Patent Application
20250144238
This application contains a computer readable Sequence Listing which has been submitted electronically in XML format with this application, and is incorporated herein by reference in its entirety. The Sequence Listing XML file submitted with this application was created on Dec. 4, 2024; is named “BCU0022US3.xml” and is 97,458 bytes in size. The Sequence Listing does not go beyond the disclosure in the application as filed.
The present disclosure relates to betaretrovirus-based compositions and methods for “tuning” betavirus ENV proteins such that they target a particular cell surface receptor; i.e., are cell and/or tissue-type specific, allowing for better site-specific transgene delivery and expression.
Precise targeting is widely recognized as one of the most significant barriers to the manufacturing and use of viral vectors for gene therapy applications. See Gene therapy coming of age: Opportunities and challenges to getting ahead published Oct. 2, 2019, by E. Capra, J. Smith, and G. Yang, incorporated herein by reference. Specifically, they state that more precise targeting can reduce potential off-target effects and improve safety, which can be especially important if the therapy integrates into or edits the genome.
Currently, a number of viral approaches are in clinical development for gene therapy approaches, including, e.g., adenovirus (AV), adeno-associated (AAV), and Lentivrus (LV). Due in part to the epidemiology of AVs in the human population and AV antigen-specific T cells, which result in the lifelong immunity, vectors based on AV tend to have compromised potencies and trigger a stronger immunological response compared to other viral vectors, such as those based on AAV.
AAV has received much attention because it lacks the essential genes needed for replication and expression of its own genome, making its safety profile attractive but additional challenges exist. Vectors based on AAV are irrevocably limited to a ˜5 kb capacity, so all components needed for proper expression need to be modified to fit into the small capsid. AAV serotypes or engineered capsids have the capacity to transduce multiple tissue/cell types, cell-type-specific, and therefore, transgene expression is by and large controlled at the level of gene transcription. However, innate immunity can detrimentally affect AAV-mediated transgene expression.
Retrovirus infection of a cell begins when the trimeric envelope protein (ENV) complex on the surface of the virion binds to a cognate receptor on a suitable host cell (
ENV is the product of the retroviral env gene. The ENV precursor protein is cleaved by cellular furins, such that the mature ENV comprises two subunits: a surface (SU) subunit and a transmembrane (TM) subunit. SU contains the viral receptor-specificity determinants, while TM comprises a type-I fusion complex that is required for virion-cell membrane fusion. Interactions between SU and TM ensure that receptor binding precedes triggering of fusion with the host cell, while additional interactions within and between subunits are involved in proper assembly of the mature, heterotrimeric spike (SU3-TM3). While superficially similar, retroviruses of each genus differ in the specific mechanisms that regulate the fusion and entry processes. For decades, the focus was on oncogenic retroviruses in the Alpharetrovirus and Gammaretrovirus genera; with the emergence of HIV-1 and AIDS, the focus switched to the Lentivirus genus.
Thanks to the modularity of retrovirus genomes, virions can be engineered to package, reverse-transcribe, and insert non-viral sequences into host-cell DNA, a feature that has been exploited in the development of numerous gene-therapy platforms—the most notable examples being the HIV-1 (genus Lentivirus) and MLV (genus Gammaretrovirus).
Lentiviral vectors have several features that make them amenable to transgene delivery for therapeutic purposes. Lentiviral vectors are integrating vectors that permit long-term transgene expression. They have a packaging capacity of up to 9 kb. High-level expression of multiple genes may be a requisite for achieving therapeutic outcomes for certain diseases. However, an obvious safety consideration in designing a lentiviral vector system for gene therapy is the unintended generation of replication-competent provirus. Also, it has been reported that even self-inactivating lentiviral vectors with strong promoter and enhancer elements can activate neighboring genes, integrate to form chimeric gene fusions, and cause aberrant splicing of cellular transcripts.
Therefore, there exists an ongoing need for improved viral delivery of transgenes for gene therapy approaches. In contrast to AV, AAV and Lentivirus, little is known about betaretroviruses (genus Betaretrovirus), and they have not been widely examined for their potential use in viral vector platforms, and gene therapy. Accordingly, betaretroviruses exist as a potential alternative to currently available viral-based gene delivery platforms.
An aspect of the present disclosure relates to betaretroviruses (genus Betaretrovirus), and a recombinant betaretroviral envelope (rENV) protein for use in an engineered delivery vehicle and gene therapy, their associated methods of use, and manufacture. In particular, the present disclosure provides compositions and methods to tune betavirus ENV proteins such that they target a particular cell surface receptor, i.e., are cell and/or tissue-type specific, allowing for better site-specific transgene delivery and expression.
The present disclosure also provides a nucleic acid comprising a polynucleotide that encodes a transcription unit for a recombinant betaretroviral envelope protein comprising a receptor binding domain (RBD), a betaretrovirus surface subunit domain, and a betaretrovirus transmembrane (TM) domain. The RBD is a receptor binding ligand (RBL), wherein the RBL is a peptide, polypeptide or protein ligand binding specifically to a receptor on a cell, a tissue and/or a mammal.
An additional aspect of the present disclosure is directed to an engineered delivery vehicle that comprises a polynucleotide encoding one or more recombinant betaretroviral envelope proteins comprising a receptor binding domain, a betaretrovirus surface subunit (SU) domain, and a betaretrovirus transmembrane domain for forming a packaged nucleic acid vector delivery vehicle.
Another aspect of the present disclosure is related to an engineered delivery vehicle that comprises a virus-like particle having a recombinant betaretroviral envelope (rENV) protein on its surface and including a packaged nucleic acid viral vector cargo. The rENV protein comprises a receptor binding domain (RBD), a betaretrovirus surface subunit (SU) domain, and a betaretrovirus transmembrane (TM) domain. The packaged nucleic acid viral vector cargo comprises a polynucleotide encoding the rENV, and a transcription unit encoding a therapeutic protein.
The present disclosure also provides an engineered delivery vehicle that comprises a polynucleotide encoding one or more betaretroviral elements or recombinant betaretroviral envelope proteins for forming the engineered delivery vehicle for packaging cargo within the delivery vehicle. In some embodiments, the betaretroviral elements or recombinant betaretroviral envelope proteins comprise a betaretroviral envelope protein, which comprises at lease one of a surface subunit (SU) domain, a betaretrovirus transmembrane (TM) domain or a combination thereof. In any aspect or embodiment described herein, the SU domain comprises a recombinant receptor binding domain (RBD) and a scaffold domain. In any aspect or embodiment described herein, the SU domain is covalently or non-covalently bound to the TM domain.
In any aspect or embodiment described herein, the RBD is a receptor binding ligand (RBL), which is a peptide, polypeptide, or protein ligand binding specifically to a receptor. In some embodiments, the RBD binds specifically to a receptor on a target cell and/or target tissue.
In any aspect or embodiment described herein, the betaretroviral envelope protein is from a mouse mammary tumor virus (MMTV), a jaagsiekte sheep retrovirus (JSRV), or an intracisternal a-type particle elements with an envelope (IAPE).
In any aspect or embodiment described herein, the cargo is a transgene encoding a therapeutic protein, which comprises an immune checkpoint modulator, an antibody or portion thereof, a fusion protein, an anticoagulant, an immunosuppressive agent, an immunostimulatory agent, an enzyme, a growth factor, a hormone, an interferon, an interleukin, a thrombolytic, an anti-angiogenic, a chemotherapeutic, an antibiotic, an antifungal, an antiviral, and any combination thereof.
In any aspect or embodiment described herein, the engineered delivery vehicle comprises a reverse transcriptase, and the engineered delivery vehicle is a virus-like particle.
In another aspect, the present disclosure provides a method of delivering cargo to a target cell and/or target tissue by contacting the target cell and/or the target tissue with a polynucleotide encoding one or more betaretroviral elements or recombinant betaretroviral envelope proteins for forming an engineered delivery vehicle for packaging cargo within the delivery vehicle.
In any aspect or embodiment described herein, the method further comprises contacting the target cell and/or the target tissue with a reverse transcriptase. In some embodiments, the delivery vehicle is a virus-like particle.
In any aspect or embodiment described herein, the target cell is a mammalian cell and the tissue is a mammalian tissue. In some embodiments, the target cell is Crandell-Rees Feline Kidney (CRFK) cell, Vero cell, or human embryonic kidney 293T cell.
In any aspect or embodiment described herein, the polynucleotide, which is delivered to the target cells and/or the target tissue by the method described herein, encodes one or more betaretroviral elements or recombinant betaretroviral envelope proteins for forming a delivery vehicle for packaging cargo within the delivery vehicle. In some embodiments, the betaretroviral elements or recombinant betaretroviral envelope proteins comprise betaretroviral envelope protein, which comprises a surface subunit domain and a betaretrovirus transmembrane domain. In any aspect or embodiment described herein, the SU domain comprises a recombinant receptor binding domain. In any aspect or embodiment described herein, the SU domain comprises a scaffold domain and a recombinant receptor binding domain. In any aspect or embodiment described herein, the SU domain is covalently or non-covalently bound to the TM domain. In any aspect or embodiment described herein, the RBD is a receptor binding ligand (RBL), which is a peptide, polypeptide, or protein ligand binding specifically to a receptor. In some embodiments, the RBD binds specifically to a receptor on a target cell and/or target tissue.
In any aspect or embodiment described herein, the method further comprises contacting the target cell and/or the target tissue with a reverse transcriptase.
An additional aspect of the present disclosure is a method of treating, preventing, or ameliorating a genetic disease, disorder, or syndrome in a subject in need thereof, comprising the step of administering to the subject a therapeutically effective amount of a polynucleotide that encodes one or more betaretroviral elements or recombinant betaretroviral envelope proteins for forming a delivery vehicle for packaging cargo within the delivery vehicle, wherein the polynucleotide treats or ameliorates at least one symptom of the genetic disease, disorder, or syndrome.
