 1×10 9 Genome copies (GC) / kg ~ approx. 1 x 10 14 In a further embodiment, the rAAV is administered to a subject at a dose of 3×10 GC / kg in a suspension of the rAAV described herein in a formulation buffer. 13 It is formulated as GC / kg.

[0118] In certain embodiments, the method of treating idiopathic DCM or a disease associated with a mutation in the LMNA gene further comprises monitoring hLaminA expression and percent cardiomyocyte transduction using endomyocardial biopsy.

[0119] The methods and compositions described herein may be used for the treatment of any of the stages of idiopathic dilated cardiomyopathy (DCM) or disease associated with mutations in the Lamin A (LMNA) gene. In certain embodiments, the patient is a toddler, an infant, or the patient is 3-6 years old, 3-12 years old, 3-18 years old, 3-20 years old. In certain embodiments, the patient is 18 years old or older. In certain embodiments, the patient is about 20-60 years old. In certain embodiments, the patient is about 40-50 years old. In certain embodiments, the patient is 60 years old or older.

[0120] In certain embodiments, the methods and compositions may be used for the treatment of adult-onset dilated cardiomyopathy with conduction defects. In certain embodiments, the methods and compositions may be used for the treatment of LMNA cardiomyopathy caused by loss-of-function mutations in the LMNA gene. In certain embodiments, the methods and compositions may be used for the treatment of LMNA cardiomyopathy that is autosomal dominant. In certain embodiments, the methods and compositions may be used for the treatment of early-onset phenotypic diseases associated with nonsense mutations in the LMNA gene (e.g., idiopathic DCM). In certain embodiments, the methods and compositions may be used for the treatment of LMNA cardiomyopathy associated with missense and truncations in the LMNA gene (e.g., idiopathic DCM). In certain embodiments, the methods and compositions may be used for the treatment of LMNA cardiomyopathy associated with Q15X mutations in the LMNA gene (e.g., idiopathic DCM). In certain embodiments, the methods and compositions may be used for the treatment of LMNA cardiomyopathy associated with N195K mutations in the LMNA gene (e.g., idiopathic DCM).

[0121] Additionally, other diseases may be associated with mutations in the LMNA gene, including muscular dystrophies, neuropathy, lipodystrophy, partial progeroid. For example, diseases associated with mutations in the LMNA gene include Emery-Dreifuss muscular dystrophy (EDMD), Maloof syndrome (MLF), congenital muscular dystrophy (MDC), limb-girdle muscular dystrophy type 1B (LGMD1B), Charcot-Marie-Tooth disease type 2B1 (CMT2B1), axonal neuropathy, familial partial lipodystrophy type 2 (FPLD2), mandibular acrodysplasia lipodystrophy (MAD), mandibular acrodysplasia type A (MADA), atypical Werner syndrome (AWS), premature aging syndrome (progeria), and Hutchinson-Gilford progeria syndrome (HGPS).

[0122] Symptoms of idiopathic dilated cardiomyopathy (DCM) or diseases associated with mutations in the Lamin A (LMNA) gene include atrioventricular (AV) conduction block, atrial arrhythmias including atrial fibrillation, atrial flutter and atrial tachycardia, ventricular arrhythmias including sustained ventricular tachycardia and ventricular fibrillation (VF). In certain embodiments, the methods and compositions described herein are used to alleviate or ameliorate one or more symptoms of idiopathic dilated cardiomyopathy (DCM) or diseases associated with mutations in the Lamin A (LMNA) gene, including atrioventricular (AV) conduction block, atrial arrhythmias including atrial fibrillation, atrial flutter and atrial tachycardia, ventricular arrhythmias including sustained ventricular tachycardia and ventricular fibrillation (VF) dilated cardiomyopathy, and / or heart failure. In certain embodiments, the methods and compositions described herein may be used to alleviate one or more symptoms of idiopathic dilated cardiomyopathy (DCM) or diseases associated with mutations in the Lamin A (LMNA) gene, including increased life expectancy and / or reduced progression to heart failure.

[0123] In certain embodiments, combination therapy or treatment may be utilized, including co-administration with another active agent. In certain embodiments, combination therapy may further include administration of a beta blocker, angiotensin-converting enzyme (ACE) inhibitor, a diuretic. The diuretic used may be acetazolamine (Diamox) or other suitable diuretic. In some embodiments, the diuretic is administered at the time of gene therapy administration. In some embodiments, the diuretic is administered prior to gene therapy administration. In some embodiments, a 3 mL injection volume of the diuretic is administered. In certain embodiments, combination therapy may further include an implantable cardioverter defibrillator (ICD), a pacemaker (PM), and / or cardiac resynchronization therapy (CRT).

