Patent Application
20230043051
Hereditary angioedema (HAE) is a rare condition characterized by recurrent episodes of swelling in face, throat, and in most extremities. HAE is a potentially life-threatening disorder characterized by unpredictable and recurrent attacks of vasodilation manifesting as subcutaneous and submucosal angioedema. In some cases, HAE is associated with low plasma levels of C1-inhibitor (type I), while in other cases the protein circulates in normal or elevated amounts but it is dysfunctional (type II). C1 inhibitor is the main regulator of plasma kallikrein activity. Symptoms of HAE attacks include swelling of the face, mouth and/or airway that occur spontaneously or are triggered by mild trauma. Edematous attacks affecting the airways can be fatal. In addition to acute inflammatory flares, excess plasma kallikrein activity has also been associated with chronic conditions, such as autoimmune diseases, including lupus erythematosus.
Various strategies for the treatment of C1-INH deficiencies or dysfunctions have been contemplated and developed, including for example inhibiting members of the contact system. For example, lanadelumab is a fully human monoclonal antibody inhibitor of plasma kallikrein that has been approved for the treatment of HAE.
Use of vectors that produce proteins in vivo is desirable for the treatment of disease, but is limited by various factors including poor protein production following delivery to a subject.
To date, available therapies have not addressed challenges including disease recurrence and the need for long-term continued administration. Thus, there is a need for a novel and lasting therapeutic approach to treat HAE.
The present invention provides recombinant adeno-associated virus (rAAV) vectors that allow for efficient and robust human C1 esterase inhibitor (C1-INH or C1EI) expression in vivo.
In one aspect, the present invention provides, among other things, a recombinant adeno-associated virus (rAAV) vector including an AAV8 capsid and a codon-optimized SERPING1 sequence encoding a C1 inhibitor (C1-INH).
In some embodiments, the codon-optimized SERPING1 sequence encoding C1-INH includes a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95% or 99% identity to SEQ ID NO: 2.
In some embodiments, the codon-optimized SERPING1 sequence encoding C1-INH includes a sequence identical to SEQ ID NO: 2.
In some embodiments, the vector further includes a liver-specific promoter.
In some embodiments, the liver-specific promoter is transthyretin promoter (TTR).
In some embodiments, the vector further includes a ubiquitous promoter.
In some embodiments, the vector further includes one or more of the following: a 5′ and a 3′ inverted terminal repeat, an intron upstream of the sequence, and a cis-acting regulatory module (CRM).
In some embodiments, the vector further comprises a WPRE sequence.
In some embodiments, the WPRE sequence is modified.
In some embodiments, the WPRE contains a mut6delATG modification.
In some embodiments, the intron is a minute virus of mice (MVM) or SV40 intron.
In some embodiments, the CRM is liver-specific CRM.
In some embodiments, the CRM is CRM8.
In some embodiments, the vector comprises at least three CRMs.
In another aspect, the present invention provides, among other things, a recombinant adeno-associated virus (rAAV) including an AAV8 capsid and an rAAV vector, and vector including:
a. a 5′ inverted terminal repeat (ITR);
b. a cis-acting regulatory module (CRM);
c. a liver specific promoter;
d. a minute virus of mice (MVM);
e. a SERPING1 sequence encoding C1 inhibitor (C1-INH);
f. a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE); and
g. a 3′ ITR.
In some embodiments, the SERPING1 sequence is a wild type sequence or a codon-optimized sequence.
In some embodiments, the codon-optimized SERPING1 sequence has at least about 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: 2.
In another aspect, the present invention provides, among other things, a method of treating a subject having hereditary angioedema (HAE), comprising administering to the subject in need thereof a rAAV of any one of the preceding embodiments.
In another aspect, the present invention provides, among other things, a method of treating a subject having hereditary angioedema (HAE), comprising administering to the subject in need thereof a recombinant adeno-associated virus (rAAV) vector comprising an AAV8 capsid, and a promoter operably linked to a nucleic acid sequence that encodes C1 inhibitor (C1-INH), and wherein administering results in an increase in C1-INH enzymatic activity in the subject.
In some embodiments, C1-INH is detected in the plasma of the subject.
In some embodiments, C1-INH is detected in the liver of the subject.
In some embodiments, C1-INH is maintained for at least 30, 60, 90, 120, 150, 180 days or more after a single administration.
In some embodiments, C1-INH activity is present in the subject following administration of the rAAV vector.
In some embodiments, the subject has C4 level restored to a pre-attack level.
In some embodiments, the AAV is administered intravenously.
In some embodiments, the AAV is administered intrathecally.
In some embodiments, the AAV is administered at dose of at least about 5×109 vg.
In some embodiments, the administering of the rAAV does not elicit immune response.
Various aspects of the invention are described in detail in the following sections. The use of sections is not meant to limit the invention. Each section can apply to any aspect of the invention. In this application, the use of “or” means “and/or” unless stated otherwise. As used herein, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise.
Adeno-associated virus (AAV): As used herein, the terms “adeno-associated virus” or “AAV” or recombinant AAV (“rAAV”) includes, but is not limited to, AAV type 1, AAV type 2, AAV type 3 (including types 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, avian AAV, bovine AAV, canine AAV, equine AAV, and ovine AAV (see, e.g., Fields et al., Virology, volume 2, chapter 69 (4th ed., Lippincott-Raven Publishers); Gao et al., J. Virology 78:6381-6388 (2004); Mori et al., Virology 330:375-383 (2004)). Typically, AAV can infect both dividing and non-dividing cells and can be present in an extrachromosomal state without integrating into the genome of a host cell. AAV vectors are commonly used in gene therapy.
