Patent Application
20230000908
The present disclosure relates to engineered cells and uses thereof.
Sepsis was once viewed as an uncontrollable inflammatory response to pathogens. However, researchers have re-evaluated therapeutic approaches for sepsis after failures of over 40 clinical trials of anti-inflammatory agents. Recent clinical data reveal that more than 60% of septic patients survive through the initial inflammatory storm but rapidly progress to a longer immunosuppressive state, which is characterized by paralysis and death of immune cells, leading to the inability to clear invading pathogens, an increased susceptibility to hospital-acquired infections, and a high mortality rate. Therefore, what is needed are novel compositions and methods for engineering cells and methods for treating diseases (for example, sepsis).
In some aspects, disclosed herein is an antigen presenting cell, comprising:
In some embodiments, the first nucleic acid and the second nucleic acid are linked by third nucleic acid.
In some embodiments, the recombinant polynucleotide is encapsulated by the vitamin-lipid.
In some embodiments, the recombinant polynucleotide comprises an RNA or a DNA.
In some embodiments, the antimicrobial peptide comprises the sequence SEQ ID NO: 1.
In some embodiments, the first nucleic acid comprises the sequence SEQ ID NO: 2.
In some embodiments, the second nucleic acid comprises the sequence SEQ ID NO: 4.
In some embodiments, the linker comprises a cathepsin B sensitive linker. In some embodiments, the third nucleic acid comprises the sequence SEQ ID NO: 6.
In some embodiments, the recombinant polynucleotide comprises the sequence SEQ ID NO: 8.
In some embodiments, the vitamin-lipid comprises a vitamin moiety, and wherein the vitamin moiety comprises vitamin B3, vitamin C, vitamin D, vitamin E, vitamin H, or a derivative thereof. In some embodiments, the vitamin moiety is vitamin C.
In some embodiments, the vitamin-lipid is selected from the group consisting of:
wherein R is
In some embodiments, the antigen presenting cell comprises a comprises a macrophage or a dendritic cell. In some embodiments, the macrophage comprises a bone marrow-derived macrophage or a monocyte-derived macrophage. In some embodiments, the dendritic cell comprises a bone marrow-derived dendritic cell, a monocyte-derived dendritic cell, a conventional dendritic cell-1, or a conventional dendritic cell-2.
In some aspects, disclosed herein is a method of treating sepsis, comprising administering to a subject one or more antigen presenting cells comprising:
In some embodiments, the antigen presenting cell is derived from the subject. In some embodiments, the subject comprises a human. In some embodiments, the human has or is suspected of having sepsis.
The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several aspects described below.
Sepsis was traditionally, viewed as an uncontrollable inflammatory response to pathogens. However, researchers have re-evaluated therapeutic approaches for sepsis after failures of over 40 clinical trials of anti-inflammatory agents. Clinical data reveal that more than 60% septic patients survive through the initial inflammatory storm but rapidly progress to a longer immunosuppressive state, which is characterized by paralysis and death of immune cells, leading to inability to clear invading pathogens, increased susceptibility to hospital-acquired infections, and high mortality rate. As a result, potential therapeutic targets have been extensively explored in order to treat sepsis such as removing the anaphylatoxin C5a or blocking the C5a receptor. Meanwhile, approaches aiming to restore immune function have been developed and tested in patients with sepsis.
Macrophages are one of the most efficient pathogen scavengers during infections. In patients with sepsis, impaired macrophages/monocytes can be primarily responsible for the insufficient antimicrobial defense. Several small clinical trials of immunostimulatory agents have indicated benefits in reversing deactivated macrophages/monocytes, thereby enhancing infection eradication. In contrast, meta-analysis of large clinical trials did not show significant changes in reducing the patient mortality. Several reasons can lead to these different clinical results. First, the immunostimulatory agents are not able to restore the function of impaired macrophages/monocytes to their original levels. Second, invaded bacteria are usually trapped in macrophage phagosomes which further fuse with lysosomes to form phagolysosomes. Within phagolysosomes, reactive oxygen species (ROS), reactive nitrogen species (RNS), and lysozymes work synergistically to clear bacteria. However, many bacteria, such as Staphylococcus aureus and Escherichia coli have evolved immune escape mechanisms for thwarting phagolysosomal killing, including scavenging ROS and RNS, and resisting lysozymes, resulting in intracellular survival and recurrent infections. Third, although combined antibiotic therapy is the standard treatment in the sepsis clinical guideline, 70%-80% of sepsis death is related to persistent infections, indicating the prevalence of antibiotic resistance and the paucity of new antimicrobial agents. As an alternative, immunotherapy based on adoptive cell transfer bypasses the need for restoring the dysfunctional immune cells and therefore provides potential benefits in immunocompromised patients.