[0124] Optionally, immunosuppressive combination therapy may be used in subjects in need thereof. Immunosuppressants for such combination therapy include, but are not limited to, glucocorticoids, steroids, antimetabolites, T-cell inhibitors, macrolides (e.g., rapamycin or rapalogs), and cytostatic agents (including alkylating agents, antimetabolites, cytotoxic antibiotics, antibodies, or agents active against immunophilins). Immunosuppressants may include nitrogen mustards, nitrosoureas, platinum compounds, methotrexate, azathioprine, mercaptopurine, fluorouracil, dactinomycin, anthracyclines, mitomycin C, bleomycin, mithramycin, IL-2 receptor (CD25)-specific antibodies or CD3-specific antibodies, anti-IL-2 antibodies, cyclosporine, tacrolimus, sirolimus, IFN-β, IFN-γ, opioids, or TNF-α (tumor necrosis factor-α) binding agents. In certain embodiments, immunosuppressive therapy may be initiated 0, 1, 2, 3, 4, 5, 6, 7 days prior to or after gene therapy administration, or earlier or later. Such immunosuppressive therapy may involve administration of one, two, or more drugs (e.g., glucocorticoids, prednerisone, mycophenolate mofetil (MMF), and / or sirolimus (i.e., rapamycin)). Such immunosuppressive drugs may be administered to a subject in need once, twice, or more times at the same dose or adjusted doses. Such therapy may involve simultaneous administration of two or more agents on the same day (e.g., prednerisone, mycophenolate mofetil (MMF), and / or sirolimus (i.e., rapamycin)). One or more of these drugs may be continued at the same dose or adjusted doses after gene therapy administration. Such therapy may be for about one week (7 days), about 60 days, or more, as needed. In certain embodiments, a tacrolimus-free regimen is selected.

[0125] In one embodiment, the rAAV described herein is administered once to a subject in need thereof. In another embodiment, the rAAV is administered two or more times to a subject in need thereof.

[0126] As used interchangeably herein, "patient" or "subject" refers to a mammal, male or female, including humans, veterinary or agricultural animals, domestic or pet animals, and animals typically used in clinical research. In one embodiment, the subject of these methods and compositions is a human patient. In one embodiment, the subject of these methods and compositions is a human patient, male or female. In certain embodiments, the subject of these methods and compositions is diagnosed with idiopathic dilated cardiomyopathy (DCM) or a disease associated with a mutation in the Lamin A (LMNA) gene, and / or symptoms of idiopathic dilated cardiomyopathy (DCM) or a disease associated with a mutation in the Lamin A (LMNA) gene.

[0127] It is to be understood that the compositions in the methods described herein are intended to apply to other compositions, regimens, aspects, embodiments, and methods described throughout the specification.

[0128] kit In certain embodiments, kits are provided that include concentrated vector suspended in a formulation (optionally frozen), optional dilution buffer, and devices and components required for intravenous administration. In another embodiment, the kit may additionally or alternatively include components for intravenous delivery. In one embodiment, the kit provides sufficient buffer to allow injection. Such buffer may allow for about 1:1 to 1:5 dilution of the concentrated vector, or more. In other embodiments, more or less buffer or sterile water is included to allow for dose titration and other adjustments by the treating physician. In yet other embodiments, the kit includes one or more components of the device. Suitable dilution buffers are available, such as saline, phosphate buffered saline (PBS), or glycerol / PBS.

[0129] It is to be understood that the compositions in the kits described herein are intended to apply to the other compositions, regimens, aspects, embodiments, and methods described throughout this specification.

[0130] The term "heterologous" when used to describe a nucleic acid sequence or protein means that the nucleic acid or protein is derived from a different organism or a different species of the same organism than the host cell or subject in which it is expressed. When used with reference to a protein or nucleic acid in a plasmid, expression cassette, or vector, the term "heterologous" indicates that the protein or nucleic acid is present with another sequence or subsequence from the protein or nucleic acid in question that is not found in the same relationship to each other in nature.