In any aspect or embodiment described herein, the method further comprises determining whether the cells or tissues of the subject express the receptor of interest by any assay or method for detecting a DNA or RNA sequence. In some embodiments, the assay or method comprises genotyping, genome sequencing, next generation sequencing, and gene expression analysis of a sample obtained from the subject.
In any aspect or embodiment described herein, the method further comprises administering a therapeutically effective amount of reverse transcriptase to the subject in need thereof. The reverse transcriptase could be encoded by a polynucleotide. The reverse transcriptase could be encoded by the polynucleotide encoding one or more betaretroviral elements or recombinant betaretroviral envelope proteins for forming an engineered delivery vehicle for packaging cargo within the delivery vehicle.
In any aspect or embodiment described herein, the polynucleotide, which is delivered to the subject in need thereof by the method, encodes one or more betaretroviral elements or recombinant betaretroviral envelope proteins for forming a delivery vehicle for packaging cargo within the delivery vehicle. In some embodiments, the betaretroviral elements or recombinant betaretroviral envelope proteins comprise betaretroviral envelope protein, which comprises a surface subunit domain and a betaretrovirus transmembrane domain. In some embodiments, the SU domain comprises a recombinant receptor binding domain and a scaffold domain. In some embodiments, the SU domain is covalently or non-covalently bound to the TM domain. In some embodiments, the RBD is a receptor binding ligand, which is a peptide, polypeptide or protein ligand binding specifically to a receptor. In some embodiments, the RBD binds specifically to a receptor on a target cell and/or target tissue.
In any aspect or embodiment described herein, the method involves administering an effective amount of polynucleotides encoding betaretroviral elements, betaretroviral envelope protein, or a recombinant betaretrovirus, or a recombinant betaretroviral envelope protein to a subject. In any aspect or embodiment described herein, the subject is a human, non-human primate, dog, cat, livestock (e.g., cattle, sheep, pig, goat, horse, donkey, mule, buffalo, oxen, llama, camel, alpaca, cow, ox, reindeer, sheep, water buffalo, yak, et.), poultry (e.g., chicken, turkey, etc.).
In some embodiments, the subject is a human, non-human primate, dog, cat, livestock, or poultry. In some embodiments, the betaretroviral envelope protein is from a mouse mammary tumor virus, a jaagsiekte sheep retrovirus, or an intracisternal a-type particle elements with an envelope.
In some embodiments, the disorder is hypercholesterolemia, and the RBL is a cyclic 8-residue peptide (VH434), specifically binding to low-density lipoprotein (LDL) receptors.
The details of one or more embodiments of the present disclosure are set forth herein. Other features, objects, and advantages of the present disclosure will be apparent from the Detailed Description, Examples, Figures, and Claims.
The accompanying drawings, which are incorporated into and form a part of the specification, illustrate several embodiments of the present disclosure and, together with the description, serve to explain the principles of the present disclosure. The drawings are only for the purpose of illustrating specific embodiments of the present disclosure and are not to be construed as limiting the present disclosure. Further objects, features, and advantages of the disclosure will become apparent from the following detailed description taken in conjunction with the accompanying figures showing illustrative embodiments of the disclosure.
Current gene therapy platforms either result in non-specific delivery, are immunogenic, or require incorporation of multiple, different proteins into each viral particle for the cell-specific binding and entry functions. The latter results in loss of efficiency due to the need to get different proteins to incorporate into the same particle in appropriate ratios. The ideal viral vector system uses a single viral protein that combines both target cell receptor-specific binding and membrane fusion functions. It is also desirable to provide a platform for viral vector development based on a viral protein that can be engineered to use pre-specified receptors of interest, without compromising virion incorporation, fusogenicity, or infectivity.
Retroviruses continue to play a major role in the development of gene therapy vector platforms. This began with early work with murine leukemia virus (MLV, genus Gammaretrovirus) and includes significant investment in developing vectors based on HIV-1 (genus Lentivirus). To our knowledge, very little attention or effort has focused on betaretroviruses. As described previously, our evolution-guided and structure-guided analyses indicates that retroviruses in the genus Betaretrovirus have envelope proteins (ENVs) with sequence and structural properties that are well-suited for solving a major barrier in targeted gene-delivery and gene-therapy applications. Specifically: 1) betaretrovirus ENV proteins exist as natural sequences that exist in thousands of copies in vertebrate genomes in the form of endogenous retrovirus (ERV) loci, 2) comparative evolutionary analyses and AI-guided structure prediction suggests that these have highly divergent receptor-binding domains, but 3) that these also share a highly conserved “core” structure. This means that in nature, a conserved core structure has been coupled to an array of structurally diverse receptor-binding regions. Based on these observations, we predict that synthetic betaretrovirus ENVs can be rationally designed and customized to bind to user-specified cell surface receptors by retaining the conserved core and modifying the variable regions. These can then be coupled with existing gene-delivery platforms, including lentivirus vectors, to achieve cell-specific or tissue-specific payload delivery, ex vivo and/or in vivo.
As described herein, evolution-guided models of betaretrovirus ENV proteins demonstrate that these share a core structure that allows the betaretroviruses to evolve to use different, unrelated receptors. Described herein is a betaretrovirus-based platform that provides similar structural flexibility, allowing for engineering receptor-specific viral entry proteins (referred to herein, as “modular betaretrovirus attachment & cell entry”, or MBRACE).
While various embodiments of the present disclosure are described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous modifications and changes to, and variations and substitutions of, the embodiments described herein will be apparent to those skilled in the art without departing from the disclosure. It is understood that various alternatives to the embodiments described herein may be employed in practicing the disclosure. It is also understood that every embodiment of the disclosure may optionally be combined with any one or more of the other embodiments described herein that are consistent with that embodiment.
Where elements are presented in list format (e.g., in a Markush group), it is understood that each possible subgroup of the elements is also disclosed, and any one or more elements can be removed from the list or group.
It is also understood that, unless clearly indicated to the contrary, in any method described or claimed herein that includes more than one act or step, the order of the acts or steps of the method is not necessarily limited to the order in which the acts or steps of the method are recited, but the disclosure encompasses embodiments in which the order is so limited.
It is further understood that, in general, where an embodiment in the description or the claims is referred to as comprising one or more features, the disclosure also encompasses embodiments that consist of, or consist essentially of, such feature(s).
It is also understood that any embodiment of the disclosure, e.g., any embodiment found within the prior art, can be explicitly excluded from the claims, regardless of whether or not the specific exclusion is recited in the specification.
Headings are included herein for reference and to aid in locating certain sections. Headings are not intended to limit the scope of the embodiments and concepts described in the sections under those headings, and those embodiments and concepts may have applicability in other sections throughout the entire disclosure.
All patent literature and all non-patent literature cited herein are incorporated herein by reference in their entirety to the same extent as if each patent literature or non-patent literature were specifically and individually indicated to be incorporated herein by reference in its entirety.
Unless defined otherwise or clearly indicated otherwise by their use herein, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this application belongs.
As used in the specification and the appended claims, the indefinite articles “a” and “an” and the definite article “the” can include plural referents as well as singular referents unless specifically stated otherwise or the context clearly indicates otherwise.
The term “exemplary” as used herein means “serving as an example, instance or illustration”. Any embodiment or feature characterized herein as “exemplary” is not necessarily to be construed as preferred or advantageous over other embodiments or features.
The term “about” or “approximately” means an acceptable error for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined. In certain embodiments, the term “about” or “approximately” means within one standard deviation. In some embodiments, when no particular margin of error (e.g., a standard deviation to a mean value given in a chart or table of data) is recited, the term “about” or “approximately” means that range which would encompass the recited value and the range which would be included by rounding up or down to the recited value as well, taking into account significant figures. In certain embodiments, the term “about” or “approximately” means within 10% or 5% of the specified value. Whenever the term “about” or “approximately” precedes the first numerical value in a series of two or more numerical values or in a series of two or more ranges of numerical values, the term “about” or “approximately” applies to each one of the numerical values in that series of numerical values or in that series of ranges of numerical values.
Whenever the term “at least” or “greater than” precedes the first numerical value in a series of two or more numerical values, the term “at least” or “greater than” applies to each one of the numerical values in that series of numerical values.
Whenever the term “no more than” or “less than” precedes the first numerical value in a series of two or more numerical values, the term “no more than” or “less than” applies to each one of the numerical values in that series of numerical values.
The terms “disease”, “disorder”, or “condition” are used interchangeably herein, refer to any alternation in state of the body or of some of the organs, interrupting or disturbing the performance of the functions and/or causing symptoms such as discomfort, dysfunction, distress, or even death to the person afflicted or those in contact with a person. A disease or disorder can also be related to a distemper, ailing, ailment, malady, disorder, sickness, illness, complaint, or affectation.
The term “in need thereof” when used in the context of a therapeutic or prophylactic treatment, means having a disease, being diagnosed with a disease, or being in need of preventing a disease, e.g., for one at risk of developing the disease. Thus, a subject in need thereof can be a subject in need of treating or preventing a disease.
As used herein, the term “in combination” refers to the use of more than one prophylactic and/or therapeutic agent simultaneously or sequentially and in a manner such that their respective effects are additive or synergistic.
As used herein, the terms “treat,” “treatment,” “treating,” or “amelioration” refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a disease, disorder, or syndrome related to a bacteria. The term “treating” includes reducing or alleviating at least one adverse effect or symptom of a disease, disorder, or syndrome. Treatment is generally “effective” if one or more symptoms or clinical markers are reduced. Alternatively, or in addition, treatment is “effective” if the progression of a disease, disorder, or syndrome is reduced or halted. That is, “treatment” includes not just the improvement of symptoms or markers, but also a cessation of, or at least slowing of, progress or worsening of symptoms compared to what would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, remission (whether partial or total), and/or decreased mortality. For example, treatment is considered effective if the condition is stabilized. The term “treatment” of a disease also includes providing relief from the symptoms or side-effects of the disease (including palliative treatment).