Administering: As used herein, the terms “administering,” or “introducing” are used interchangeably in the context of delivering rAAV vectors encoding a therapeutic into a subject, by a method or route which results in delivery of the rAAV vector. Various methods are known in the art for administering rAAV vectors, including for example intravenously, subcutaneously or transdermally. Transdermal administration of rAAV vector can be performed by use of a “gene gun” or biolistic particle delivery system. In some embodiments, the rAAV vectors are administered via non-viral lipid nanoparticles.
Animal: As used herein, the term “animal” refers to any member of the animal kingdom. In some embodiments, “animal” refers to humans, at any stage of development. In some embodiments, “animal” refers to non-human animals, at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and/or worms. In some embodiments, an animal may be a transgenic animal, genetically-engineered animal, and/or a clone.
Approximately or about: As used herein, the term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).
Functional equivalent or derivative: As used herein, the term “functional equivalent” or “functional derivative” denotes, in the context of a functional derivative of an amino acid sequence, a molecule that retains a biological activity (either function or structural) that is substantially similar to that of the original sequence. A functional derivative or equivalent may be a natural derivative or is prepared synthetically. Exemplary functional derivatives include amino acid sequences having substitutions, deletions, or additions of one or more amino acids, provided that the biological activity of the protein is conserved. The substituting amino acid desirably has chemico-physical properties which are similar to that of the substituted amino acid. Desirable similar chemico-physical properties include, similarities in charge, bulkiness, hydrophobicity, hydrophilicity, and the like.
In vitro: As used herein, the term “in vitro” refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within a multi-cellular organism.
In vivo: As used herein, the term “in vivo” refers to events that occur within a multi-cellular organism, such as a human and a non-human animal. In the context of cell-based systems, the term may be used to refer to events that occur within a living cell (as opposed to, for example, in vitro systems).
IRES: As used herein, the term “IRES” refers to any suitable internal ribosome entry site sequence.
Polypeptide: The term, “polypeptide,” as used herein refers a sequential chain of amino acids linked together via peptide bonds. The term is used to refer to an amino acid chain of any length, but one of ordinary skill in the art will understand that the term is not limited to lengthy chains and can refer to a minimal chain comprising two amino acids linked together via a peptide bond. As is known to those skilled in the art, polypeptides may be processed and/or modified.
Protein: The term “protein” as used herein refers to one or more polypeptides that function as a discrete unit. If a single polypeptide is the discrete functioning unit and does not require permanent or temporary physical association with other polypeptides in order to form the discrete functioning unit, the terms “polypeptide” and “protein” may be used interchangeably. If the discrete functional unit is comprised of more than one polypeptide that physically associate with one another, the term “protein” refers to the multiple polypeptides that are physically coupled and function together as the discrete unit.
Regulatory element: As used herein, the term “regulatory element” refers to transcriptional control elements, in particular non-coding cis-acting transcription control elements, capable of regulating and/or controlling transcription of a gene. Regulatory elements comprise at least one transcription factor binding site, for example at least one binding site for a tissue specific transcription factor. In embodiments described herein, regulatory elements have at least one binding site for a liver-specific transcription factor. Typically, regulatory elements increase or enhance promoter-driven gene expression when compared to the transcription of the gene from the promoter alone, without the regulatory elements. Thus, regulatory elements particularly comprise enhancer sequences, although it is to be understood that the regulatory elements enhancing transcription are not limited to typical far upstream enhancer sequences, but may occur at any distance of the gene they regulate. As is understood in the art, sequences regulating transcription may be situtated either upstream (e.g., in the promoter region) or downstream (e.g., in the 3′UTR) of the gene that is regulated in vivo, and may be located in the immediate vicinity of the gene or further away. Regulatory elements can comprise either naturally occurring sequences, combinations of (parts of) such regulatory elements or several copies of a regulatory element, e.g., non-naturally occurring sequences. Accordingly, regulatory elements include naturally occurring and optimized or engineered regulatory elements to achieve a desired expression level.
Subject: As used herein, the term “subject” refers to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate). A human includes pre- and post-natal forms. In many embodiments, a subject is a human being. A subject can be a patient, which refers to a human presenting to a medical provider for diagnosis or treatment of a disease. The term “subject” is used herein interchangeably with “individual” or “patient.” A subject can be afflicted with or is susceptible to a disease or disorder but may or may not display symptoms of the disease or disorder.
Substantially: As used herein, the term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result. The term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.
Substantial homology: The phrase “substantial homology” is used herein to refer to a comparison between amino acid or nucleic acid sequences. As will be appreciated by those of ordinary skill in the art, two sequences are generally considered to be “substantially homologous” if they contain homologous residues in corresponding positions. Homologous residues may be identical residues. Alternatively, homologous residues may be non-identical residues will appropriately similar structural and/or functional characteristics. For example, as is well known by those of ordinary skill in the art, certain amino acids are typically classified as “hydrophobic” or “hydrophilic” amino acids, and/or as having “polar” or “non-polar” side chains. Substitution of one amino acid for another of the same type may often be considered a “homologous” substitution.