[0131] As described above, the terms "increase," "decrease," "reduce," "mitigate," "improve," "delay," or any grammatical variations thereof, or any similar term, unless otherwise specified, refer to a variation of about 5-fold, about 2-fold, about 1-fold, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10%, about 5% compared to a corresponding reference (e.g., an untreated control or a normal subject without idiopathic dilated cardiomyopathy (DCM) or a disease associated with a mutation in the Lamin A (LMNA) gene).

[0132] The term "expression" is used herein in its broadest sense and includes the production of RNA, or the production of RNA and protein. With respect to RNA, the terms "expression" or "translation" specifically relate to the production of peptides or proteins. Expression can be transient or stable.

[0133] As used herein, the term "administering" or any grammatical variations thereof refers to delivering a composition described herein to a subject.

[0134] The words "comprise", "comprises", and "comprising" are to be interpreted inclusively rather than exclusively. The phrases "consist", "consisting", and variations thereof are to be interpreted exclusive rather than inclusive. Although various embodiments herein have been presented using the word "comprising", in other circumstances, the relevant embodiment is also intended to be described as being included using the words "consisting of" or "consisting essentially of". As used throughout this specification and claims, the terms "comprising", "containing", "including", and variations thereof include other components, elements, integers, steps, etc. Conversely, the term "consisting" and variations thereof exclude other components, elements, integers, steps, etc.

[0135] It should be noted that the terms "a" or "an" refer to one or more. Thus, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably herein.

[0136] As used herein, the terms "about" or "to" refer to ±10% variation from and variations therebetween, unless otherwise specified, referenced integers and values. For example, "about" 500 μM includes ±50 (i.e., 450 to 550, including integers therebetween). For other values, particularly when referring to percentages (e.g., 90% to the taste), the term "about" includes all values ​​within the range, including both integers and fractions.

[0137] As noted above, the term "about" when used to modify a numerical value means a variation of ±10% (±10%, e.g., ±1, ±2, ±3, ±4, ±5, ±6, ±7, ±8, ±9, ±10, or values ​​therebetween) from the given reference, unless otherwise specified.

[0138] In certain cases, the term "E+#" or "e+#" is used to refer to the exponent. For example, "5E10" or "5e10" refers to 5×10 10 These terms may be used interchangeably.

[0139] With regard to the description of various embodiments herein, it is contemplated that each of the compositions described herein is, in another embodiment, useful in the methods of the invention. In addition, it is also contemplated that each of the compositions described herein that are useful in the methods is, in another embodiment, itself an embodiment of the invention.

[0140] Unless otherwise defined herein, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs and by reference to published documents which provide such persons with general guidance to many of the terms used in this application. EXAMPLES

[0141] The following examples are provided to illustrate certain aspects of the claimed invention, but the invention is not limited to these examples.

[0142] There is a high and unmet need in adult-onset dilated cardiomyopathy (DCM) with conduction defects, and full penetrance is observed with high transplantation and mortality rates. In some cases, DCM is caused by loss-of-function mutations in the Lamin A (LMNA) gene, which is amenable to gene replacement therapy. Since DCM has an autosomal dominant inheritance pattern, the presence of residual normal protein reduces the risk of an immune response to the transgene delivered in the case of gene replacement therapy. There are approximately 40,000 symptomatic LMNA-associated cardiomyopathy (or DCM) patients in the United States, so the addressable patient population is large.

[0143] Missense and truncating mutations are common in DCM families. Nonsense mutations are observed in more severe and / or early onset phenotypes (Hasselberg et al. European Heart Journal (2018) 39, 853-860; Suguru Nishiuchi. Circulation: Cardiovascular Genetics. Gene-Based Risk Stratification for Cardiac Disorders in LMNA Mutation Carriers, Volume: 10, Issue: 6). In some cases of DCM, carriers of very early truncating mutations in the LMNA gene have demonstrated a lack of dominant-negative function (e.g., Q15X). There are LMNA knockout and LMNA mutant mouse models available to address the function of Lamin A. For example, mice with the N195K DCM mutation have a less severe phenotype than KO mice.

[0144] Here, we developed an AAV vector expressing mature human lamin A (hLamin A) from a cardiac-selective promoter that rescues the lethal phenotype of LMNA knockout mice.

[0145] Example 1. Production of rAAV containing Lamin A. In the studies herein, rAAVs were generated and comparative studies were performed that contained engineered mature human lamin A (also referred to as hLaminA, hLamin A, huLaminA, huLamin A, hLMNA) sequences and mature hLamin A. In some cases, rAAVs containing a GFP gene-specific promoter were generated and used to evaluate promoter-driven cardiac transgene expression in mice.