As used herein, the term “gene expression” includes both gene transcription, whereby DNA (or RNA in the case of some RNA-containing viruses) corresponding to a gene is transcribed to generate an RNA molecule and RNA translation, whereby an RNA molecule is translated to generate a protein encoded by the gene. As used herein, the term “protein expression” is used to refer both to gene expression comprising transcription of DNA (or RNA) to form an RNA molecule and subsequent processing and translation of the RNA molecule to form protein and to gene expression comprising translation of mRNA to form protein.
As used herein, a “subject”, “patient”, “individual” and like terms are used interchangeably and refers to a host organism that can be an animal, a plant, or a single-cell microbe.
As used herein, the term “administering,” refers to the placement of an agent (e.g., a bacteriophage) as disclosed herein into a subject by a method or route that results in at least partial delivery of the agent at a desired site.
The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of” or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e., “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.”
As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from anyone or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a nonlimiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.
In the claims, as well as in the specification above, all transitional phrases, such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood/construed to be open-ended (i.e., to mean including but not limited to) unless otherwise noted. Only the transitional phrases “consisting of” and “consisting essentially of” shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.
The terms first, second, etc., as used herein are not meant to denote any particular ordering, but simply for convenience to denote a plurality of, for example, compositions. All methods described herein can be performed in a suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. Thus, in certain methods described herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited unless the context indicates otherwise.
The articles “a” and “an” as used herein and in the appended claims are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article unless the context clearly indicates otherwise. By way of example, “an element” means one element or more than one element. The use of the terms “a” and “an” and “the” and similar referents (especially in the context of the claims) are to be construed to cover both the one or more than one (e.g., “at least one”, “plurality”, or “one or more”) of the grammatical object of the article, unless otherwise indicated herein or clearly contradicted by context. By way of example, “an element” means one element or more than one element.
Recitation of ranges of values are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. The endpoints of all ranges are included within the range and independently combinable.
The use of any and all examples, or exemplary language (e.g., “such as”), is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention as used herein.
The terms “decrease”, “reduce”, “reduction”, “lower” or “lowering,” or “inhibit” are all used herein generally to mean a decrease by a statistically significant amount. For example, “decrease”, “reduce”, “reduction”, or “inhibit” means a decrease by at least 10% as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%), or at least about 80%, or at least about 90% or up to and including a 100% decrease (e.g., absent level or non-detectable level as compared to a reference level), or any decrease between 10-100%) as compared to a reference level. In the context of a marker or symptom, by these terms is meant a statistically significant decrease in such level. The decrease can be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, and is preferably down to a level accepted as within the range of normal for an individual without a given disease.
The term “polynucleotide”, “nucleic acid”, or “nucleic acid sequence” refers to a polymer composed of nucleotide units. Polynucleotides can contain naturally occurring nucleic acids (e.g., deoxyribonucleic acid [“DNA”] and ribonucleic acid [“RNA”]), or/and nucleic acid analogs. For example, the term “nucleic acid” or “polynucleotide” includes single-, double- or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine or pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide base. Polynucleotides containing one or more nucleic acid or nucleotide analogs are sometimes called “aptamers”. Nucleic acid or nucleotide analogs include without limitation those which have a non-naturally occurring base/nucleobase, have a sugar or non-sugar moiety other than 2′-deoxyribose or ribose, or engage in linkages with other nucleotides other than the naturally occurring phosphodiester bond, or a combination thereof. Non-limiting examples of nucleic acid or nucleotide analogs include xeno (biotic) nucleic acids (XNAs) having a backbone other than the naturally occurring sugar-phosphate backbone present in DNA or RNA (e.g., bridged nucleic acids [BNAs], cyclohexene nucleic acids [CeNAs], 2′-deoxy-2′-fluoroarabino nucleic acids [FANAs], glycol nucleic acids [GNAs], 1,5-anhydrohexitol nucleic acids [HNAs], locked nucleic acids [LNAs], 2′-O-methyl ribonucleotides, morpholino nucleic acids [MNAs], peptide nucleic acids [PNAs], and threose nucleic acids [TNAs]), phosphorothioates, phosphorodithioates, phosphorotriesters, phosphoramidates, boranophosphates, methylphosphonates, chiral-methyl phosphonates, and the like. DNA and RNA polynucleotides can be synthesized using a DNA or RNA polymerase or an automated DNA or RNA synthesizer. Polynucleotides containing nucleic acid analogs can be synthesized using, e.g., an engineered DNA or RNA polymerase that recognizes the nucleic acid analogs, a phosphoramidite strategy, or an automated peptide synthesizer in the case of PNAs. The term “nucleic acid molecule” typically refers to a larger polynucleotide. The term “oligonucleotide” typically refers to a shorter polynucleotide. In certain embodiments, an oligonucleotide contains no more than about 50 nucleotides. In some embodiments, when a polynucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes the corresponding, or the complementary, RNA sequence (i.e., A, U, G, C) in which “U” replaces “T”.
Conventional notation is used herein to describe polynucleotide sequences: the left-hand end of a single-stranded polynucleotide sequence is the 5′ end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5′ direction. The direction of 5′ to 3′ addition of nucleotides to a newly forming (daughter) DNA strand, or to a nascent RNA transcript, is referred to as the direction of replication or transcription, respectively. The DNA strand having a complementary, antiparallel sequence as a daughter DNA strand or a primary RNA transcript (the “coding strand”) is called the “template strand”. “Upstream” is toward the 5′ end of an RNA molecule, and “downstream” is toward its 3′ end. When considering double-stranded DNA, “upstream” is toward the 5′ end of the coding strand for the gene in question and “downstream” is toward the 3′ end of the coding strand, so the 3′ end of the template strand is upstream of the gene in question and the 5′ end of the template strand is downstream of the gene.
The term “primer” refers to a polynucleotide that is capable of specifically hybridizing to a designated polynucleotide template and providing a point of initiation for synthesis of a complementary polynucleotide. Such synthesis occurs when the polynucleotide primer is placed under conditions in which synthesis is induced, i.e., in the presence of nucleotides, a complementary polynucleotide template, and an agent for polymerization such as a DNA or RNA polymerase. A primer is typically single-stranded but may be double-stranded. Primers are typically deoxyribonucleic acids, but a wide variety of synthetic and naturally occurring primers are useful for many applications. A primer is complementary to the template to which it is designed to hybridize and serves as a site for the initiation of synthesis but need not reflect the exact sequence of the template. In such a case, specific hybridization of the primer to the template depends on the stringency of the hybridization conditions. A primer can be labeled with an agent that promotes isolation (e.g., biotin), separation (e.g., biotin) or detection (e.g., a fluorescent, chromogenic or radioactive moiety) of the coding strand in single-stranded form or the coding strand hybridized to the template strand.
The term “gene therapy” refers to a therapeutic technique that involves the modification, replacement, or introduction of genetic material into a patient's cells to treat or prevent disease. Gene therapies typically utilize vectors, such as viral or non-viral delivery systems, to transport functional copies of genes, RNA molecules, or gene-editing components to target cells. Once delivered, the genetic material may integrate into the patient's genome, produce therapeutic proteins, or repair existing DNA errors.
The term “cancer disease” or “cancer” includes a disease characterized by aberrantly regulated cellular growth, proliferation, differentiation, adhesion, and/or migration. “Cancer cell” means an abnormal cell that grows by rapid, uncontrolled cellular proliferation and continues to grow after the stimuli that initiated the new growth cease.
“Metastasis” refers to the process by which cancer cells spread from their original site to other parts of the body. This complex phenomenon involves several stages, including the detachment of malignant cells from the primary tumor, invasion into the extracellular matrix, penetration through the endothelial basement membranes to access blood vessels and body cavities, and eventual transport through the bloodstream to infiltrate target organs. The establishment of new tumors at these sites relies on angiogenesis, the formation of new blood vessels. Notably, metastasis can occur even after the primary tumor has been removed, as residual cancer cells may persist and acquire metastatic capabilities. In the context of this invention, “metastasis” specifically refers to “distant metastasis,” which indicates the spread of cancer cells far from the primary tumor and regional lymph nodes.
The term “contacting” refers to the act of making contact or bringing into immediate or close proximity, whether at the cellular or molecular level. This can involve initiating a physiological reaction, chemical reaction, or physical change, and may occur in various environments, such as in a solution, reaction mixture, or in vitro and in vivo conditions.
Reference throughout the specification to “some embodiments”, “an embodiment”, and so forth, means that a particular element described in connection with the embodiment is included in at least one embodiment described herein, and may or may not be present in other embodiments. In addition, it is to be understood that the described elements may be combined in any suitable manner in the various embodiments. A “combination thereof” is open and includes any combination comprising at least one of the listed components or properties optionally together with a like or equivalent component or property not listed.
“Pharmaceutical compositions” are compositions comprising at least one active agent, and at least one other substance, such as a carrier, excipient, or diluent. Pharmaceutical compositions meet the U.S. FDA's GMP (good manufacturing practice) standards for human or non-human drugs.
“Pharmaceutically acceptable salts” include derivatives of the disclosed compounds in which the parent compound is modified by making inorganic and organic, non-toxic, acid or base addition salts thereof. The salts of the present compounds can be synthesized from a parent compound that contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting free acid forms of these compounds with a stoichiometric amount of the appropriate base (such as Na, Ca, Mg, or K hydroxide, carbonate, bicarbonate, or the like), or by reacting free base forms of these compounds with a stoichiometric amount of the appropriate acid. Such reactions are typically carried out in water or in an organic solvent, or in a mixture of the two. Generally, non-aqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred, where practicable. Salts of the present compounds further include solvates of the compounds and of the compound salts.