Disclosed herein is adoptive transfer using macrophages loaded with antimicrobial peptides/cathepsin B in the lysosomes (MACs). To construct the MACs, the antimicrobial peptides/cathepsin B (AMP-CatB) mRNA was designed. Vitamin C lipid nanoparticles (VcLNPs) were identified with more efficient delivery of mRNA than Lipofectamine 3000 and electroporation in both the RAW264.7 cell line and bone marrow derived macrophages (BMDMs). The VcLNPs allow specific accumulation of AMP-CatB in the macrophage lysosomes, the key location for antibacterial activities. It is also disclosed herein that adoptive MAC transfer eliminates MDR bacteria including Staphylococcus aureus and Escherichia coli, and restores mouse body conditions in the immunocompromised sepsis models, which provides a strategy for overcoming MDR bacteria-induced sepsis and shed light on the development of nanoparticle cell therapy for infectious diseases. Reference will now be made in detail to the embodiments of the invention, examples of which are illustrated in the drawings and the examples. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs.
Terms used throughout this application are to be construed with ordinary and typical meaning to those of ordinary skill in the art. However, Applicant desires that the following terms be given the particular definition as defined below.
As used herein, the article “a,” “an,” and “the” means “at least one,” unless the context in which the article is used clearly indicates otherwise.
The term “comprising” and variations thereof as used herein is used synonymously with the term “including” and variations thereof and are open, non-limiting terms. Although the terms “comprising” and “including” have been used herein to describe various embodiments, the terms “consisting essentially of” and “consisting of” can be used in place of “comprising” and “including” to provide for more specific embodiments and are also disclosed.
As used herein, the terms “may,” “optionally,” and “may optionally” are used interchangeably and are meant to include cases in which the condition occurs as well as cases in which the condition does not occur. Thus, for example, the statement that a formulation “may include an excipient” is meant to include cases in which the formulation includes an excipient as well as cases in which the formulation does not include an excipient.
The terms “about” and “approximately” are defined as being “close to” as understood by one of ordinary skill in the art. In one non-limiting embodiment, the terms are defined to be within 10%. In another non-limiting embodiment, the terms are defined to be within 5%. In still another non-limiting embodiment, the terms are defined to be within 1%.
The term “nucleic acid” as used herein means a polymer composed of nucleotides, e.g. deoxyribonucleotides (DNA) or ribonucleotides (RNA).
The terms “ribonucleic acid” and “RNA” as used herein mean a polymer composed of ribonucleotides.
The terms “deoxyribonucleic acid” and “DNA” as used herein mean a polymer composed of deoxyribonucleotides.
The term “oligonucleotide” denotes single- or double-stranded nucleotide multimers. Suitable oligonucleotides may be prepared by the phosphoramidite method described by Beaucage and Carruthers, Tetrahedron Lett., 22: 1859-1862 (1981), or by the triester method according to Matteucci, et al., J. Am. Chem. Soc., 103:3185 (1981), both incorporated herein by reference, or by other chemical methods using either a commercial automated oligonucleotide synthesizer or VLSIPS™ technology. When oligonucleotides are referred to as “double-stranded,” it is understood by those of skill in the art that a pair of oligonucleotides exist in a hydrogen-bonded, helical array typically associated with, for example, DNA. In addition to the 100% complementary form of double-stranded oligonucleotides, the term “double-stranded,” as used herein is also meant to refer to those forms which include such structural features as bulges and loops, described more fully in such biochemistry texts as Stryer, Biochemistry, Third Ed., (1988), incorporated herein by reference for all purposes.
The term “polynucleotide” refers to a single or double stranded polymer composed of nucleotide monomers.
The term “polypeptide” refers to a compound made up of a single chain of D- or L-amino acids or a mixture of D- and L-amino acids joined by peptide bonds.
The term “promoter” or “regulatory element” refers to a region or sequence determinants located upstream or downstream from the start of transcription and which are involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. Promoters need not be of bacterial origin, for example, promoters derived from viruses or from other organisms can be used in the compositions, systems, or methods described herein.