[0146] rAAV is produced using a triple transfection technique that utilizes (1) a cis plasmid encoding the AAV2 rep protein and the AAVhu68 VP1 cap gene, (2) a cis plasmid containing adenovirus helper genes not provided by the packaging cell line expressing adenovirus E1a, and (3) a trans plasmid containing a vector genome for packaging into AAV capsids. See, e.g., US2020 / 0056159. The trans plasmid is designed to contain either the vector genome, including huLamin A.

[0147] The vector genome contains an AAV 5' inverted terminal repeat (ITR) and an AAV 3' ITR at the extreme 5' and 3' ends, respectively. The ITRs flank the sequence of an expression cassette packaged into the AAV capsid with a sequence encoding mature lamin A. The expression cassette further comprises a regulatory sequence operably linked to the fusion protein coding sequence, the regulatory control sequence comprising a CMV IE enhancer, a spacer sequence, and a hybrid cardiac promoter comprising chicken cardiac troponin T (chTnT), and the expression cassette further comprises a rabbit beta-globin (RBG) polyA. SEQ ID NO: 1 refers to the expression cassette of CMV-IE.chTnT.mature_hLaminA.rBG. SEQ ID NO: 2 refers to the vector genome of rAAV.CMV-IE.chTnT.mature_hLaminA.rBG, hereinafter also referred to as rAAV.hLaminA.

[0148] Additionally, the inventors investigated the effectiveness of achieving cardiac-specific AAV transduction using various AAV capsids. The inventors investigated AAVhu95 capsid and AAVhu96 capsid. AAVhu95 capsid and AAVhu96 are clade F AAV capsids isolated from human tissue. AAVhu95 capsid and AAVhu96 capsid are closely related to AAVhu68 capsid and have similar production yields. Furthermore, upon investigation, AAVhu95 capsid showed moderate improvement in cardiac transduction over AAVhu68 capsid. Briefly, in this study, a single NHP was transfected with 1000 mAbs of each unique barcode (3x10 13 Mice were intravenously administered a combination of three vectors (AAVhu68, AAVhu95, and AAVhu96) expressing GFP (GFP / vector). Figure 1A shows the relative levels of gene transfer into NHP hearts relative to AAVhu68 plotted as fold change in RNA sequencing reads (prevalence of RNA reads in tissues versus administered vector concentration). Similarly, mice were intravenously administered 10 µg / mL of the indicated vector (AAVhu95 or AAVhu68) expressing GFP. 12 GC. Animals were sacrificed 14 days after vector administration, and vector RNA copies and GFP fluorescence-positive area were quantified from heart samples. Figure 1B shows the level of transduction in mouse hearts after administration of AAVhu68 or AAVhu95 containing a gene encoding green fluorescent protein, plotted as a percentage of GFP-positive area. Figure 1C shows the level of transduction in mouse hearts after administration of AAVhu68 or AAVhu95 containing a gene encoding green fluorescent protein, plotted as number of copies / ng RNA.

[0149] Example 2. Pilot study investigating the efficacy of rAAV containing Lamin A in a Lamin A knockout mouse model In this study, we used AAVhu95 and AAVhu68 capsids (in the first mouse studies) that encode mature lamin A under the regulatory control of a hybrid cardiac promoter that contains the CMV IE enhancer, spacer sequence, and a cardiac-selective promoter (cardiac troponin T or TnT). The use of an engineered sequence that encodes mature lamin A avoids the potential toxicity of uncleaved farnesylated prelamin. Additionally, the farnesylated portion of lamin A (not included in mature lamin A) appears to be unnecessary for function. Furthermore, lamin C-only mice are phenotypically normal.