A “therapeutically effective amount” means an amount effective, when administered to a patient, to provide a therapeutic benefit such as an amelioration of at least a symptom of the disorder, decrease the frequency or severity of symptoms, or effect a change in a clinical marker for a disease or disorder, slowing the progression of a disease or disorder, halting the progression of a disease or disorder, or reversing the course of a disorder.
The term “virus-like particle” (VLP) refers to a structure that in at least one attribute resembles a virus, but which has not been demonstrated to be infectious. A VLP may be a nonreplicating, noninfectious viral shell that contains a viral capsid but lacks all or part of the viral genome, in particular, the replicative components of the viral genome. VLPs are generally composed of one or more viral proteins, such as, but not limited to those proteins referred to as capsid, coat, shell, surface, and structural proteins.
A “reverse transcriptase” is an enzyme used to generate complementary DNA (cDNA) from an RNA template, a process termed reverse transcription. A reverse transcriptase is used by betaretrovirus to replicate their genomes.
The terms “cargo”, “cargo molecular”, “packaged nucleic acid viral vector cargo” and “payload” are used interchangeably and refer to, e.g., a molecule, agent or compound that is carried or shuttled by another agent to a site or location, and confers some beneficial effect upon administration to a subject. The beneficial effect includes enablement of diagnostic determinations; amelioration of a disease, symptom, disorder, or pathological condition; reducing or preventing the onset of a disease, symptom, disorder or condition; and generally counteracting a disease, symptom, disorder or pathological condition. The cargo may be a transgene encoding a therapeutic protein. The therapeutic protein may be an immune checkpoint modulator, an antibody or portion thereof, a fusion protein, an anticoagulant, an immunosuppressive agent, an immunostimulatory agent, an enzyme, a growth factor, a hormone, an interferon, an interleukin, a thrombolytic, an anti-angiogenic, a chemotherapeutic, an antibiotic, an antifungal, an antiviral, and any combination thereof.
An “engineered delivery vehicle” and a “packaged nucleic acid vector delivery vehicle” are used interchangeably and comprises one or more components encoded in polynucleotides in the engineered delivery system described herein. As described elsewhere herein, such components include, but are not necessarily limited to, polynucleotides encoding one or more betaretroviral elements, betaretroviral envelope proteins, or recombinant betaretroviruses, or recombinant betaretroviral envelope proteins for forming a delivery vehicle, or an engineered delivery vehicle for packaging cargo within the delivery vehicle and deliver the cargo into to target cells and tissues.
A genetic “element” within a vector refers to specific nucleic acid (e.g., DNA, RNA), nucleotide, polynucleotide, open reading frame (ORF), or framework region (FR) that play distinct roles in gene delivery, expression, or regulation of gene(s). Representative genetic “element” includes promoters, enhancers, polyadenylation (poly-A) signals, splicing sites, replication origins and selectable markers.
A “transcription unit” refers to specific nucleic acid (e.g., DNA, RNA), nucleotide, polynucleotide, open reading frame (ORF), or framework region (FR) that play distinct roles in gene delivery, expression, or regulation of gene(s). Representative genetic “transcription unit” includes promoters, enhancers, polyadenylation (poly-A) signals, splicing sites, replication origins and selectable markers.
A “betaretroviral element” refers to a specific sequence (e.g., polynucleotides, polypeptide and polynucleotides encoding them) derived from betaretroviruses that enables the vector to deliver and integrate genetic material into the host genome. The sequence comprises nucleic acid, nucleotide, polynucleotide, open reading frame (ORF), or framework region (FR) derived from betaretroviruses. The sequence also comprises amino acid or polypeptide encoded by nucleic acid, nucleotide, polynucleotide, open reading frame, or framework region derived from betaretroviruses. Representative betaretroviral elements or recombinant betaretroviral envelope proteins include but are not limited to betaretroviral envelope (ENV) protein, surface subunit (SU) domain of envelope protein, scaffold domain of envelope protein, transmembrane (TM) domain of envelope protein, viral receptor-specificity determinants of SU domain, type-I fusion complex of TM domain, and receptor binding domains (RBDs), signal peptide, glycosylation sites, furin cleavage site, fusion peptide, heptad repeats, membrane spanning region, cytoplasmic domain.
The engineered polynucleotide may further include regulatory elements to control expression of the engineered delivery vehicle. The term “regulatory element” refers to promoters, enhancers, Kozak sequences, Internal Ribosome Entry Sites (IRES), other expression control elements and cellular localization signals. Such regulatory elements are described, for example, in Goeddel, Gene Expression Technology: Methods In Enzymology 185, Academic Press, San Diego, Calif. (1990).
Definitions of common terms in cell biology and molecular biology can be found in “The Merck Manual of Diagnosis and Therapy”, 19th Edition, published by Merck Research Laboratories, 2006 (ISBN 0-911910-19-0); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); Immunology by Werner Luttmann, published by Elsevier, 2006. Definitions of common terms in molecular biology can also be found in Benjamin Lewin, Genes X, published by Jones & Bartlett Publishing, 2009 (ISBN-10:0763766321); Kendrew et al. (eds.), Molecular Biology and Biotechnology: A Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8) and Current Protocols in Protein Sciences 2009, Wiley Intersciences, Coligan et al., eds.
Unless otherwise stated, the present invention was performed using standard procedures, as described, for example in Sambrook et al., Molecular Cloning: A Laboratory Manual (3 ed.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2001); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (1995); Current Protocols in Protein Science (CPPS) (John E. Coligan, et. al., ed., John Wiley and Sons, Inc.), Current Protocols in Cell Biology (CPCB) (Juan S. Bonifacino et. al. ed., John Wiley and Sons, Inc.), and Culture of Animal Cells: A Manual of Basic Technique by R. Ian Freshney, Publisher: Wiley-Liss; 5th edition (2005), Animal Cell Culture Methods (Methods in Cell Biology, Vol. 57, Jennie P. Mather and David Barnes editors, Academic Press, Ist edition, 1998) which are all incorporated by reference herein in their entireties.
Viral vectors are the focus of intensive research and development in both the academic and biotech sectors, primarily because of the hope that gene therapy can be used to correct a wide spectrum of inherited and somatic genetic disorders. Targetable viral vectors can also be useful tools to research in organismal model systems, where precise delivery of genetic modifications is desirable in order to study or understand developmental processes. Although viral vectors are widely used for such applications, these suffer from a lack of precise targeting, and often have to be re-engineered for each novel application.
Many viruses use cell surface receptors to bind to host cells and initiate fusion between the virion membrane and the host cell membrane, resulting in delivery of the virion contents into the host cell. This natural process is the cornerstone of many approaches to gene therapy. Current viral vectors used for gene delivery (e.g., for gene therapy) rely on the natural receptor specificities of well-studied viruses; prominent examples include HIV-1 and MLV (retroviruses), VSV (a rhadbdovirus), and AAV (a parvovirus). Depending on the tissue distribution of the virus' receptor, infection can result in delivery of the payload into many irrelevant cell types and tissues, in addition to the cell type or tissue of interest. Conversely, a cell or tissue target of choice may not express a receptor conveniently used by any known viruses for entry, precluding use of a viral delivery system for treatment. To solve these issues, significant modifications of existing viral vector platforms must be used; however, these often reduce the efficiency of vector production and result in diminished delivery capacity (infectivity) of the engineered viral particles.
The ideal viral vector is one that can be rationally designed and modified to use pre-specified receptors, based on the target cell or tissue type and the nature of the disease or condition being treated. To construct a model of betaretrovirus entry proteins and understand how they evolved to use different receptors, betaretrovirus Env proteins and related endogenous retrovirus (ERV) sequences found abundantly in mammalian genomes that encode betaretrovirus-related proteins were used. The resulting model suggests that all betaretrovirus entry proteins evolved from a common core structure. This structure provided evolutionary flexibility, which allowed the betaretroviruses to spread to many different mammalian hosts and to adapt to different cellular receptors. Taking a cue from nature, we proposed that this evolutionary flexibility also means that betaretrovirus entry proteins can be customized in the laboratory to recognize pre-specified receptors. We refer to this as modular betaretrovirus attachment & cell entry (MBRACE).
An aspect of the present disclosure relates to betaretroviruses (genus Betaretrovirus), and betaretrovirus ENV proteins for use in viral vector platforms, an engineered delivery vehicle and gene therapy, their associated methods of use and manufacture. In particular, the present disclosure provides compositions and methods to tune betavirus ENV proteins such that they target a particular cell surface receptor; i.e., are cell and/or tissue-type specific, allowing for better site-specific transgene delivery and expression.
The advantage of MBRACE is the ability to customize a single viral protein for precise delivery across multiple applications. This will increase the efficiency of production, and reduce some of the burden of repeated, iterative evaluation processes incurred for each new application.
In an aspect, the compositions and methods described herein are used for rational design of viral entry proteins to use pre-specified receptors for cell entry, for example, receptor/biomarker-specific targeting of viral vectors for gene therapy. In certain embodiments, the compositions and methods are useful for ex vivo, in vitro, as well as in vivo gene delivery.
Thus, in an aspect, the description provides a recombinant betaretrovirus vector or betaretrovirus ENV protein as described herein.
In any aspect or embodiment described herein, the recombinant betaretrovirus vector as described or claimed herein comprising expressing a recombinant betaretrovirus ENV protein and a transgene, and the transgene encodes a therapeutic protein.
In any aspect or embodiment described herein, the disclosure provides a nucleic acid comprising a polynucleotide that encodes a transcription unit for a recombinant betaretroviral envelope protein comprising a receptor binding domain (RBD), a betaretrovirus surface subunit domain, and a betaretrovirus transmembrane (TM) domain. The RBD is a receptor binding ligand (RBL), wherein the RBL is a peptide, polypeptide or protein ligand binding specifically to a receptor on a cell, a tissue and/or a mammal.