The term “recombinant” refers to a human manipulated nucleic acid (e.g. polynucleotide) or a copy or complement of a human manipulated nucleic acid (e.g. polynucleotide), or if in reference to a protein (i.e, a “recombinant protein”), a protein encoded by a recombinant nucleic acid (e.g. polynucleotide). In some embodiments, a recombinant expression cassette comprising a promoter operably linked to a second nucleic acid (e.g. polynucleotide) may include a promoter that is heterologous to the second nucleic acid (e.g. polynucleotide) as the result of human manipulation (e.g., by methods described in Sambrook et al., Molecular Cloning—A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1989) or Current Protocols in Molecular Biology Volumes 1-3, John Wiley & Sons, Inc. (1994-1998)). In another example, a recombinant expression cassette may comprise nucleic acids (e.g. polynucleotides) combined in such a way that the nucleic acids (e.g. polynucleotides) are extremely unlikely to be found in nature. For instance, human manipulated restriction sites or plasmid vector sequences may flank or separate the promoter from the second nucleic acid (e.g. polynucleotide). One of skill will recognize that nucleic acids (e.g. polynucleotides) can be manipulated in many ways and are not limited to the examples above.
The term “expression cassette” or “vector” refers to a nucleic acid construct, which when introduced into a host cell, results in transcription and/or translation of an RNA or polypeptide, respectively. In some embodiments, an expression cassette comprising a promoter operably linked to a second nucleic acid (e.g. polynucleotide) may include a promoter that is heterologous to the second nucleic acid (e.g. polynucleotide) as the result of human manipulation (e.g., by methods described in Sambrook et al., Molecular Cloning—A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1989) or Current Protocols in Molecular Biology Volumes 1-3, John Wiley & Sons, Inc. (1994-1998)).
The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity over a specified region when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (see, e.g., NCBI web site or the like). Such sequences are then said to be “substantially identical.” This definition also refers to, or may be applied to, the compliment of a test sequence. The definition also includes sequences that have deletions and/or additions, as well as those that have substitutions. As described below, the preferred algorithms can account for gaps and the like. Preferably, identity exists over a region that is at least about 10 amino acids or 20 nucleotides in length, or more preferably over a region that is 10-50 amino acids or 20-50 nucleotides in length. As used herein, percent (%) nucleotide sequence identity is defined as the percentage of amino acids in a candidate sequence that are identical to the nucleotides in a reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared can be determined by known methods.
For sequence comparisons, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Preferably, default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nuc. Acids Res. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al. (1990) J. Mol. Biol. 215:403-410). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) or 10, M=5, N=−4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915) alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparison of both strands.
The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01.
The phrase “codon optimized” as it refers to genes or coding regions of nucleic acid molecules for the transformation of various hosts, refers to the alteration of codons in the gene or coding regions of polynucleic acid molecules to reflect the typical codon usage of a selected organism without altering the polypeptide encoded by the DNA. Such optimization includes replacing at least one, or more than one, or a significant number, of codons with one or more codons that are more frequently used in the genes of that selected organism.
The term “nucleobase” refers to the part of a nucleotide that bears the Watson/Crick base-pairing functionality. The most common naturally-occurring nucleobases, adenine (A), guanine (G), uracil (U), cytosine (C), and thymine (T) bear the hydrogen-bonding functionality that binds one nucleic acid strand to another in a sequence specific manner.
Nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, “operably linked” means that the DNA or RNA sequences being linked are near each other, and, in the case of a secretory leader, contiguous and in reading phase. However, operably linked nucleic acids (e.g. enhancers and coding sequences, or multiple coding sequences) do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice. In some embodiments, a promoter is operably linked with a coding sequence when it is capable of affecting (e.g. modulating relative to the absence of the promoter) the expression of a protein from that coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter).
The term “gene” or “gene sequence” refers to the coding sequence or control sequence, or fragments thereof. A gene may include any combination of coding sequence and control sequence, or fragments thereof. Thus, a “gene” as referred to herein may be all or part of a native gene. A polynucleotide sequence as referred to herein may be used interchangeably with the term “gene”, or may include any coding sequence, non-coding sequence or control sequence, fragments thereof, and combinations thereof. The term “gene” or “gene sequence” includes, for example, control sequences upstream of the coding sequence (for example, the ribosome binding site).