As is well known in this art, amino acid or nucleic acid sequences may be compared using any of a variety of algorithms, including those available in commercial computer programs such as BLASTN for nucleotide sequences and BLASTP, gapped BLAST, and PSI-BLAST for amino acid sequences. Exemplary such programs are described in Altschul, et al., basic local alignment search tool, J. Mol. Biol., 215(3): 403-410, 1990; Altschul, et al., Methods in Enzymology, Altschul, et al., “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs”, Nucleic Acids Res. 25:3389-3402, 1997; Baxevanis, et al., Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins, Wiley, 1998; and Misener, et al., (eds.), Bioinformatics Methods and Protocols (Methods in Molecular Biology, Vol. 132), Humana Press, 1999. In addition to identifying homologous sequences, the programs mentioned above typically provide an indication of the degree of homology. In some embodiments, two sequences are considered to be substantially homologous if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of their corresponding residues are homologous over a relevant stretch of residues. In some embodiments, the relevant stretch is a complete sequence. In some embodiments, the relevant stretch is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more residues.
Substantial identity: The phrase “substantial identity” is used herein to refer to a comparison between amino acid or nucleic acid sequences. As will be appreciated by those of ordinary skill in the art, two sequences are generally considered to be “substantially identical” if they contain identical residues in corresponding positions. As is well known in this art, amino acid or nucleic acid sequences may be compared using any of a variety of algorithms, including those available in commercial computer programs such as BLASTN for nucleotide sequences and BLASTP, gapped BLAST, and PSI-BLAST for amino acid sequences. Exemplary such programs are described in Altschul, et al., Basic local alignment search tool, J. Mol. Biol., 215(3): 403-410, 1990: Altschul, et al., Methods in Enzymology; Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997; Baxevanis et al., Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins, Wiley, 1998; and Misener, et al., (eds.), Bioinformatics Methods and Protocols (Methods in Molecular Biology, Vol. 132), Humana Press, 1999. In addition to identifying identical sequences, the programs mentioned above typically provide an indication of the degree of identity. In some embodiments, two sequences are considered to be substantially identical if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of their corresponding residues are identical over a relevant stretch of residues. In some embodiments, the relevant stretch is a complete sequence. In some embodiments, the relevant stretch is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more residues.
Suffering from: An individual who is “suffering from” a disease, disorder, and/or condition has been diagnosed with or displays one or more symptoms of the disease, disorder, and/or condition.
Therapeutically effective amount: As used herein, the term “therapeutically effective amount” of a therapeutic agent means an amount that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and/or condition, to treat, diagnose, prevent, and/or delay the onset of the symptom(s) of the disease, disorder, and/or condition. It will be appreciated by those of ordinary skill in the art that a therapeutically effective amount is typically administered via a dosing regimen comprising at least one unit dose.
Treating: As used herein, the term “treat,” “treatment,” or “treating” refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of and/or reduce incidence of one or more symptoms or features of a particular disease, disorder, and/or condition. Treatment may be administered to a subject who does not exhibit signs of a disease and/or exhibits only early signs of the disease for the purpose of decreasing the risk of developing pathology associated with the disease.
The recitation of numerical ranges by endpoints herein includes all numbers and fractions subsumed within that range (e.g. 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.9, 4 and 5). It is also to be understood that all numbers and fractions thereof are presumed to be modified by the term “about.”
Various aspects of the invention are described in detail in the following sections. The use of sections is not meant to limit the invention. Each section can apply to any aspect of the invention. In this application, the use of “or” means “and/or” unless stated otherwise. As used herein, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise.
Various aspects of the invention are described in detail in the following sections. The use of sections is not meant to limit the invention. Each section can apply to any aspect of the invention. In this application, the use of “or” means “and/or” unless stated otherwise.
The present disclosure describes efficient and robust recombinant adeno-associated virus (rAAV) vectors for in vivo production of C1-INH for the treatment of diseases associated with a C1-INH deficiency, such as HAE.
HAE is characterized by decreased levels of C1-INH with concomitant upregulation of bradykinin. It is inherited in an autosomal dominant pattern and affects 1:10,000 to 1:50,000 people. The underlying cause of HAE (type I and II) is attributed to autosomal dominant inheritance of mutations in the C1 esterase inhibitor gene (C1EI gene or SERPING1 gene), mapped to chromosome 11. Autosomal dominant mutations in the SERPING1 gene can be more than 300, yet it is amenable to gene therapy. Eighty-five percent of HAE cases are type I in which there is a deficiency in the amount of C1 esterase inhibitor produced (see e.g., Gower et al., World Allergy Organ J., 4: S9-S21 (2011); Cungo et al., Trends Mol Med, 15: 69-78 (2009); Gooptu et al., Annu Rev Biochem, 78: 147-176 (2009); and Zuraw et al., J Allergy Clin Immunol Pract, 1: 458-467 (2013)). The remainder of cases are characterized by the expression of a dysfunctional C1 esterase inhibitor.
The frequency, duration and severity of attacks associated with HAE vary, with 30% of patients reporting a frequency of greater than one attack/month, 40% report 6 to 11 attacks/year and the remaining 30% are infrequently symptomatic. Usually, symptoms are transient progressing over 12 to 36 hours and subsiding within 2 to 5 days; however, some attacks may last up to one week. Although HAE episodes are self-limiting, the unpredictable occurrence of attacks places considerable strain on patients, often heavily impacting quality of life, and can be fatal.