In any aspect or embodiment described herein, the disclosure provides an engineered delivery vehicle that comprises a polynucleotide encoding one or more recombinant betaretroviral envelope proteins comprising a receptor binding domain, a betaretrovirus surface subunit (SU) domain, and a betaretrovirus transmembrane domain for forming a packaged nucleic acid vector delivery vehicle.
In any aspect or embodiment described herein, the disclosure provides an engineered delivery vehicle that comprises a virus-like particle having a recombinant betaretroviral envelope (rENV) protein on its surface and including a packaged nucleic acid viral vector cargo. The packaged nucleic acid viral vector cargo comprises a polynucleotide encoding the rENV, and a transcription unit encoding a therapeutic protein.
In any aspect or embodiment described herein, the recombinant betaretrovirus ENV protein as described or claimed herein, comprises a surface subunit (SU) domain including a recombinant receptor binding domain (RBD) and a scaffold domain, and a betaretrovirus transmembrane (TM) domain, and the SU domain is covalently or non-covalently bound to the TM domain.
In any aspect or embodiment described herein, the recombinant betaretrovirus ENV protein as described or claimed herein, comprises an RBD that binds specifically to a surface receptor on a target cell and/or target tissue. In certain embodiments, the RBD is a portion of a protein known to interact with an extracellular receptor domain on a target cell or target tissue. For example, in certain embodiments, the RBD is a domain of a viral protein that interacts with a receptor on the surface of a target cell or target tissue, e.g., RBD can be the receptor biding domain of HIV, which interacts with CD4 and co-receptors on T cells.
In any aspect or embodiment described herein, the recombinant betaretrovirus ENV protein as described or claimed herein, comprises an RBD that is a receptor binding ligand (RBL), e.g., a peptide, polypeptide or protein ligand, that binds specifically to a receptor of interest. For example, the RBD can be the extracellular domain or portion thereof of a cell surface protein or peripheral membrane protein that interacts with or specifically binds to a receptor on the target cell or target tissue of interest. In other embodiments, the RBD is a receptor binding ligand (RBL), e.g., a peptide, polypeptide or protein that is a ligand of a receptor on the target cell or target tissue of interest. For example, the RBL can be a peptide, polypeptide, or protein, including an antibody or antigen binding portion or fragment thereof of an antibody or immunoglobulin, a chemokine, cytokine or the like. As would be understood by the skilled artisan, any number of protein-based ligand molecules could be utilized in order to target the described viral vector to the intended cellular or tissue target, which are contemplated and encompassed by the present disclosure.
In an additional aspect, the disclosure provides betaretrovirus-based vaccines. For example, vectored delivery of biomolecules to a specific cell or tissue type, e.g., nucleic acid-based vaccine.
As described herein, MBRACE is a novel method for customized engineering of viral vectors to use a pre-specified receptor of interest. Briefly, a viral vector is modified for delivery to a desired cell- or tissue-type by choosing an appropriate receptor as a biomarker, and then engineering a betaretrovirus entry protein to use that particular biomarker as an entry receptor instead of its native receptor. This provides a means for precise targeting of the appropriate cell or tissue for delivery of the contents of the viral particle. The starting structure for development can be an existing betaretrovirus entry protein, or an ERV-encoded betaretrovirus entry protein. The diversity of betaretroviral proteins available can also be exploited by pre-selecting those with any desirable properties relevant to the particular application as a starting point for customization.
Current gene therapy platforms either result in non-specific delivery, or require incorporation of multiple, different proteins into each viral particle for the cell-specific binding and entry functions. The latter results in loss of efficiency due to the need to get different proteins to incorporate into the same particle in appropriate ratios. The ideal system is a single viral protein that combines both the target cell receptor-specific binding and membrane fusion functions into a single protein. MBRACE is an improvement over current approaches by creating a single, customizable, viral protein for target cell-specific binding and entry.
As described herein, developed herein is a surprising and unexpected framework region (FR) able to fold into a common “scaffold” that can link divergent receptor-binding domains with the highly conserved fusion apparatus provided by the TM subunit. The adaptability of the core betaretrovirus SU structure can be exploited by coupling a betaretrovirus “scaffold” with a ligand or ligand mimic for a heterologous receptor (
While a conserved scaffold might have evolved to accommodate divergent variable region (VAR) domains (and receptor preferences), it is reasonable to assume that the scaffolds and VAR domains of the same viral lineage or structural class have acquired adaptations to work together optimally. This suggests that recombining heterologous ligands and scaffolds may result in suboptimal or nonfunctional ENVs. To address this issue, the large number of potential scaffolds is leveraged, in silico modeling is used to identify optimal scaffold-ligand combinations, and cell culture systems are used to iteratively test and refine combinations for optimal performance.
Sequences for nucleic acids containing betaretroviral element or recombinant betaretroviral envelope protein are shown in SEQ ID NO: 1-21, which can be utilized in the methods and compositions described herein.
Bos taurus ERV-K113 Env polyprotein-like
Sequences for amino acids containing betaretroviral element or recombinant betaretroviral envelope protein, which can be utilized in the compositions and methods described herein, are shown in SEQ ID NO: 22-42.
MBRACE is based on re-targeting an MBRACE scaffold ENV to bind to user-specific receptor(s). Customization begins by choosing a suitable MBRACE ENV scaffold and modifying its aminoacid sequence to contain an inserted, in-frame peptide ligand that binds the desired cell-surface receptor. In-frame ligand sequences can include (but are not limited to) natural ligands of the receptor, including cellular ligands or heterologous receptor binding domains from other viruses; peptides derived by in vitro evolution, screening or selection for binding to the receptor (e.g. by phage-display); and specialized binding proteins, such as DARPins or ScFVs, that are specific for the receptor. Once a candidate ligand is identified, an MBRACE ENV scaffold sequence is modified to include the ligand sequence inserted in-frame at predetermined positions corresponding to surface-exposed, variable loops of the receptor-binding subunit of the ENV scaffold (the surface, or SU, subunit). Structural prediction (e.g. AlphaFold) is used to pre-screen the chimeric sequences for proper folding of the ENV scaffold and exposure of the inserted ligand on its apical surface. At this step, ENVs drawn from multiple MBRACE structural classes can also be tested in silico, to first identify candidate scaffolds that are structurally most compatible with the inserted ligand. Once one or more candidates are identified, the contiguous scaffold+ligand amino-acid sequence is synthesized and cloned into a standard eukaryotic expression vector. The candidate(s) are then compared to the starting scaffold ENV(s) by transfection, SDS-PAGE and immunoblot, to ensure that expression levels and post-translational modifications are comparable to the parental ENV. Candidates are functionally profiled for fusogenicity (by cell-cell fusion assay) and infectivity (using single-cycle, pseudotyped virions). The former is also used to determine whether fusion is pH-dependent, while the latter also tests for efficient transduction of a reporter gene, such as GFP or luciferase. Receptor-positive and receptor-null cells are used to confirm receptor-specific fusion and entry.
Notes: * in-frame ligand can be any peptide/polypeptide sequence that binds a receptor of interest, including (but not limited to) natural or predicted peptide ligands, peptides derived by selection or screening (e.g. by phage-display), heterologous receptor binding domains, DARPins, and receptor-specific antibody-derived sequences, such as single-chain Fv (scFv).
In one aspect, embodiments disclosed herein relate to polynucleotides encoding betaretroviral elements, betaretroviral envelope proteins, or recombinant betaretroviruses, or recombinant betaretroviral envelope proteins for forming an engineered delivery vehicle. In one aspect, embodiments disclosed herein relate to betaretroviral vectors comprising betaretroviral elements, betaretroviral envelope proteins, or recombinant betaretroviruses, or recombinant betaretroviral envelope proteins for forming an engineered delivery vehicle. In another aspect, embodiments disclosed herein are directed to use of such polynucleotides in methods of loading and/or packaging desired cargo molecules. In another aspect, embodiment disclosed herein are directed to such cargo carrying delivery vehicles and methods of using said engineered delivery vehicle to deliver cargo molecules to target cells and tissues.
In some embodiments, the engineered delivery vehicle disclosed herein comprises polynucleotides that encode one or more betaretroviral elements, betaretroviral ENV proteins, or recombinant betaretroviruses for forming an engineered delivery vehicle for packaging cargo or cargo molecules within the delivery vehicle. The polynucleotide may further include regulatory elements such as promoters, enhancers, repressors, inducers, internal ribosome entry site (IRES) to control expression of the vehicle forming system. The polynucleotides are designed for delivery to a cell, a cellular system, and/or tissue to allow expression of the delivery system components and formation of said delivery vehicles including packaging of desired cargo molecules into said delivery vehicles.
In some embodiments, the one or more betaretroviral elements or recombinant betaretroviral envelope proteins for forming a delivery vehicle comprise a betaretroviral envelope protein. The envelope protein further includes one or more receptor binding domain, which is capable of specifically binding to a target cell.
The engineered delivery vehicle may further comprise cargo domain elements, such as transgene encoding a therapeutic protein, and the therapeutic protein comprises an immune checkpoint modulator, an antibody or portion thereof, a fusion protein, an anticoagulant, an immunosuppressive agent, an immunostimulatory agent, an enzyme, a growth factor, a hormone, an interferon, an interleukin, a thrombolytic, an anti-angiogenic, a chemotherapeutic, an antibiotic, an antifungal, an antiviral, and any combination thereof.
In some embodiments, the engineered delivery vehicle may include regulatory element(s) that control expression of the vehicle-forming system.
In some embodiments, the engineered delivery vehicle within the scope of the present invention may be provided in any form, including but not limited to solid, semi-solid, emulsion, or colloidal particles. As such, any of the delivery systems described herein, including but not limited to, e.g., lipid-based systems, liposomes, micelles, microvesicles, exosomes, or gene gun may be provided as particle delivery systems within the scope of the present invention.