The term “nanoparticle” as used herein refers to a particle or structure which is biocompatible with and sufficiently resistant to chemical and/or physical destruction by the environment of such use so that a sufficient number of the nanoparticles remain substantially intact after delivery to the site of application or treatment and whose size is in the nanometer range. For the purposes of the present invention, a nanoparticle typically ranges from about 1 nm to about 1000 nm, from about 50 nm and about 500 nm, from about 50 nm and about 350 nm, from about 100 nm and about 250 nm, or from about 110 nm and about 150 nm.
The phrase “symptoms of sepsis” refers to any symptom characteristic of a subject with sepsis including but not limited to, arterial hypotension, metabolic acidosis, fever, decreased systemic vascular resistance, tachypnea and organ dysfunction. Sepsis can result from septicemia (i.e., organisms, their metabolic end-products or toxins in the blood stream), including bacteremia (i.e., bacteria in the blood), as well as toxemia (i.e., toxins in the blood), including endotoxemia (i.e., endotoxin in the blood). The term “sepsis” also encompasses fungemia (i.e., fungi in the blood), viremia (i.e., viruses or virus particles in the blood), and parasitemia (i.e., helminthic or protozoan parasites in the blood). Thus, phenotypes associated with septicemia and septic shock (acute circulatory failure resulting from septicemia often associated with multiple organ failure and a high mortality rate) are symptoms of sepsis.
As used herein, the terms “treating” or “treatment” of a subject includes the administration of a therapeutic agent to a subject with the purpose of curing, healing, alleviating, relieving, altering, remedying, ameliorating, improving, stabilizing or affecting a disease or disorder, or a symptom of a disease or disorder. The terms “treating” and “treatment” can also refer to reduction in severity and/or frequency of symptoms, elimination of symptoms and/or underlying cause, and improvement or remediation of damage.
“Therapeutic agent” refers to any composition that has a beneficial biological effect. Beneficial biological effects include both therapeutic effects, e.g., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, e.g., prevention of a disorder or other undesirable physiological condition (e.g., sepsis). The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, cells, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like. When the terms “therapeutic agent” is used, then, or when a particular agent is specifically identified, it is to be understood that the term includes the agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, proagents, conjugates, active metabolites, isomers, fragments, analogs, etc.
“Therapeutically effective amount” or “therapeutically effective dose” of a composition refers to an amount that is effective to achieve a desired therapeutic result. In some embodiments, a desired therapeutic result is the treatment of sepsis. In some embodiments, a desired therapeutic result is restoring immune system in a sepsis patient. In some embodiments, a desired therapeutic result is reduction or clearance of a pathogen. Therapeutically effective amounts of a given therapeutic agent will typically vary with respect to factors such as the type and severity of the disorder or disease being treated and the age, gender, and weight of the subject. The term can also refer to an amount of a therapeutic agent, or a rate of delivery of a therapeutic agent (e.g., amount over time), effective to facilitate a desired therapeutic effect. The precise desired therapeutic effect will vary according to the condition to be treated, the tolerance of the subject, the agent and/or agent formulation to be administered (e.g., the potency of the therapeutic agent, the concentration of agent in the formulation, and the like), and a variety of other factors that are appreciated by those of ordinary skill in the art. In some instances, a desired biological or medical response is achieved following administration of multiple dosages of the composition to the subject over a period of days, weeks, or years.
“Pharmaceutically acceptable carrier” (sometimes referred to as a “carrier”) means a carrier or excipient that is useful in preparing a pharmaceutical or therapeutic composition that is generally safe and non-toxic, and includes a carrier that is acceptable for veterinary and/or human pharmaceutical or therapeutic use. The terms “carrier” or “pharmaceutically acceptable carrier” can include, but are not limited to, phosphate buffered saline solution, water, emulsions (such as an oil/water or water/oil emulsion) and/or various types of wetting agents.
As used herein, the term “carrier” encompasses any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations. The choice of a carrier for use in a composition will depend upon the intended route of administration for the composition. The preparation of pharmaceutically acceptable carriers and formulations containing these materials is described in, e.g., Remington's Pharmaceutical Sciences, 21st Edition, ed. University of the Sciences in Philadelphia, Lippincott, Williams & Wilkins, Philadelphia, Pa., 2005. Examples of physiologically acceptable carriers include saline, glycerol, DMSO, buffers such as phosphate buffers, citrate buffer, and buffers with other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN™ (ICI, Inc.; Bridgewater, N.J.), polyethylene glycol (PEG), and PLURONICS™ (BASF; Florham Park, N.J.). To provide for the administration of such dosages for the desired therapeutic treatment, compositions disclosed herein can advantageously comprise between about 0.1% and 99% by weight of the total of one or more of the subject compounds based on the weight of the total composition including carrier or diluent.