To date, therapeutic agents are indicated for long-term prophylaxis, therapy for acute attacks and short-term prophylaxis (i.e., prior to dental surgery), and include agents such as Danazol, which has a high adverse effect profile, C1 inhibitor replacement protein, bradykinin receptor antagonists, kallikrein inhibitors, fresh frozen plasma and purified C1 inhibitor. These therapies can alleviate symptoms and maximize quality of life; however, disease recurrence and the need for long-term continued administration remains a major obstacle to therapy (see e.g., Aberer, Ann Med, 44: 523-529 (2012); Charignon et al., Expert Opin Pharmacother, 13: 2233-2247 (2012); Papadopoulou-Alataki, Curr Opin Allergy Clin Immunol, 10: 20-25 (2010); Parikh et al., Curr Allergy Asthma Rep, 11: 300-308 (2011); Tourangeau et al., Curr Allergy Asthma Rep, 11: 345-351 (2011); Bowen et al., Ann Allergy Asthma Immunol, 100: S30-S40 (2008); Frank, Immunol Allergy Clin North Am, 26: 653-668 (2006); Cicardi et al., J Allergy Clin Immunol, 99: 194-196 (1997); Kreuz et al., Transfusion 49: 1987-1995 (2009); Bork et al., Ann Allergy Asthma Immunol, 100: 153-161 (2008); and Cicardi et al., J Allergy Clin Immunol, 87: 768-773 (1991)).
The present invention provides, among other things, methods and compositions for treating HAE using recombinant adeno-associated virus (rAAV) vectors including codon-optimized SERPING1 sequence that encode hC1-INH. In particular, the present invention provides a method of treating HAE by administering a rAAV comprising a codon-optimized sequence SERPING1 encoding a human C1-INH at an effective dose such that at least one symptom or feature of HAE is reduced in intensity, severity, or frequency. The gene therapy method described herein was particularly effective in expressing therapeutic level of hC1-INH.
rAAV SERPING1 Vector Design
In some aspects, provided herewith is a recombinant adeno-associated virus (rAAV) vector encoding C1-INH. The rAAV vector comprises a capsid and a SERPING1 sequence.
Schematic that illustrate exemplary rAAV vectors of the present disclosure are illustrated in
The SERPING1 sequence of the vector can be a wild-type or a codon-optimized variant. Accordingly, in some embodiments, the rAAV vector comprises a wild-type SERPING1 nucleotide sequence. In some embodiments, the rAAV vector comprises a codon-optimized SERPING1 sequence.
A suitable C1-INH of the present invention is any protein or a portion of a protein that can substitute for at least partial activity of naturally-occurring C1-INH or rescue one or more phenotypes or symptoms associated with C1-INH-deficiency.
In some embodiments, a suitable C1-INH nucleotide sequence for the present invention comprises a SERPING1 sequence encoding human C1-INH protein. The naturally-occurring human C1-INH nucleotide sequence is shown in GenBank: AF435921.1. The corresponding human C1-INH amino acid sequence is shown in Table 1:
Various kinds of promoters can be used in the rAAV vector described herein. These include, for example, ubiquitous, tissue-specific, and regulatable (e.g. inducible or repressible) promoters. In some embodiments, the promoter is a liver-specific promoter. Examples of liver-specific promoters are known in the art and include, for example, human transthyrethin promoter (TTR), modified hTTR, (hTTR mod.), α-Antitrypsin promoter, human factor IX pro/liver transcription factor-responsive oligomers, LSP, CMV/CBA promoter (1.1 kb), CAG promoter (1.7 kb), mTTR, modified mTTR, mTTR pro, mTTR enhancer, and the basic albumin promoter. Liver specific promoters are described, for example, in Zhijian Wu et al., Molecular Therapy vol. 16, no 2, February 2008, the contents of which are incorporated herein by reference.
In some embodiments, the promotor is a ubiquitous promoter. In some embodiments, the promoter is a chicken beta actin promoter.
In some embodiments, the rAAV vector contains additional enhancer or regulatory elements to promote transcription and/or translation of the mRNA (e.g., enhancer sequences, Kozak sequences, polyadenylation sequences, transcriptional termination sequences, IRES and the like). In some embodiments, the vector comprises a 5′ and a 3′ inverted terminal repeat (ITR). In some embodiments, the vector comprises one or more enhancer elements. In some embodiments, the vector comprises a polyA tail.
In some embodiments, the rAAV vector comprises one or more small elements, such as an intron. Various introns are known in the art. Suitable introns for the rAAV vector described herein include for example an MVM intron, a truncated F.IX intron, a chimeric β globin SD/immunoglobulin heavy chain SA intron, SV40 and/or an alpha globin 1st intron. In some embodiments, the rAAV vector comprises an MVM intron. In some embodiments, the rAAV vector comprises an SV40 intron. In some embodiments, the intron can be an Exon1-Intron1-partially Exon2 from the SERPING1 gene.
In some embodiments, the rAAV vector comprises woodchuck hepatitis virus post-transcriptional control element (WPRE) as WPRE increases transgene expression of viral vectors in a host of tissues. Various optimized or variant forms of WPRE are known in the art, and include for example WPRE wild-type, WPRE3, and WPREmut6delATG among others. WPRE and associated WPRE variants are described in U.S. Pat. Nos. 10,179,918; 7,419,829; 9,731,033; 8,748,169; 7,816,131; 8,865,881; 6,287,814; U.S. Patent Publication No. 2016/0199412; U.S. Patent Publication No. 2017/0114363; U.S. Patent Publication No. 2017/0360961; U.S. Patent Publication No. 2019/0032078; U.S. Patent Publication No. 2018/0353621; International Publication No. WO2017201527; International Publication No. WO2018152451; International Publication No. WO2013153361; International Publication No. WO2014144756; European Patent No. EP1017785; and European Patent Publication No. 3440191. Each of the foregoing publications are incorporated herein by reference in its entirety.