In some embodiments, recombinant vectors can comprise polynucleotides of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant vectors include one or more regulatory elements, which may be selected on the basis of the host cells to be used for expression, that is operatively linked to the nucleic acid sequence to be expressed.
In some embodiments, the disclosed engineered delivery vehicle includes polynucleotides encoding one or more betaretroviral elements, betaretroviral envelope proteins, or recombinant betaretroviruses, or recombinant betaretroviral envelope proteins for forming a delivery vehicle for packaging cargo within the delivery vehicle. In some embodiments, the cargo is a transgene encoding a therapeutic protein. Cargoes that can be delivered in accordance with the systems and methods described herein include, but are not necessarily limited to, biologically active agents, therapeutic agents, imaging agents, and monitoring agents. Representative cargo or cargo molecules may include, but are not limited to, an immune checkpoint modulator, an antibody or portion thereof, a fusion protein, an anticoagulant, an immunosuppressive agent, an immunostimulatory agent, an enzyme, a growth factor, a hormone, an interferon, an interleukin, a thrombolytic, an anti-angiogenic, a chemotherapeutic, an antibiotic, an antifungal, an antiviral, and any combination thereof. The delivery particles described herein may be used and further comprise a number of different cargo molecules for delivery.
In some embodiments, the cargo or cargo molecules may comprise a therapeutic agent. The terms “therapeutic agent”, “therapeutic capable agent” or “treatment agent” are used interchangeably and refer to a molecule or compound that confers some beneficial effect upon administration to a subject. The beneficial effect includes enablement of diagnostic determinations; amelioration of a disease, symptom, disorder, or pathological condition; reducing or preventing the onset of a disease, symptom, disorder or condition; and generally counteracting a disease, symptom, disorder or pathological condition.
In some embodiments, the cargo is a genetic modulating agent.
In some embodiments, the engineered delivery vehicle further comprises a reverse transcriptase.
In some embodiments, the engineered delivery vehicle further comprises an RNA; a DNA; a single-stranded RNA; or a single-stranded DNA.
In some embodiments, the polynucleotide having nucleotide sequences is at least 80% identical to any one of SEQ ID NO: 1-21.
In some embodiments, the polynucleotide having nucleotide sequences comprises a sequence of any one of SEQ ID NO: 1-21.
In some embodiments, the polynucleotide encoding the rENV protein comprises a nucleotide sequence with at least 80% sequence identity to any one of SEQ ID NO: 1-21 or a portion thereof.
In some embodiments, the polynucleotide encoding the rENV protein comprises a nucleotide sequence encoding an SU domain, or a TM domain or both having at least 80% sequence identity to an SU domain or TM domain of SEQ ID NO: 1-21.
In some embodiments, the polynucleotide encoding the rENV has a nucleotide sequence comprising a sequence of any one of SEQ ID NO: 1-21 or a portion thereof.
In some embodiments, the polynucleotide encoding the one or more rENV proteins comprises a nucleotide sequence encoding an SU domain, or a TM domain or both having at least 80% sequence identity to an SU domain or TM domain of SEQ ID NO: 1-21.
In some embodiments, the rENV protein comprises an SU domain, TM domain, variable domain or scaffold domain of a protein having at least 80% sequence identity to any one of SEQ ID NO: 22-42 or a portion thereof.
In some embodiments, the rENV protein comprise an SU domain, TM domain, variable domain or scaffold domain of a protein having a sequence of any one of SEQ ID NO: 22-42 or a portion thereof.
In some embodiments, the engineered delivery vehicle comprises a polynucleotide encoding the one or more rENV proteins and comprises a nucleotide sequence with at least 80% sequence identity to any one of SEQ ID NO: 1-21 or a portion thereof.
In some embodiments, the engineered delivery vehicle comprises a polynucleotide encoding the one or more rENV proteins and comprises a nucleotide sequence encoding an SU domain, or a TM domain or both having at least 80% sequence identity to an SU domain or TM domain of SEQ ID NO: 1-21.
In some embodiments, the engineered delivery vehicle comprises a polynucleotide encoding the one or more rENV proteins and comprises a nucleotide sequence comprising a sequence of any one of SEQ ID NO: 1-21 or a portion thereof.
In some embodiments, the engineered delivery vehicle further comprises an RNA; a DNA; a single-stranded RNA; a single-stranded DNA.
In some embodiments, the engineered delivery vehicle comprises a rENV protein comprising an SU domain, TM domain, variable domain or scaffold domain of a protein having at least 80% sequence identity to any one of SEQ ID NO: 22-42 or a portion thereof.
In some embodiments, the engineered delivery vehicle comprises a rENV protein comprising an SU domain, TM domain, variable domain or scaffold domain of a protein having a sequence of any one of SEQ ID NO: 22-42 or a portion thereof.
The method of claim 33, wherein rENV protein is from a mouse mammary tumor virus (MMTV), a jaagsiekte sheep retrovirus (JSRV), or an intracisternal a-type particle elements with an envelope (IAPE).
In some embodiments, the rENV protein is encoded by the polynucleotide having nucleotide sequences at least 80% identical to any one of SEQ ID NO: 1-21.
In some embodiments, the rENV protein is encoded by the polynucleotide having nucleotide sequences comprises a sequence of any one of SEQ ID NO: 1-21.
In some embodiments, the rENV protein comprises an SU domain, TM domain, variable domain or scaffold domain of a protein having at least 80% sequence identity to any one of SEQ ID NO: 22-42 or a portion thereof.
In some embodiments, the rENV protein comprises an SU domain, TM domain, variable domain or scaffold domain of a protein having a sequence of any one of SEQ ID NO: 22-42 or a portion thereof.
Provided are methods of delivering cargo to a target cell and/or target tissue by contacting the target cell and/or the target tissue with an engineered delivery vehicle that comprises polynucleotides encoding betaretroviral elements, betaretroviral envelope protein, a recombinant betaretrovirus, or recombinant betaretroviral envelope protein for packaging cargo within the engineered delivery vehicle. Therefore, the disclosed methods can be utilized as research tools in molecular biology or a related field.
In some embodiments, a method of contacting a cell may comprise, for example, contacting a cell in a culture with a polynucleotide encoding a surface subunit domain of envelope protein, a polynucleotide encoding a recombinant receptor binding domain of envelope protein, a scaffold domain of envelope protein, and/or betaretrovirus transmembrane domain of envelope. In some embodiments, the RBD is a receptor binding ligand, which binds specifically to a receptor on a target cell and/or target tissue.
In some embodiments, contacting a cell comprises adding a polynucleotide encoding at least one betaretrovirus envelope protein surface subunit sequence, at least one betaretrovirus envelope protein receptor binding domain sequence, at least one betaretrovirus envelope protein scaffold domain sequence, at least one betaretrovirus envelope protein transmembrane, a reverse transcriptase, or a composition (e.g., a pharmaceutical composition) to a supernatant of a cell culture (e.g., a cell culture on a tissue culture plate or dish).
In some embodiments, a cell described herein is a cell isolated or derived from a subject. In some embodiments, a cell is a mammalian cell (e.g., a cell isolated or derived from a mammal). In some embodiments, a cell is a human cell. In some embodiments, a cell is isolated or derived from a particular tissue of a subject. In some embodiments, a cell is in vitro. In some embodiments, a cell is ex vivo. In some embodiments, a cell is in vivo, e.g., a cell is within a subject (e.g., within a tissue or organ of a subject). In some embodiments, a cell is a primary cell. In some embodiments, a cell is from a cell line (e.g., an immortalized cell line). In some embodiments, a cell is a cancer cell or an immortalized cell.
An additional aspect of the present disclosure is a method of treating, preventing, or ameliorating a genetic disease, disorder, or syndrome in a subject. The method comprises administering an effective amount of polynucleotide encoding at least one betaretroviral elements, betaretroviral envelope proteins, or recombinant betaretroviruses, or recombinant betaretroviral envelope proteins for forming an engineered delivery vehicle for packaging cargo within the delivery vehicle to the subject (e.g., patient) in need thereof. The method effectuates the amelioration of at least one symptom of the disease, disorder, or syndrome in the subject. In some embodiments, envelope protein includes a surface subunit domain including a recombinant receptor binding domain and a scaffold domain. The envelope protein also includes a betaretrovirus transmembrane domain. The subunit domain is covalently or non-covalently bound to the transmembrane domain. In some embodiments, the RBD is a receptor binding ligand, which binds specifically to a receptor on a target cell and/or target tissue.
In some embodiments described herein, the method involves administering an effective amount of polynucleotides encoding betaretroviral elements, betaretroviral envelope protein, or a recombinant betaretrovirus, or a recombinant betaretroviral envelope protein to a subject.
In some embodiments described herein, the subject is a human, non-human primate, dog, cat, livestock (e.g., cattle, sheep, pig, goat, horse, donkey, mule, buffalo, oxen, llama, camel, alpaca, cow, ox, reindeer, sheep, water buffalo, yak, et.), poultry (e.g., chicken, turkey, etc.).
In some embodiments described herein, the method involves determining whether cells or tissues of the subject express the receptor of interest. The step of determining whether cells or tissues of the subject express the receptor of interest can be performed by any assay or method for detecting a DNA or RNA sequence. The assays or methods include, but are not limited to, genotyping, genome sequencing, next generation sequencing, gene expression analysis, immunohistochemistry, Southern blotting, Western blotting, and polymerase chain reaction (PCR)-based amplification, reverse transcription polymerase chain reaction, real-time reverse transcription polymerase chain reaction (RT-PCR), polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP), mutant allele-specific PGR amplification (MASA) assays, oligonucleotide ligation assays, hybridization assays, TaqMan assays, and microarray analyses.