As used throughout, by a “subject” (or a “host”) is meant an individual. Thus, the “subject” can include, for example, domesticated animals, such as cats, dogs, etc., livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.) mammals, non-human mammals, primates, non-human primates, rodents, birds, reptiles, amphibians, fish, and any other animal. The subject can be a mammal such as a primate or a human.
In some aspects, disclosed herein is an antigen presenting cell, comprising:
In some aspects, disclosed herein is an antigen presenting cell, comprising:
In some aspects, disclosed herein is an antigen presenting cell, comprising:
In some embodiments, the antigen presenting cell comprises a macrophage or a dendritic cell.
It should be understood that the term “antigen presenting cells” or “APCs” used herein refers to a heterologous group of immune cells that can process and present antigens for stimulating responses of certain lymphocytes (e.g., T cells and B cells). Classical APCs include, for example, dendritic cells, macrophages, B cells, and neutrophils.
It is understood herein that macrophages are commonly known as phagocytosing immune cells (Meszaros et al, 1999). They also secrete factors such as chemokines or cytokines. In addition to phagocytosis and antigen presentation, these cells may play a supportive role through a varied repertoire of plasma membrane and secreted molecules (Gordon 1995, BioEssays, Volume17, Issue11), as previously shown for erythroblasts, hepatocytes and neurons (Sadahira & Morr, Pathol Int. 1999 October; 49(10):841-8.) (Takeishi, Hirano, et al. Arch Histol Cytol. 1999 December; 62(5):413-22.) (Polazzi, Gianni, et al. Glia. 2001 December; 36(3):271-80.). By “macrophages” is meant cells exhibiting properties usually described for macrophages, including phagocytosis, expression of defined cell surface markers such as CD64, CD14 and HLA-DR antigen expression. Macrophages according to the invention can be isolated from tissues or preferentially by ex vivo differentiation from blood monocytes (also referred herein as “monocyte-derived macrophage”), bone marrow precursor cells (also referred herein as “bone marrow-derived macrophage”) or from any other possible precursor, and by using any differentiation method, precursors and methods being known by any person skilled in the art. Accordingly, in some embodiments, the macrophage comprises a bone marrow-derived macrophage. In some embodiments, the macrophage comprises a monocyte-derived macrophage. In some embodiments, the macrophage comprises an iPSC-derived macrophage. In some embodiments, the macrophage comprises a macrophage cell line, including, for example, RAW264.7, THP-1, U937, IC-21, J774A.1, MV-4-11, or KGl.
It should be also understood herein that “dendritic cell” or “DC”, as used herein, refers to a type of antigen presenting cell, which is typically identified by the expression of one or more of the following markers on its cell surface: CD1a, CD1b, and CD1c, CD4, CD11c, CD33, CD40, CD80, CD86, CD83 and HLA-DR. In some embodiments, the dendritic cell is a mature DC. In some embodiments, the dendritic cell is an immature DC. DCs according to the invention can be isolated from tissues or preferentially by ex vivo differentiation from blood monocytes (also referred herein as “monocyte-derived dendritic cell”), bone marrow precursor cells (also referred herein as “bone marrow-derived dendritic cell”) or from any other possible precursor, and by using any differentiation method, precursors and methods being known by any person skilled in the art.
Accordingly, in some embodiments, the dendritic cell comprises a bone marrow-derived dendritic cell. In some embodiments, the dendritic cell comprises a monocyte-derived dendritic cell. In some embodiments, the dendritic cell comprises an iPSC-derived dendritic cell. In some embodiments, the dendritic cell is a conventional dendritic cell 1 (or cDC1, lymphoid DC), which is typically identified by the expression of one or more of the following markers on its cell surface: CD141, CLEC9A, and XCR1. In some embodiments, the dendritic cell is a conventional dendritic cell 2 (or cDC2, myeloid DC), which is typically identified by the expression of one or more of the following markers on its cell surface: CD1c and CD172a. In some embodiments, the dendritic cell is plasmacytoid DC (or pDC), which is typically identified by the expression of one or more of the following markers on its cell surface: CD123, CD303, and CD304.