[0150] Briefly, newborn littermates of Lamin A knockout (KO) and wild-type (WT) mice (N-7-10 per group) were inoculated with 5 × 10 10 GC(5e10 GC, approx. 3×10 13Mice were injected intravenously (via temporal vein injection) with either vehicle (PBS) or AAVhu68.huLaminA at a dose of 100 GC / kg (3e13 GC / kg) (approximate dose assuming 5e13 GC / kg of body weight per g). Mice were examined for survival and weighed throughout the study on the days indicated. Survival and echocardiographic parameters were recorded for up to 12 weeks. Treatment showed survival rescue, with cardiac parameters not clearly improved by echo (possible deficit in severe model pre-treatment at birth). In vivo spectral and tissue Doppler interval measurements along with two-dimensional B-mode and M-mode transthoracic echocardiography were performed in all mice at P42 using a Vevo 3100 ultrasound imaging system (FUJIFILM Visual Sonics) and a 40 Mhz transducer. Mice were anesthetized via 2% isoflurane in an induction chamber for 3 minutes and then moved to an animal monitoring platform with ECG capability and thermoregulation controlled by feedback from a rectal thermometer. Mice were then fitted with a nose cone (1.5% isoflurane to maintain an anesthetic plane). Nair was used to remove hair before imaging. Investigators performing echo acquisition and analysis were blinded to the study groups. Analysis was performed using Vevo LAB software (FUJIFILM VisualSonics).

[0151] Figure 2A shows the 5×10 10 GC (approx. 3×10 13 Figure 2B shows Kaplan-Meier survival plots of lamin knockout (KO) and wild-type (WT) mice neonatally administered either vehicle control or AAVhu68.hLaminA intravenously at a dose of 5 x 10 10 GC (approx. 3×10 13Figure 2C shows the measured body weights of lamin knockout (KO) and wild-type (WT) mice neonatally administered either vehicle control or AAVhu68.hLaminA intravenously at a dose of 100 mg / kg (GC / kg). Figure 2C shows a representative Western blot confirming the expression of lamin A in the heart and the lack of expression of lamin A in the liver following administration of AAVhu68.hLamin A in knockout mice. These results confirm the expression of lamin A in cardiac tissue and demonstrate increased survival in knockout mice receiving AAV-LMNA treatment. At necropsy, liver and cardiac tissues were collected and analyzed by immunohistochemical staining with anti-human lamin antibodies to investigate the expression of mature huLamin A (Figures 3A-C).

[0152] Figure 3A shows the 5×10 10 GC (approx. 3×10 13 Figure 3B shows representative microscopic images from immunohistochemistry (IHC) analysis of anti-human lamin staining of FRG mouse liver tissue after neonatal intravenous administration of AAVhu68.hLaminA at a dose of 5 × 10 GC / kg. Figure 3C shows representative microscopic images from immunohistochemistry (IHC) analysis of anti-human lamin staining of mouse heart tissue after neonatal administration of vehicle control. Figure 3D shows representative microscopic images from immunohistochemistry (IHC) analysis of anti-human lamin staining of FRG mouse liver tissue after neonatal intravenous administration of AAVhu68.hLaminA at a dose of 5 × 10 GC / kg. 10 GC (approx. 3×10 13 Figure 7 shows representative microscopy images from immunohistochemistry (IHC) analysis of mouse cardiac tissue stained with anti-human lamin following neonatal intravenous administration of AAVhu68.hLaminA at a dose of 1000 mg / kg (1000 mg / kg). Figure 7A shows representative images of histology analysis of cardiac tissue in knockout mice following administration of PBS in knockout mice. Figure 7B shows representative images of histology analysis of cardiac tissue in knockout mice following administration of AAV-LMNA in knockout mice, confirming expression of lamin A in ventricular cardiac cells.