In some embodiments, the rAAV vector comprises one or more cis regulatory elements (CREs). CREs are modified backbone elements of a construct. Various optimized or variant forms of CRE are known in the art, and include for example CRE4 and CRE6 among others. The following publications describe different variants of CRE, and each of which is incorporated herein by reference: International Publication Nos. WO2016146757, WO2014064277, WO2014063753, and WO2009130208.
In some embodiments, the rAAV vector comprises a cis-actin regulatory module (CRM). Various kinds of CRM are suitable for use in the vectors described herein and include for example liver-specific CRM, neuronal-specific CRM and/or CRM8. In some embodiments, the vector includes more than one CRM. For example, in some embodiments, the vector comprises two, three, four, five or six CRM. In some embodiments, the vector comprises three CRM, for example three CRM8.
In some embodiments, the rAAV vector is sequence optimized to increase transcript stability, for more efficient translation, and to reduce immunogenicity. In some embodiments, the rAAV vector is sequence optimized to increase transcript stability, for more efficient translation, and/or to reduce immunogenicity. In some embodiments, the SERPING1 is sequence optimized.
In some embodiments, the rAAV vector is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11 vector. In some embodiments, the rAAV vector is AAV1. In some embodiments, the rAAV vector is AAV 2. In some embodiments, the rAAV vector is AAV 3. In some embodiments, the rAAV vector is AAV 4. In some embodiments, the rAAV vector is AAV 5. In some embodiments, the rAAV vector is AAV 6. In some embodiments, the rAAV vector is AAV 7. In some embodiments, the rAAV vector is AAV 8. In some embodiments, the rAAV vector is AAV 9. In some embodiments, the rAAV vector is AAV 10. In some embodiments, the rAAV vector is AAV 11. In some embodiments, the rAAV vector is sequence optimized. In some embodiments, the rAAV capsid is modified
Exemplary element sequences are shown in Table 2 below. In some embodiments, the rAAV vector comprises a rAAV vector element comprising a nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identity with a vector element sequence shown in Table 2. In some embodiments, the rAAV vector comprises a vector element nucleotide sequence identical to a vector element nucleotide sequence shown in Table 2.
Sequence identity of codon-optimized SERPING1 sequences (HA03, HA04, HA05, and HA06) with the wild type SERPING1 sequence (HA01), and sequence identity of one codon-optimized SERPING1 sequence with the other codon-optimized sequence are given below in Table 3.
Use of rAAV Vectors Encoding C1-INH for Treatment of Disease
Described herein are methods of treating a disease associated with C1-INH deficiency. Accordingly, in some embodiments, the rAAV vectors described herein are suitable for treating a subject that has a C1-INH deficiency, such as patients suffering from HAE. The method of treating includes administering to the subject in need thereof a recombinant adeno-associated virus (rAAV) vector as described herein.
The rAAV vector described herein can be used to treat any disease associated with C1-INH deficiency or disorder.
In some embodiments, the rAAV vector remains episomal following administration to a subject in need thereof. In some embodiments, the rAAV vector does not remain episomal following administration to a subject in need thereof. For example, in some embodiments, the rAAV vector integrates into the genome of the subject. Such integration can be achieved, for example, by using various gene-editing technologies, such as, zinc finger nucleases (ZFNs), Transcription activator-like effector nucleases (TALENS), ARCUS genome editing, and/or CRISPR-Cas systems.
In some embodiments, a pharmaceutical composition comprising a rAAV vector described herein is used to treat subjects in need thereof. The pharmaceutical composition containing a rAAV vector or particle of the invention contains a pharmaceutically acceptable excipient, diluent or carrier. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions and the like. The pharmaceutical composition can be in a lyophilized form. Such carriers can be formulated by conventional methods and are administered to the subject at a therapeutically effective amount.
The rAAV vector is administered to a subject in need thereof via a suitable route. In some embodiments, the rAAV vector is administered by intravenous, intraperitoneal, subcutaneous, or intradermal routes. In one embodiment, the rAAV vector is administered intravenously. In embodiments, the intradermal administration comprises administration by use of a “gene gun” or biolistic particle delivery system. In some embodiments, the rAAV vector is administered via a non-viral lipid nanoparticle. For example, a composition comprising the rAAV vector may comprise one or more diluents, buffers, liposomes, a lipid, a lipid complex. In some embodiments, the rAAV vector is comprised within a microsphere or a nanoparticle, such as a lipid nanoparticle or a inorganic nanoparticle.
In some embodiments, functional C1-INH is detectable in plasma of the subject at about 1 to 6 weeks post administration of the rAAV vector. In some embodiments, functional C1-INH is detectable in plasma of the subject at about one week. In some embodiments, functional C1-INH is detectable in plasma of the subject at about 2 weeks. In some embodiments, functional C1-INH is detectable in plasma of the subject at about 3 weeks. In some embodiments, functional C1-INH is detectable in plasma of the subject at about 4 weeks. In some embodiments, functional C1-INH is detectable in plasma of the subject at about 5 weeks. In some embodiments, functional C1-INH is detectable in plasma of the subject at about 6 weeks. In some embodiments, functional C1-INH is detectable in hepatocytes of the subject at about 1 to 6 weeks post administration of the rAAV vector. In some embodiments, functional C1-INH is detectable in hepatocytes of the subject greater than 7 weeks post administration of the rAAV vector.