In some embodiments, the antigen presenting cell is derived from a subject selected from the group consisting of a mouse, a rat, a human, or a non-human primate. In some embodiments, the antigen presenting cell is derived from a mouse. In some embodiments, the antigen presenting cell is derived from a rat. In some embodiments, the antigen presenting cell is derived from a human. In some embodiments, the antigen presenting cell is derived from a non-human primate.
While a number of nanoparticles are described herein, additional nanoparticles known in the art can also be used herein.
In some aspects, disclosed herein is a lipid-based nanoparticle, comprising:
In some aspects, disclosed herein is a lipid-based nanoparticle, comprising:
In some aspects, disclosed herein is a lipid-based nanoparticle, comprising:
a vitamin-lipid.
The term “vitamin-lipid” used herein refers to a compound comprising a vitamin moiety and a lipid, wherein the lipid can be a lipid-like moiety. The term “vitamin-lipid” is also meant to refer to those forms described more fully in WO2019/027999, incorporated herein by reference for all purposes. Accordingly, the vitamin-lipid can be, for example, a compound of Formula A:
wherein m is an integer from 1 to 20,
wherein n is an integer from 1 to 3; and
In one embodiments, the vitamin-lipid can be a compound of Formula I
The vitamin moiety can be, for example, vitamin B3, vitamin C, vitamin D, vitamin E, vitamin H, or a derivative thereof. In some embodiments, the vitamin moiety is vitamin C or a derivative thereof. The vitamin moiety is shown below:
In some embodiments, vitamin-lipid comprises:
In some embodiments, the vitamin-lipid is selected from the group consisting of:
or a salt thereof.
In some embodiments, the vitamin-lipid is selected from the group consisting of
or a salt thereof.
In some embodiments, the lipid-based nanoparticle comprises a vitamin-lipid in a molar ratio of about 10% to about 60%. In some embodiments, the lipid-based nanoparticle comprises a vitamin-lipid in a molar ratio of about 10% to about 40%. In some embodiments, the lipid-based nanoparticle comprises a vitamin-lipid in a molar ratio of about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 50%, about 60%, about 70%, or about 80%.
In one embodiments, the lipid-based nanoparticle comprises a vitamin-lipid in a molar ratio of about 30%.
In some embodiments, the lipid-based nanoparticle further comprises a non-cationic lipid, which can include, but not limited to, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), 1,2-dioleyl-sn-glycero-3-phosphotidylcholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1,2-dioleoyl-5/7-glycero-3-phospho-(1′-rac-glycerol) (DOPG), or combinations thereof.
In some embodiments, the lipid-based nanoparticle further comprises a polyethylene glycol-lipid (PEG-lipid), which can include, but not limited to, PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, and PEG-modified dialkylglycerols. Representative polyethylene glycol-lipids include DMG-PEG, DLPE-PEGs, DMPE-PEGs, DPPC-PEGs, and DSPE-PEGs.
In some embodiments, the lipid-based nanoparticle further comprises PEGylated cholesterol, DC-Choi (N,N-dimethyl-N— ethylcarboxamidocholesterol), 1,4-bis(3-N-oleylamino-propyl)piperazine, or combinations thereof.
In some embodiments, the lipid-based nanoparticle further comprises a recombinant polynucleotide, wherein the polynucleotide can be encapsulated by the vitamin-lipid.
In some embodiments, the recombinant polynucleotide comprises an RNA or a DNA. In some embodiments, the recombinant polynucleotide is an RNA. In some embodiments, the recombinant polynucleotide is a mRNA. In some embodiments, the recombinant polynucleotide is a DNA.
The term “antimicrobial peptide” as disclosed herein comprises a peptide or a derivative thereof having antimicrobial activity against one or more selected from a group consisting of bacteria such as Gram-positive bacteria, Gram negative bacteria, etc. and fungi such as yeast, molds, etc. In some embodiments, the antimicrobial peptide comprises the sequence SEQ ID NO: 1, or a fragment or functionally active variant thereof. In some embodiments, the antimicrobial peptide is selected from the group comprising a polypeptide sequence at least 60% (for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NO: 1. In some embodiments, the antimicrobial peptide is selected from Protegrin 1, C16G2, Omiganan, β-Defensins, hLF1-11, LL37, or MSI-78, or a fragment or functionally active variant thereof. These antimicrobial peptide sequences and additional antimicrobial peptides are known in the art, for example, see U.S. Pat. No. 8,754,039, which is incorporated by reference herein in its entirety.