[0153] Next, we investigated the efficacy of treatment with rAAV.hLaminA on cardiac rhythm in Lamin A KO mice. Electrocardiograms and echocardiograms were performed according to protocols well established and published in the literature. Figure 4A shows a representative ECG analysis showing the RR interval (sec) over time in Lamin A KO mice administered AAV (0-23). ​​Figure 4B shows a representative ECG analysis showing the RR interval (sec) over time in Lamin A KO mice administered AAV (24-48). Figure 4C shows a representative ECG analysis showing the RR interval (sec) over time in WT mice administered vehicle (PBS) (0-12). Figure 4D shows a representative ECG analysis showing the RR interval (sec) over time in WT mice administered vehicle (PBS) (13-26). In comparison, we observed bradycardia in vehicle (PBS)-treated lamin A KO mice (Figure 5A-Figure 5D). Figure 5A shows a representative electrocardiogram analysis showing the RR interval (seconds) over time in vehicle (PBS)-treated lamin A KO mice (0-18). Figure 5B shows a representative electrocardiogram analysis showing the RR interval (seconds) over time in vehicle (PBS)-treated lamin A KO mice (19-38). Figure 5C shows a representative electrocardiogram analysis showing the RR interval (seconds) over time in vehicle (PBS)-treated lamin A KO mice (zoomed in 5A, time 0-10). Figure 5D shows a representative electrocardiogram analysis showing the RR interval (seconds) over time in vehicle (PBS)-treated lamin A KO mice (zoomed in 5B, time 7.00-7.45). Figure 6A shows echocardiographic results in wild-type (WT) and knockout (KO) mice administered vehicle (PBS) or AAV, plotted as ejection fraction (%) (One-way Anova nonparametric Kruskal-Wallis with Dunn's multiple comparison test: P<0.05, **P<0.01, ***P<0.01, ****P<0.0001).Figure 6B shows echocardiographic results in wild-type (WT) and knockout (KO) mice administered vehicle (PBS) or AAV plotted as fractional shortening (%) (Kruskal-Wallis one-way anova nonparametric with Dunn's multiple comparison test: P<0.05, **P<0.01, ***P<0.01, ****P<0.0001). Figure 6C shows echocardiographic results in wild-type (WT) and knockout (KO) mice administered vehicle (PBS) or AAV plotted as stroke volume (μL) (Kruskal-Wallis one-way anova nonparametric with Dunn's multiple comparison test: P<0.05, **P<0.01, ***P<0.01, ****P<0.0001).

[0154] Additionally, we investigated the efficacy of AAVhu95.CMVe.chTNTp.LaminA.rBG (AAVhu95-LMNA) in mice. Briefly, mice were treated with AAVhu95-LMNA and expression was examined by Western blot on day 5. Figure 8 shows a representative Western blot analysis of LMNA expression in mice administered AAVhu95-LMNA. The antibody used in the Western blot analysis showed low affinity to endogenous mouse LMNA.

[0155] Additionally, we investigated the efficacy of AAVhu95-LMNA in heterozygous mice. Figure 9A shows the results of LMNA telemetry studies plotted as a percentage of WT-PBS of LMNA expression in wild-type and heterozygous knockout mice after administration of either PBS (control) or AAV-LMNA. Figure 9B shows the results of LMNA telemetry studies plotted as a percentage of WT-PBS of LMNC expression in wild-type and heterozygous knockout mice after administration of either PBS (control) or AAV-LMNA. Figure 9C shows a representative Western blot analysis of heart samples of lamin A and lamin C expression in mice (wild-type and heterozygous knockout mice) administered AAVhu95-LMNA. These results show that lamin A expression is significantly increased in the hearts of AAVhu95-LMNA-treated mice at day 120.

[0156] In summary, in the knockout mouse phenotype model, we observed a low survival rate over the last 6 weeks, some mice lost weight, we also observed a trend towards decreased fractional shortening (FS) and ejection fraction (EF) in echocardiograms, and decreased stroke volume (SV), and we also observed abnormally shaped nuclei and suspicion of arrhythmia as the cause of cardiac arrest. In comparison, in AAV-treated mice, we observed an increase in the survival rate of knockout mice and confirmed an increase in LMNA expression in treated heterozygous knockout mice.

[0157] Example 3. Safety and expression studies of rAAV.hLaminA in mice and non-human primates (NHPs). In this study, we will perform telemetry pharmacology studies and evaluate arrhythmias as a cause of death. Furthermore, we will perform additional echocardiogram (ECG) and echo studies in aged mice heterozygous for laminopathies.

[0158] Next, we will conduct safety and expression studies in non-human primates. In this study, we will use AAVhu95 expressing human lamin A (e.g., AAVhu95.CMV-IE.chTnT.hLaminA.rBG or AAVhu95.hLaminA or AAVhu95-LMNA). Briefly, NHPs (N=2) will be administered 3×10 13 The mice will be administered intravenously with AAVhu95.hLaminA at a dose of 1000 mg / kg GC. The study period will be 60 days (2 months) during which continuous cage-side observations, vitals, physical examinations will be performed, and samples will be taken for evaluation of CBC, serum chemistry, and troponin I levels. Additionally, echoes and ECGs will be performed on day 0 (i.e., pre-dose baseline), month 1, and month 2 of the study period. At the end of the study period, necropsies will be performed and tissues will be collected for histopathology and in situ hybridization analysis (ISH) for transgene expression in the heart.

[0159] Expression cannot be performed by protein IHC due to cross-reactivity of human / NHP lamin A. ISH was performed but showed low expression compared to what would be expected at this dose level. It is not conclusive whether this is because hu95 performs less well in NHPs compared to mouse or compared to the benchmark hu68.