In some embodiments, functional C1-INH is detectable in plasma of the subject at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 12 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, or 10 years after administration of the rAAV vector. Accordingly, in some embodiments, functional C1-INH is detectable in plasma of the subject at least 3 months after administration of the rAAV vector. In some embodiments, functional C1-INH is detectable in plasma of the subject at least 6 months after administration of the rAAV vector. In some embodiments, functional C1-INH is detectable in plasma of the subject at least 12 months after administration of the rAAV vector. In some embodiments, functional C1-INH is detectable in plasma of the subject at least 2 years after administration of the rAAV vector. In some embodiments, functional C1-INH is detectable in plasma of the subject at least 3 years after administration of the rAAV vector. In some embodiments, functional C1-INH is detectable in plasma of the subject at least 4 years after administration of the rAAV vector. In some embodiments, functional C1-INH is detectable in plasma of the subject at least 5 years after administration of the rAAV vector. In some embodiments, functional C1-INH is detectable in plasma of the subject at least 6 years after administration of the rAAV vector. In some embodiments, functional C1-INH is detectable in plasma of the subject at least 7 years after administration of the rAAV vector. In some embodiments, functional C1-INH is detectable in plasma of the subject at least 8 years after administration of the rAAV vector. In some embodiments, functional C1-INH is detectable in plasma of the subject at least 9 years after administration of the rAAV vector. In some embodiments, functional C1-INH is detectable in plasma of the subject at least 10 years after administration of the rAAV vector. In some embodiments, functional C1-INH is detectable in plasma of the subject for the remainder of the subject's life following administration of the rAAV vector.
In some embodiments, the administered rAAV comprising SERPING1 results in the production of active C1-INH to the same extent as found following administration of purified C1-INH protein delivered intravenously. In some embodiments, the administered rAAV comprising SERPING1 results in production of a greater amount of active C1-INH as compared to administration of purified C1-INH protein delivered intravenously.
In some embodiments, the administered rAAV comprising SERPING1 results in the increase of C1-INH in the subject. In some embodiments, the increase of C1-INH is detected in plasma of the subject. In some embodiments, the increase of C1-INH is detected in liver tissue of the subject. In some embodiments, the increase of C1-INH can be detected in one or more tissues/organs including gall bladder, spleen, ovary, urinary bladder, fat, placenta, lung, prostate, heart, lymph node, and endometrium. In some embodiments, the administered rAAV comprising SERPING1 increases C1-INH in the subject by about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, or about 10% in comparison to the subject's baseline C1-INH level prior to administering the rAAV comprising SERPING1. Accordingly, in some embodiments, the administered rAAV comprising SERPING1 increases C1-INH in the subject by about 95%. In some embodiments, the administered rAAV comprising SERPING1 increases C1-INH in the subject by about 90%. In some embodiments, the administered rAAV comprising SERPING1 increases C1-INH in the subject by about 85%. In some embodiments, the administered rAAV comprising SERPING1 increases C1-INH in the subject by about 80%. In some embodiments, the administered rAAV comprising SERPING1 increases C1-INH in the subject by about 75%. In some embodiments, the administered rAAV comprising SERPING1 increases C1-INH in the subject by about 70%. In some embodiments, the administered rAAV comprising SERPING1 increases C1-INH in the subject by about 65%. In some embodiments, the administered rAAV comprising SERPING1 increases C1-INH in the subject by about 60%. In some embodiments, the administered rAAV comprising SERPING1 increases C1-INH in the subject by about 55%. In some embodiments, the administered rAAV comprising SERPING1 increases C1-INH in the subject by about 50%. In some embodiments, the administered rAAV comprising SERPING1 increases C1-INH in the subject by about 45%. In some embodiments, the administered rAAV comprising SERPING1 increases C1-INH in the subject by about 40%. In some embodiments, the administered rAAV comprising SERPING1 increases C1-INH in the subject by about 35%. In some embodiments, the administered rAAV comprising SERPING1 increases C1-INH in the subject by about 30%. In some embodiments, the administered rAAV comprising SERPING1 increases C1-INH in the subject by about 25%. In some embodiments, the administered rAAV comprising SERPING1 increases C1-INH in the subject by about 20%. In some embodiments, the administered rAAV comprising SERPING1 increases C1-INH in the subject by about 15%. In some embodiments, the administered rAAV comprising SERPING1 increases C1-INH in the subject by about 10%.
In some embodiments, following administration of the AAV vector to the subject the levels of functional C1-INH detectable in the circulation are between about 2 and 20 times greater than the amount of functional C1-INH detectable in the subject before administration of the rAAV comprising SERPING1.
In some embodiments, following administration of the AAV vector to the subject the levels of detectable active C1-INH meets or exceeds human therapeutic level. In some embodiments, the levels of active C1-INH post administration of the rAAV vector is about between 2 and 35 times the human therapeutic level. In some embodiments, the levels of active C1-INH post administration is about 2 times the human therapeutic level. In some embodiments, the levels of active C1-INH post administration is about 3 times the human therapeutic level. In some embodiments, the levels of active C1-INH post administration is about 4 times the human therapeutic level. In some embodiments, the levels of active C1-INH post administration is about 5 times the human therapeutic level. In some embodiments, the levels of active C1-INH post administration is about 6 times the human therapeutic level. In some embodiments, the levels of active C1-INH post administration is about 6 times the human therapeutic level. In some embodiments, the levels of active C1-INH post administration is about 7 times the human therapeutic level. In some embodiments, the levels of active C1-INH post administration is about 8 times the human therapeutic level. In some embodiments, the levels of active C1-INH post administration is about 9 times the human therapeutic level. In some embodiments, the levels of active C1-INH post administration is about 10 times the human therapeutic level. In some embodiments, the levels of active C1-INH post administration is about 15 times the human therapeutic level. In some embodiments, the levels of active C1-INH post administration is about 20 times the human therapeutic level. In some embodiments, the levels of active C1-INH post administration is about 25 times the human therapeutic level. In some embodiments, the levels of active C1-INH post administration is about 30 times the human therapeutic level. In some embodiments, the levels of active C1-INH post administration is about 35 times the human therapeutic level.