The antimicrobial peptide of any preceding aspect can be encoded by the first nucleic acid of the recombinant polynucleotide. In some embodiments, the first nucleic acid comprises the sequence SEQ ID NO: 2, or a fragment or functionally active variant thereof. In some embodiments, the first nucleic acid is selected from the group comprising a polynucleotide sequence at least 60% (for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NO: 2.
Cathepsin B disclosed herein comprises a polypeptide sequence set forth in SEQ ID NO: 3, or a fragment or functionally active variant thereof. In some embodiments, cathepsin B is selected from the group comprising a polypeptide sequence at least 60% (for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NO: 3.
Cathepsin B of any preceding aspect can be encoded by the second nucleic acid of the recombinant polynucleotide. In some embodiments, the second nucleic acid comprises the sequence SEQ ID NO: 4, or a fragment or functionally active variant thereof. In some embodiments, the second nucleic acid is selected from the group comprising a polynucleotide sequence at least 60% (for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NO: 4.
In some aspects, disclosed herein is an antigen presenting cell, comprising:
In some aspects, disclosed herein is an antigen presenting cell, comprising:
In some embodiments, the second nucleic acid encoding a cathepsin peptide comprises Cathepsin A, Cathepsin B, Cathepsin C, Cathepsin D, Cathepsin E, Cathepsin F, or Cathepsin G, or a fragment or functionally active variant thereof. These sequences are known in the art and can be found at the National Center for Biotechnology Information website (www.ncbi.nlm.nih.gov). In some embodiments, the sequences are from a mammal. In some embodiments, the sequences are from a mouse. In some embodiments, the sequences are from a primate. In some embodiments, the sequences are from a human.
In some embodiments, the linker comprises a cathepsin B sensitive linker. In some embodiments, the linker comprises a polypeptide sequence set forth in SEQ ID NO: 5, or a fragment or functionally active variant thereof. In some embodiments, the linker is selected from the group comprising a polypeptide sequence at least 60% (for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NO: 5.
The linker of any preceding aspect can be encoded by the third nucleic acid. In some embodiments, the third nucleic acid comprises the sequence SEQ ID NO: 6, or a fragment or functionally active variant thereof. In some embodiments, the third nucleic acid is selected from the group comprising a polynucleotide sequence at least 60% (for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NO: 6.
In some embodiments, the linker comprises a polypeptide sequence set forth in SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12, or a fragment or functionally active variant thereof. In some embodiments, the linker is selected from the group comprising a polypeptide sequence at least 60% (for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12. In some embodiments, the linker may also be Phe-Lys.
In some embodiments, the recombinant polynucleotide of any preceding aspect comprises the sequence SEQ ID NO: 7, or a fragment or functionally active variant thereof. In some embodiments, the recombinant polynucleotide is selected from the group comprising a polynucleotide sequence at least 60% (for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NO: 7.
In some embodiments, the recombinant polynucleotide encodes a recombinant polypeptide comprising a sequence set forth in SEQ ID NO: 8, or a fragment or functionally active variant thereof. In some embodiments, the recombinant polypeptide is selected from the group comprising a polynucleotide sequence at least 60% (for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NO: 8.
In some embodiments, the first nucleic acid and the second nucleic acid are linked by the third nucleic acid.
In some aspects, disclosed herein is a method of treating sepsis, comprising administering to a subject one or more antigen presenting cells comprising:
In some aspects, disclosed herein is a method of treating sepsis, comprising administering to a subject one or more antigen presenting cells comprising:
In some aspects, disclosed herein is a method of treating sepsis, comprising administering to a subject one or more antigen presenting cells comprising:
In some aspects, disclosed herein is a method of treating sepsis, comprising administering to a subject one or more antigen presenting cells comprising:
As disclosed above, the term “sepsis” also encompasses bacteremia (i.e., bacteria in the blood), toxemia (i.e., toxins in the blood), fungemia (i.e., fungi in the blood), viremia (i.e., viruses or virus particles in the blood), and parasitemia (i.e., helminthic or protozoan parasites in the blood). Thus, phenotypes associated with septicemia and septic shock (acute circulatory failure resulting from septicemia often associated with multiple organ failure and a high mortality rate) are symptoms of sepsis. Accordingly, disclosed herein is a method of treating, inhibiting, or reducing sepsis or the symptoms of sepsis (for example, reducing load of bacteria, toxin, fungi, virus or parasites in the blood, and/or restoring, maintaining or improving immune system of the affected subject.