[0160] Heterozygous mice do not have a lethal phenotype. This study was performed to determine whether the phenotype could be seen by ECG and ultimately rescued by treatment. Six-month-old HET mice were injected with 3 × 10 13Mice received AAVhu95M199.CMVe.chTNTp.laminA.rBG IV (tail vein) at a dose of 1000 GC / kg, or PBS as a control, and WT PBS was used as a control. Regular ECG recordings were performed on these mice via an implanted telemeter. Tissue was harvested at approximately D120 and used for Western blots. Due to technical issues (electrodes did not stay in place) and WT controls also had abnormal readings over time, there were inconclusive results from the telemetry. This study confirmed good expression at D120 and suggested that the lack of expression in NHPs was not due to the capsid, unless hu95 behaves differently in mice (good cardiotropy) vs. NHPs.

[0161] References 1. Hershberger, R., Hedges, D. & Morales, A. Dilated cardiomyopathy: the complexity of a diverse genetic architecture. Nat Rev Cardiol 10,531-547(2013). 2.Parks SB, Kushner JD, Nauman D, et al.Lamin A / C mutation analysis in a cohort of 324 unrelated patients with idiopathic or familial dilated cardiomyopathy.Am Heart J 2008;156:161-9. 3.Hasselberg et al.European Heart Journal(2018)39,853-860. 4.Kang et al.BMB Reports 2018;51:327-37. 5. Charron, P., et al., What Should Cardiologists know about Lamin Disease?, Arrhythmia&Electrophysiology Review 2012;1(1):22-8. 6.Suguru Nishiuchi,Circulation:Cardiovascular Genetics.Gene-Based Risk Stratification for Cardiac Disorders in LMNA Mutation Carriers,Volume:10,Issue:6. 7.Hasselberg et al.European Heart Journal(2018)39,853-860. 8.Kumar, S. et al., Long-Term Arrhythmic and Nonarrhythmic Outcomes of Lamin A / C Mutation Carriers, J Am Coll Cardiol.2016 Nov,68(21)2299-2307. 9.Ollila L.,et al.Clinical disease presentation and ECG characteristics of LMNA mutation carriers,Open Heart 2017;4:e000474. 10.Rubinstein LV, Gail MH, Santner TJ, Planning the duration of a comparative clinical trial with loss to follow-up and a period of continued observation, (1981) J Chron Dis 8:67-74.

[0162] All documents cited herein are incorporated herein by reference and are hereby incorporated by reference. U.S. Provisional Patent Application No. 63 / 293,680, filed December 24, 2021, is incorporated herein by reference in its entirety. Although the present invention has been described with reference to specific embodiments, it will be understood that modifications can be made without departing from the spirit of the invention. Such modifications are intended to fall within the scope of the appended claims.

Claims

1. 1. A recombinant adeno-associated virus (rAAV) comprising an adeno-associated virus (AAV) capsid and a vector genome packaged in the AAV capsid, wherein the vector genome comprises an AAV 5' inverted terminal repeat (ITR), an expression cassette, and an AAV 3' ITR, wherein the expression cassette comprises an engineered open reading frame (ORF) for a mature human lamin A (hLaminA) coding sequence that encodes mature hLaminA lacking a preprotein carboxy (C)-terminal tail, the ORF being operably linked to regulatory control sequences that direct expression of the mature hLaminA protein in a cell, wherein the regulatory control sequences comprise a promoter, optionally an enhancer, and a polyadenylation (polyA) sequence. (a) the engineered ORF comprises a coding sequence having the nucleic acid sequence of SEQ ID NO:4, or a nucleic acid sequence at least 90% identical to SEQ ID NO:4 that encodes mature hLaminA lacking the preprotein carboxy (C)-terminal tail; or (b) the mature hLaminA has the amino acid sequence of SEQ ID NO:5; (c) the mature hLaminA coding sequence comprises the nucleic acid sequence of SEQ ID NO: 4; and / or (d) the regulatory control sequence comprises a promoter that is a cardiac promoter, optionally, the cardiac promoter is a cardiac troponin T (cTnT) promoter; The rAAV of claim 1.