In some embodiments, the rAAV. SERPING1 vector is delivered as a single dose per subject. In some embodiments, the subject is delivered the minimal effective dose (MED). As used herein, MED refers to the rAAV SERPING1 vector dose required to achieve C1-INH activity resulting in increased C1-INH levels in a subject.
The vector titer is determined on the basis of the DNA content of the vector preparation. In some embodiments, quantitative PCR or optimized quantitative PCR is used to determine the DNA content of the rAAV SERPING1 vector preparations. In one embodiment, the dosage is about 1×1011 vector genomes (vg)/kg body weight to about 2×1013 vg/kg, inclusive of endpoints.
In some embodiments, the dosage is at least 5×109 vg/kg or above.
In some embodiments, the rAAV SERPING1 vector compositions can be formulated in dosage units to contain an amount of replication-defective virus that is in the range of about 1.0×109 vg to about 1.0×1015 vg. As used herein, the term “dosage” can refer to the total dosage delivered to the subject in the course of treatment, or the amount delivered in a single (of multiple) administration.
In some embodiments, the dosage is sufficient to increase plasma C1-INH levels in the patient by 25% or more. In some embodiments, rAAV SERPING1 is administered in combination with one or more therapies for the treatment of HAE.
Other features, objects, and advantages of the present invention are apparent in the examples that follow. It should be understood, however, that the examples, while indicating embodiments of the present invention, are given by way of illustration only, not limitation. Various changes and modifications within the scope of the invention will become apparent to those skilled in the art from the examples.
Exemplary methods and designs of generating rAAV expression constructs (rAAV vectors) comprising coding sequences of human C1-esterase inhibitor (C1-INH) and variations of the same are provided in this Example. In this study, hSERPING1 was used as a coding sequence for human C1-INH (hC1-INH), and recombinant AAV vector (rAAV8) was used as a vector. The basic design of a rAAV vector comprises an expression cassette flanked by inverted terminal repeats (ITRs): a 5′-ITR and a 3′-ITR These ITRs mediate the replication and packaging of the vector genome by the AAV replication protein Rep and associated factors in vector producer cells. Typically, an expression cassette contains a promoter, a coding sequence, a polyA tail and/or a tag, as shown in
In another embodiment, a WPRE sequence was inserted downstream of the coding region. This element creates a tertiary structure that increases the mRNA stability. A schematic representation of the expression constructs described herein is shown in
Condon-Optimized Constructs
Additionally, the coding sequences for the SERPING1 were codon-optimized based on multiple parameters, such as codon adaptation index (CAI), CpG site count, GC content, and repetitious base sequences. High CAI was preferred to utilize more frequently used codons and to potentially increase transgene product expression level from the vector. CpG island sequences, which can elicit immune response, were reduced. Repetitious bases were also removed. Any number of variations of the above scheme can be performed. For example, more than one promoter may be used. Additionally, different combinations of regulatory region, promotor, intron, and exon can be contemplated.
Different expression constructs designed for treating human angioedema (HAE constructs) are listed in Table 4, and have been depicted in
J constructs, in general, are shown in
S constructs, in general, are shown in
N constructs, in general, are shown in
U constructs, in general, are shown in
P constructs, in general, are shown in
This example illustrates the potency of the AAV8.SERPING1 vector in hC1-INH expression in vitro.
HepG2 cells (liver cells) were transfected with rAAV vectors (AAV8.SERPING1) expressing hC1-INH or with a control vector (as a negative control), and supernatants were collected after 72 h. The rAAV vector construct is depicted in
The level of hC1-INH expression in HepG2 cells was determined by measuring the amount of hC1-INH present in the supernatants using ELISA. As shown in
This example illustrates the potency of rAAV (i.e AAV8.hSERPING1) vector in dose-dependent expression of hC1-INH in vivo.
To demonstrate in vivo expression, an AAV8.hSERPING1vector encoding hC1-INH, depicted as M01 in
The efficacy of the rAAV was determined by monitoring levels of hC1-INH in plasma. The level of hC1-INH in plasma was expressed in terms of the percentage of hC1-INH level present in a normal mice. Results are depicted in
Furthermore, the transduction and transcription efficiencies of the rAAV were also determined by injecting the vectors into mice intravenously. Mice (C57/bl/6) received the rAAV at one of the three doses—1×1011 vg/kg, 4×1011 vg/kg, and 4×1012 vg/kg, and a fourth group of mice received the vehicle only. The rAAV vector construct is depicted as M01 in
The results from this example show that rAAV.AAV8.hSERPING1 vectors express C1-INH in vivo in a dose-dependent manner. This result also reveals the presence of physiological human C1-INH levels in wild-type mice.
This example illustrates in vivo efficacies of some codon-optimized rAAV8.C1-INH constructs in expressing C1-INH in plasma.