In some embodiments, the envelope protein comprises a cargo-binding domain. The cargo or cargo molecule is a transgene encoding a therapeutic protein, which includes but is not limited to an immune checkpoint modulator, an antibody or portion thereof, a fusion protein, an anticoagulant, an immunosuppressive agent, an immunostimulatory agent, an enzyme, a growth factor, a hormone, an interferon, an interleukin, a thrombolytic, an anti-angiogenic, a chemotherapeutic, an antibiotic, an antifungal, an antiviral, and any combination thereof.
In some embodiments, the cargo is delivered in the form of virus-like particle.
In some embodiments, disclosed herein is a method of treating a subject with hypercholesterolemia or disorders associated with mutations in low-density lipoprotein (LDL) receptors.
In some embodiments, disclosed herein is a method of treating a subject has a genetic disease, disorder, or syndrome. The disease, disorder, or syndrome includes but is not limited to Cystic Fibrosis, Sickle Cell Anemia, Down Syndrome (Trisomy 21), Huntington's Disease, Duchenne Muscular Dystrophy, Hemophilia, Tay-Sachs Disease, Marfan Syndrome, Fragile X Syndrome, Phenylketonuria, Turner Syndrome, Klinefelter Syndrome, Albinism, Neurofibromatosis, Angelman Syndrome, Prader-Willi Syndrome, Williams Syndrome, Rett Syndrome, Wilson's Disease, Spinal Muscular Atrophy, Xeroderma Pigmentosum, Beckwith-Wiedemann Syndrome, Noonan Syndrome, Leigh Syndrome, Ehlers-Danlos Syndrome.
In some embodiments, disclosed herein is a method of treating cancer, especially those associated with genetic mutations, in a subject. The cancer can be acidophil carcinoma, acinar cell carcinoma, acral lentiginous melanomas, acute granulocytic leukemia, acute lymphoblastic leukemia, acute lymphocytic leukemia, acute myelogenous leukemia, acute myeloid leukemia, adenocarcinoma, adenoid cystic carcinoma, adenomatoid tumor, adenosquamous carcinoma, adrenal carcinoma, adrenal cortex carcinoma, adrenal cortical carcinoma, alveolar (bronchiolar) carcinoma, alveolar rhabdomyosarcoma, amelanotic melanoma, ameloblastic fibrosarcoma, ameloblastic odontosarcoma, ameloblastoma, ampullary carcinoma, androblastoma, angioma, angiosarcoma, apocrine adenocarcinoma, astroblastoma, astrocytoma, basal cell carcinoma, basophil carcinoma, basophilic leukemia, B-cell lymphoma, benign chondroma, bladder cancer, bladder carcinoma, botryoid sarcoma, embryonal rhabdomyosarcoma, brain or spinal cord cancer, branchiolo-alveolar adenocarcinoma, breast cancer, breast carcinoma, brenner tumor, bronchial adenoma, bronchogenic carcinoma, carcinoid tumors, carcinosarcoma, central nervous system cancer, cerebellar sarcoma, ceruminous adenocarcinoma, cervical carcinoma, cervical hyperplasia, cholangiocarcinoma, osteogenic sarcoma, osteosarcoma, chondroblastoma, chondromatous hamartoma, chondromyxofibroma, chondrosarcoma, chordoma, choriocarcinoma, chromophobe carcinoma, chronic granulocytic leukemia, chronic lymphocytic leukemia, chronic myeloblastic leukemia, chronic myelogenous leukemia, clear cell adenocarcinoma, colon cancer, colon carcinoma, colorectal cancer, congenital tumors, cystadenocarcinoma, dermatofibroma, dysgerminoma, embryonal carcinoma, endocrine cancer, endometrial carcinoma, endometroid carcinoma, cosinophilic leukemia, ependymoma, epithelioid cell melanoma, erythroleukemia, esophageal carcinoma, essential thrombocytosis, ewing's sarcoma, extra-mammary paraganglioma, eye cancer, fibrillary astrocytoma, fibroadenoma, fibroma, fibrosarcoma, fibrous histiocytoma, follicular adenocarcinoma, ganglioneuroblastoma, gastrinoma, genitourinary cancer, genitourinary carcinoma, germinoma, pincaloma, giant and spindle cell carcinoma, giant cell tumor of bone, glioblastoma, glioma, glomangiosarcoma, glucagonoma, granular cell carcinoma, granular cell tumor, granuloma, granulosa cell tumor, granulosa-thecal cell tumor, hairy cell leukemia, hamartoma, head-neck cancer, hemangioendothelioma, hemangioma, gall bladder carcinoma, hemangiopericytoma, hemangiosarcoma, hematopoietic cancer, hepatoblastoma, hepatocellular adenoma, hepatocellular carcinoma, infiltrating duct carcinoma, inflammatory carcinoma, insulinoma, interstitial cell carcinoma, intraepithelial carcinoma, juxtacortical osteosarcoma, Kaposi's sarcoma, keloids, kidney cancer, leiomyoma, leiomyosarcoma, melanoma, leukemia, leydig cell tumor, lipid cell tumor, lipoma, hepatoma, liposarcoma, liver cancer, lobular carcinoma, lung carcinoma, lung cancer, lymphangiosarcoma, lymphoepithelial carcinoma, lymphoid leukemia, lymphoma, lymphosarcoma cell leukemia, malignant carcinoid carcinoma, malignant fibrous histiocytoma, malignant giant cell tumor chordoma, malignant histiocytosis, malignant hypercalcemia, malignant lymphoma, malignant melanoma, malignant pancreatic insulinoma, malignant teratoma, malignant tissue, mantle cell lymphoma, mast cell leukemia, mast cell sarcoma, medullary carcinoma, medulloblastoma, megakaryoblastic leukemia, meningioma, meningiosarcoma, mesenchymal chondrosarcoma, mesenchymoma, mesonephroma, mesothelioma, metastatic colorectal cancer, mucinous adenocarcinoma, mucinous cystadenocarcinoma, mucoepidermoid carcinoma, mullerian mixed tumor, multiple myeloma, mycosis fimgoides, mycosis fungoides, myeloid leukemia, myeloid sarcoma, myeloma, myxoma, myxosarcoma, nephroblastoma, neuroblastoma, neurofibroma, neurofibrosarcoma, nodular melanomas, nonencapsulating sclerosing carcinoma, non-Hodgkin's lymphoma, non-small cell lung cancer (NSCLC), odontogenic tumor, olfactory neurogenic tumor, oligodendroblastoma, oligodendroglioma, osteitis deformans, osteochronfroma, osteocartilaginous exostoses, non-hodgkin's lymphomas, ovarian cancer, ovarian carcinoma, ovarian stromal tumor, oxyphilic adenocarcinoma, ductal adenocarcinoma, pancreatic cancer, pancreatic carcinoma, papillary adenocarcinoma, papillary and follicular adenocarcinoma, papillary carcinoma, papillary cystadenocarcinoma, papillary serous cystadenocarcinoma, papillary transitional cell carcinoma, paragranuloma, pheochromocytoma, phyllodes tumor, pilomatrix carcinoma, placental cancer, plasma cell leukemia, polycythemia vera, pre-tumor cervical dysplasia, primary brain carcinoma, primary macroglobulinemia, prostate cancer, prostatic carcinoma, protoplasmic astrocytoma, neuroblastoma, rectal cancer, renal cell carcinoma, retinoblastoma, rhabdomyoma, rhabdomyosarcoma, sarcoma, schwannoma, sebaceous adenocarcinoma, sertoli cell carcinoma, Sertoli-Leydig cell tumors, signet ring cell carcinoma, skin appendage carcinoma, skin cancer, small cell carcinoma, small cell lung cancer (SCLC), small lymphocytic (SL) NHL, soft tissue cancer, soft-tissue sarcoma, solid carcinoma, spinal cord neurofibroma, squamous cell carcinoma, stomach carcinoma, stomach cancer, stromal sarcoma, superficial spreading melanoma, synovial sarcoma, teratocarcinoma, teratoma, testicular carcinoma, the cancer is colon cancer, thecoma, thymoma, thyroid carcinoma, trabecular adenocarcinoma, transitional cell carcinoma, tubular adenoma, villous adenoma, Wilm's tumor, xanthoma or a combination thereof.
In some embodiments, the polynucleotides encoding betaretroviral elements, betaretroviral envelope protein, or a recombinant betaretrovirus, or a recombinant betaretroviral envelope protein disclosed herein are used as a gene therapy to transport functional copies of genes, RNA molecules, or gene-editing components to subjects, to modify, replace or introduce genetic materials into the subject's cell to treat or prevent genetic disease, disorder, or syndrome.
In some embodiments, the polynucleotides encoding betaretroviral elements, betaretroviral envelope protein, or a recombinant betaretrovirus, or a recombinant betaretroviral envelope protein disclosed herein are used as a delivery vehicle for packaging cargo within the delivery vehicle to the subject (e.g., patient) in need thereof.
In some embodiments, the polynucleotides encoding betaretroviral elements, betaretroviral envelope protein, or a recombinant betaretrovirus, or a recombinant betaretroviral envelope protein disclosed herein can be administered via any suitable route, which may depend on, e.g., the medical condition being treated and its location and the pharmacokinetics of the inhibitor. Potential routes of administration include without limitation oral, parenteral (including intradermal, subcutaneous, intramuscular, intravascular, intravenous, intra-arterial, intraperitoneal, intracavitary, intramedullary, intrathecal and topical), and topical (including dermal/epicutaneous, transdermal, mucosal, transmucosal, intranasal [e.g., by nasal spray or drop], ocular/intraocular [e.g., by eye drop], pulmonary [e.g., by oral or nasal inhalation], buccal, sublingual, rectal [e.g., by suppository], and vaginal [e.g., by suppository]).