3. The regulatory control sequence (a) a hybrid cardiac promoter comprising a cytomegalovirus immediate early (CMV IE) enhancer, a spacer sequence, and a cTNT promoter; (b) comprises a polyA sequence that is a rabbit beta-globin (rBG) polyA sequence; and / or (c) further comprising a mutant WPRE element; 3. The rAAV of claim 1 or 2.

4. 2. The rAAV of claim 1, wherein the expression cassette comprises a hybrid cardiac promoter comprising a CMV IE enhancer, a spacer sequence, and a cTnT promoter, the hybrid promoter being operably linked to the hLaminA coding sequence of SEQ ID NO: 4, and the expression cassette further comprises an rBG polyA sequence.

5. The rAAV of claim 1 , wherein the AAV capsid is a clade F AAV.

6. 2. The rAAV of claim 1, suitable for use in the treatment of idiopathic dilated cardiomyopathy (DCM) or a disease associated with a mutation in the lamin A (LMNA) gene, Optionally, the disease is associated with a mutation in the LMNA gene and is selected from muscular dystrophy, neuropathy, lipodystrophy, partial progeroid; Further optionally, the disease associated with a dysfunctional LMNA gene is further selected from Emery-Dreifuss muscular dystrophy (EDMD), Malouf syndrome (MLF), congenital muscular dystrophy (MDC), limb-girdle muscular dystrophy type 1B (LGMD1B), Charcot-Marie-Tooth disease type 2B1 (CMT2B1), axonal neuropathy, familial partial lipodystrophy type 2 (FPLD2), mandibuloacral dysplasia lipodystrophy (MAD), mandibuloacral dysplasia type A (MADA), atypical Werner syndrome (AWS), premature aging syndrome (progeria), and Hutchinson-Gilford progeria syndrome (HGPS). rAAV.

7. A composition comprising the rAAV stock of claim 1 and an aqueous suspension medium.

8. A pharmaceutical composition comprising the rAAV of claim 1 in a formulation buffer.

9. 9. The composition of claim 7 or the pharmaceutical composition of claim 8, formulated for delivery via intravenous (IV) injection, comprising: Optionally, the pharmaceutical composition is formulated to have a pH of about 6.5 to about 7.5 or about 6.8 to about 7.

2. Composition or pharmaceutical composition.

10. 1. A recombinant nucleic acid molecule comprising an expression cassette and an engineered open reading frame (ORF) for mature human lamin A (hLaminA), wherein the ORF has a mature hLaminA coding sequence that is a nucleic acid sequence encoding a functional mature human hLaminA lacking the preprotein carboxy (C)-terminal tail, the ORF being operably linked to regulatory control sequences, the expression cassette being flanked by a 5' inverted terminal repeat (ITR) and a 3' ITR, the engineered hLaminA gene encoding a functional mature hLaminA lacking the preprotein carboxy (C)-terminal tail, and the hLaminA coding sequence comprising the nucleic acid sequence of SEQ ID NO:

4.

11. 11. The recombinant nucleic acid molecule of claim 10, wherein the regulatory control sequence comprises a hybrid cardiac promoter comprising a CMV IE enhancer, a spacer sequence, and a chicken cTnT promoter.

12. A packaging host cell comprising the nucleic acid molecule of claim 10 or 11.

13. an AAV rep coding sequence operably linked to a sequence that expresses rep proteins in said packaging host cell, an AAV capsid coding sequence operably linked to a sequence that expresses AAV capsid proteins in said packaging host cell, and helper virus functions necessary to enable packaging of said expression cassette and ITRs into said AAV capsid; and / or The AAV capsid is AAVhu68, AAVhu96, AAVhu96, or AAV9; The packaging host cell of claim 12.

14. 2. An rAAV production system useful for producing the rAAV of claim 1, comprising: (a) a nucleic acid sequence encoding an AAV capsid protein; (b) a vector genome comprising an AAV 5' inverted terminal repeat (ITR), an expression cassette, and an AAV 3' ITR; (c) AAV rep and helper functions sufficient to enable packaging of the vector genome into the AAV capsid; a cell culture comprising the expression cassette comprises an engineered open reading frame (ORF) for mature human lamin A (hLaminA), the ORF having a mature hLaminA coding sequence that is a nucleic acid sequence encoding a functional mature hLaminA lacking a preprotein carboxy (C)-terminal tail, the ORF being operably linked to regulatory control sequences that direct expression of the mature hLaminA in a cell, the regulatory control sequences comprising a promoter, optionally an enhancer, and a polyadenylation (polyA) sequence; rAAV production system.

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