In order to test the effect of codon-optimization of hSERPING1 sequences on the in vivo efficacy of rAAV8 vectors, four codon-optimized constructs were prepared. The codon-optimized constructs were—J01, J02, J03, and J04. Mice (C57bl/6) were injected with rAAV vectors comprising either M01A (a wild-type C1-INH) or codon-optimized C1-INH sequences. All five constructs are depicted in
Mice administered with control vector (wild-type) as well as codon-optimized constructs expressed hC1-INH. Different constructs showed a moderate effect of codon optimization on their ability to express hC1-INH.
The codon-optimized construct, J04, expressed hC1-INH in a dose-dependent manner throughout the study (i.e at day 7, at day 14, and at day 28), as demonstrated in
The results from this example show that codon optimization moderately improves the efficacy of a construct over the wild-type (control vector). The codon-optimized constructs also express hC1-INH in a dose-dependent manner. Based on this study, J02 was selected for subsequent in-life studies.
Effect of WPRE
This example illustrates the in vivo efficacy of a vector comprising a codon-optimized AAV8.C1-INH-co2 sequence in combination with three different WPREs in expressing C1-INH in plasma.
Mice were injected with rAAV vectors comprising different codon-optimized constructs: (1) J02 (no WPRE element); (2) S07 (containing WPRE3); and S04 (containing WPREmut6delATG). The rAAV vector comprising WPRE is depicted in
Mice administered with the construct comprising WPRE expressed about 2.5 fold more hC1-INH compared to a codon-optimized constructs without WPRE.
The results from this example show that the incorporation of WPRE improves the efficacy of the codon-optimized constructs.
This example illustrates the in vivo efficacy of a rAAV8 vector comprising a HA06 (hSERPING1) sequence, a WPRE, and an intron for the expression of hC1-INH in plasma.
In order to test the effect of WPRE and intron on the in vivo efficacy of a rAAV8 vector comprising a HA06 sequence, combinations of different WPREs and introns were used. Introns used in this study were either an MVM intron or an exon 1-intron 1-partially exon 2, while the WPREs used were either a WPRE3 or a WPREmut6delATG. Recombinant AAV vectors comprising different combination of WPREs, introns, and codon-optimized hSERPING1 sequences used in this study are depicted in
Mice administered with the construct comprising MVM intron, HA06 (SERPING1) sequence and WPRE3 (for example, S07) expressed more hC1-INH compared to the remaining animals that received other constructs at the same dose. Mice that received J04 at 2×1012 vg/kg showed the highest expression of hC1-INH.
The results from this example show that the incorporation of MVM intron and WPRE3 improves the efficacy of the HA06 (SERPING1) sequences.
This example illustrates the in vivo efficacy of constructs comprising a HA06 (hSERPING1) sequence, a WPRE and a novel promoter in plasma.
In order to test the effect of WPRE and other novel promoters on the in vivo efficacy of rAAV8 vectors, different constructs with different novel promoter elements were used. Seven different constructs—J02, J04, S04, N01, N02, N03, and C22—were tested. C22 included in this test is a control vector. All seven constructs are depicted in
Mice administered with the construct comprising HA06 (SERPING1) sequence and WPREmut6delATG (for example, S04) expressed more hC1-INH compared to the remaining animals that received other constructs at the same dose. Mice that received J04 at 1.2×1013 vg/kg showed the highest expression of hC1-INH. The results from this example show that the incorporation of WPREmut6delATG improves the efficacy of the HA06 (SERPING1) sequence.
In another example, mice (C57bl/6; male) received S04 at two doses—2×1012 vg/kg and 2×1013 vg/kg, and plasma was collected at Day 14 and Day 28. Another group of mice received buffer to serve as a control. Results are depicted in
The results from this example show that S04 expresses hC1-INH in a dose dependent manner at both time-points.
This example illustrates the in vivo efficacy of rAAV8 vectors comprising constructs that include codon-optimized hSERPING1 sequences in plasma.
Ten different codon-optimized constructs—U01, U02, U03, U04, U05, U06, U07, U08, U09, and U10—were prepared. All ten constructs are depicted in
Mice administered with U06 expressed hC1-INH equivalent to that of group that received S04 at the same dose.
This example illustrates the in vivo efficacy of rAAV8 vectors comprising constructs that include HA06 (hSERPING1) sequence, a WPRE and novel promoter elements in plasma.
In order to test the effect of WPRE and novel promoters on the in vivo efficacy of rAAV8 vectors, different constructs with or without WPRE and novel promoter elements were used. Seven different constructs—J02, S03, S04, S06, S07, N04, and N05—were tested. All seven constructs are depicted in
Mice administered with the construct comprising MVM intron (for example, S07) expressed more hC1-INH compared to the group that received a construct without an MVM (for example, S06). In addition, the group that received construct comprising a shorter WPRE, a WPRE3, (for example, S06 and S07) expressed more hC1-INH compared to the group that received a construct comprising modified WPRE, WPREmut6delATG (for example, S03 and S04).
The results from this example show that the incorporation of MVM intron and a shorter form of WPRE, WPRE3, improve the efficacy of the HA06 (SERPING1) sequence. The construct, S07 that incorporates both a MVM intron and WPRE3 demonstrates the highest efficacy to express hC1-INH over 175 days.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the following claims:
The present application claims priority to U.S. Provisional Application Ser. No. 62/924,877, filed Oct. 23, 2019, the disclosure of which is hereby incorporated by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2020/056984 | 10/23/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62924877 | Oct 2019 | US |