In some embodiments, polynucleotides encoding betaretroviral elements, betaretroviral envelope protein, or a recombinant betaretrovirus, or a recombinant betaretroviral envelope protein are administered at an effective amount to a subject once. In some embodiments, polynucleotides encoding betaretroviral elements, betaretroviral envelope protein, or a recombinant betaretrovirus, or a recombinant betaretroviral envelope protein are administered at an effective amount to a subject multiple times (e.g., twice, three times, four times, five times, six times, or more). Repeated administration to a subject may be conducted at a regular interval (e.g., daily, every other day, twice per week, weekly, twice per month, monthly, every six months, once per year, or less or more frequently) as necessary to treat (e.g., improve or alleviate) one or more symptoms of a genetic disease, disorder, or condition in the subject.
Administration of the delivery vehicle to the subject may involve suitable carriers, excipients, and other agents within formulations that enhance transfer, delivery, tolerance, and similar factors. Numerous appropriate formulations are detailed in a standard reference for pharmaceutical chemists: Remington's Pharmaceutical Sciences (15th ed., Mack Publishing Company, Easton, Pa. (1975)), specifically in Chapter 87 by Seymour Blaug. Examples of such formulations include powders, pastes, ointments, jellies, waxes, oils, lipid-based vesicles (cationic or anionic) such as Lipofectin™, DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and mixtures containing carbowax. Any of these formulations may be suitable for use in treatments and therapies under this invention, provided the active ingredient remains active within the formulation and is physiologically compatible and tolerable for the chosen administration route.
Carriers include excipients and diluents and must be of sufficiently high purity and sufficiently low toxicity to render them suitable for administration to the patient being treated.
The carrier can be inert, or it can possess pharmaceutical benefits of its own. The amount of carrier employed in conjunction with the compound is sufficient to provide a practical quantity of material for administration per unit dose of the compound. Classes of carriers include, but are not limited to binders, buffering agents, coloring agents, diluents, disintegrants, emulsifiers, flavorants, glidents, lubricants, preservatives, stabilizers, surfactants, tableting agents, and wetting agents. Some carriers may be listed in more than one class, for example vegetable oil may be used as a lubricant in some formulations and a diluent in others. Exemplary pharmaceutically acceptable carriers include sugars, starches, celluloses, powdered tragacanth, malt, gelatin; talc, and vegetable oils. Optional active agents may be included in a pharmaceutical composition, which do not substantially interfere with the activity of the compound of the present invention.
In some embodiments, the engineered delivery vehicle disclosed herein may be delivered to a subject together with a liposome. Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, and the like.
In some embodiments, a delayed or sustained release of polynucleotides encoding betaretroviral elements, betaretroviral envelope protein, or a recombinant betaretrovirus, or a recombinant betaretroviral envelope protein for forming an engineered delivery vehicle for packaging cargo within the delivery vehicle can also be formulated as, e.g., a depot that can be implanted in or injected into a subject, e.g., intramuscularly, intracutaneously or subcutaneously. A depot formulation can be designed to deliver the nucleic acid over an extended period of time, e.g., over a period of at least about 1 week, 2 weeks, 3 weeks, 1 month or 3 months. For example, the nucleic acid can be formulated with a polymeric material (e.g., polyethylene glycol [PEG], polylactic acid [PLA] or polyglycolic acid [PGA], or a copolymer thereof [e.g., PLGA or PLA-PEG]), with a hydrophobic material (e.g., as an emulsion in an oil) and/or an ion-exchange resin, as a more lipophilic derivative (e.g., as an ester of or a salt with a fatty acid such as a C8-C20 fatty acid [e.g., decanoic acid]), or as a sparingly soluble derivative (e.g., a sparingly soluble salt).
In some embodiments, polynucleotides encoding betaretroviral elements, betaretroviral envelope protein, or a recombinant betaretrovirus, or a recombinant betaretroviral envelope protein for forming a delivery vehicle for packaging cargo within the delivery vehicle can also be contained or dispersed in a matrix material. The matrix material can comprise a polymer (e.g., ethylene-vinyl acetate) and controls the release of the compound by controlling dissolution and/or diffusion of the compound from, e.g., a reservoir, and can enhance the stability of the compound while contained in the reservoir. Such a release system can be designed as a sustained-release system, can be configured as, e.g., a transdermal or transmucosal patch, a microneedle transdermal patch, and can contain an excipient that can accelerate the compound's release, such as a water-swellable material (e.g., a hydrogel) that aids in expelling the compound out of the reservoir.
The following examples are intended only to illustrate the disclosure. Other procedures, methodologies, techniques, reagents and conditions may alternatively be used as appropriate.
Presented are exemplary ENVs. There are likely thousands of available betaretroviral ENV proteins available in mammalian genomes for integration in the compositions and methods described herein. As the library of characterized ENVs grows, starting scaffolds for optimization are chosen based on structural similarity to the ligand of interest or in silico predicted “best fit” between a ligand and scaffold. Also, as the cognate receptors for these ENVs are identified, they may also prove to be useful without further modification.
We took note of the fact that three divergent betaretrovirus ENVs, from mouse mammary tumor virus (MMTV), jaagsiekte sheep retrovirus (JSRV), and “intracisternal a-type particle elements with an envelope” (or IAPE), use unrelated cell-surface receptors representing different protein families (
At present, there are no high-resolution structures of a betaretrovirus ENV. There is one report describing loss-of-function mutations in the MMTV ENV 6, pointing to a region of SU that may be involved in receptor binding. A second study based on JSRV and ENTV ENV chimeras identified residues that may explain differential affinity for HYAL-27. These studies indicate approximate regions of SU that may affect receptor-specificity but lack the resolution to define receptor binding domains (RBDs) and their functional intermolecular interactions within the ENV complex. Investigating the relationship between Env structure and receptor-recognition for betaretroviruses is limited by this lack of structural insight. To overcome this limitation, we exploited the vast genomic “archive” of betaretrovirus sequences present as endogenous retrovirus (ERV) loci in mammalian genomes to create an evolution-guided, working model of a canonical betaretrovirus SU domain. Briefly, we used iterative PHI-BLAST and a short, conserved seed pattern (XWAYXPXPPX, where X=any amino-acid) identified from an alignment of the MMTV, JSRV and HML2 SUs to identify ERV loci with intact betaretrovirus-related Env ORFs. When a candidate ORF was identified, surrounding sequences (˜10 kB 5′ and 3′ to the hit) were extracted to identify additional proviral elements and to confirm taxonomic relationships to the Betaretroviruses. Together, these ERV ORFs represent hundreds of millions of years of betaretrovirus evolutionary history. We next used amino acid sequence alignments and secondary structure prediction to identify conserved and variable regions in the linear sequences. We focused specifically on the putative SU regions, where the major receptor-binding elements are likely to be located. By sequence inspection, we noted conservation of multiple, short sequence stretches in SU, interspersed between stretches of considerable sequence and length variation (
Next, we used the artificial intelligence program AlphaFold to generate three-dimensional structural models of each of the input SU protein sequences. Alphafold predicts that all the betaretrovirus SU sequences examined fold into structures consisting of two domains, one comprising the FR sequences and the other comprising the VAR sequences. The FR domain takes on a similar “chalice-shaped” structure for all the SUs analyzed, while the VAR domain structures differ significantly (
Phylogenetic clustering based on amino-acid alignments and structural clustering based on RMSD values of superimposed structures both suggest that the betaretrovirus SU domains comprise three classes (
Based on these models, we propose that VARs comprise the unique receptor binding domains (RBD) of each class, while the FRs fold into a conserved “scaffold” common to all betaretrovirus ENVs. The contrast between structurally superimposable scaffolds and dissimilar VAR domains likely reflects millions of years of diversifying selection on the VAR domain (e.g., adaptation to different receptors), while the essential, receptor-independent functions of the FR domain are constrained and subject to purifying selection (e.g., assembly into heterotrimeric spikes, incorporation into virions, and membrane fusion). In this scenario, separate folding of the two linked domains may have provided the structural flexibility that allowed receptor-switching and host-switching events during co-evolution of betaretroviruses and their mammalian hosts, which in turn contributed to the broad distribution of betaretroviruses and betaretroviral ERVs among modern mammals (including humans).
To our knowledge, endogenous retrovirus (ERV) sequences have not previously been exploited as a source of entry proteins for gene therapy applications. We have already identified, synthesized and characterized a small panel of divergent betaretroviral ENV proteins among ERV loci in vertebrate genome assemblies, and confirmed that several of these can be expressed in cell culture, and produce proteins which behave as predicted for betaretrovirus ENV proteins (
To be useful for viral vectors, these ENVs must be capable of directing membrane fusion, which we measure using a cell-cell fusion assay and a “split” green fluorescent protein (GFP) reporter system (
We also screen candidate ENVs using a lentivirus vector system, which produces lentivirus particles that package the gene for green fluorescent protein (GFP). This assay confirms that the synthetic ENV is compatible with a lentivirus vector system, and that the combination can be used to deliver a genetic “payload” (i.e., a GFP reporter gene) (
One approach to redirecting the receptor specificity of a betaretrovirus ENV is to modify the apical surface (the receptor binding surface) of the ENV SU subunit to display a synthetic peptide ligand for a known receptor. AI-based methods for structure prediction can be used for in silico screening of modified ENVs displaying such peptides, followed by experimental confirmation and iterative redesign (
These pilot experiments confirm that ERV ORFs found in vertebrate genomes and predicted to encode betaretrovirus ENV proteins can be used to create synthetic ENV-expression constructs. The predicted ENV proteins can be expressed in transfected cells, screened in cell-cell fusion assays for fusogenicity, and used to deliver a heterologous gene to cells using pseudotyped lentivirus particles (
The following references are incorporated herein by reference in their entirety for all purposes.
This application claims benefit of priority from U.S. Provisional Application No. 63/597,134, filed on Nov. 8, 2023, titled: Modular Betaretrovirus Receptor Attachment and Cell Entry Proteins, the entire contents of which are incorporated by reference herein for all purposes.
| Number | Date | Country | |
|---|---|---|---|
| 63597134 | Nov 2023 | US |