In some embodiments, the antigen presenting cell of any preceding aspects is derived from the subject. In some embodiments, the antigen presenting cell is derived from the subject. In some embodiments, the antigen presenting cell is derived from a different subject. In some embodiments, the subject is a human. In some embodiments, the human has or is suspected of having sepsis.
In some embodiments, the method of treating sepsis comprises administering to a subject one or more antigen presenting cells of any preceding aspect, wherein the one or more antigen presenting cells are prepared and administered together with a pharmaceutically acceptable carrier.
As the timing of sepsis can often not be predicted, it should be understood the disclosed methods of treating, preventing, reducing, and/or inhibiting sepsis, can be used prior to or following the onset of septic symptoms, to treat, prevent, inhibit, and/or reduce sepsis. Where, the disclosed methods can be performed any time prior to sepsis. In one aspect, the disclosed methods can be employed 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 months, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 days, 60, 48, 36, 30, 24, 18, 15, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2 hours, 60, 45, 30, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 minute prior to sepsis; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 75, 90, 105, 120 minutes, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 18, 24, 30, 36, 48.60 hours, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 45, 60, 90 or more days, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more months, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 years after sepsis.
The antigen presenting cells of the present invention can be administered to the appropriate subject in any manner known in the art, e.g., orally intramuscularly, intravenously, sublingual mucosal, intraarterially, intrathecally, intradermally, intraperitoneally, intranasally, intrapulmonarily, intraocularly, intravaginally, intrarectally or subcutaneously. They can be introduced into the gastrointestinal tract or the respiratory tract. Parenteral administration, if used, is generally characterized by injection.
In some aspects, disclosed herein is a method of treating a disease, comprising administering to a subject one or more antigen presenting cells comprising:
In some aspects, disclosed herein is a method of treating cancer, comprising administering to a subject one or more antigen presenting cells comprising:
In some embodiments, the disease is selected from lysosomal storage disorder, aspartylglucosaminuria, Gaucher disease, GM1 gangliosidosis, or mucopolysaccharidoses.
In some aspects, disclosed herein is a method of treating a neurodegenerative disease, comprising administering to a subject one or more antigen presenting cells comprising:
In some embodiments, the neurodegenerative disease is selected from Alzheimer's disease, Parkinson's disease, or Huntington disease.
In still other embodiments, disclosed herein is a method of treating a disease, comprising administering to a subject a therapeutically effective amount of the immune cells of any preceding aspect. In some embodiments, the disease is selected from lysosomal storage disorder, aspartylglucosaminuria, Gaucher disease, GM1 gangliosidosis, or mucopolysaccharidoses.
In some aspects, disclosed herein is an immune cell, comprising:
In some embodiments, the recombinant polynucleotide is encapsulated by the vitamin-lipid.
In some embodiments, the recombinant polynucleotide comprises an RNA or a DNA. In some embodiments, the recombinant polynucleotide is an RNA. In some embodiments, the recombinant polynucleotide is a mRNA. In some embodiments, the recombinant polynucleotide is a DNA.
In some embodiments, the immune protein comprises a chimeric antigen receptor or a cytotoxic cytokine.
In some embodiments, the immune protein is a chimeric antigen receptor. In some embodiments, the comprises an antigen binding domain, a transmembrane domain, a costimulatory signaling region, or a CD3 zeta signaling domain.
In some embodiments, the antigen binding domain binds to a tumor antigen.
In some embodiments, the immune protein is a cytotoxic cytokine, including, for example, interferon gamma, Tumor Necrosis Factor, granzyme A, granzyme B, or perforin.
In some embodiments, disclosed herein is a method of treating a cancer, comprising administering to a subject a therapeutically effective amount of the immune cells of any preceding aspect.
In still other embodiments, disclosed herein is a method of treating a disease, comprising administering to a subject a therapeutically effective amount of the immune cells of any preceding aspect. In some embodiments, the disease is selected from lysosomal storage disorder, aspartylglucosaminuria, Gaucher disease, GM1 gangliosidosis, or mucopolysaccharidoses.
In some embodiments, disclosed herein is a method of treating a neurodegenerative disease, comprising administering to a subject a therapeutically effective amount of the immune cells of any preceding aspect. In some embodiments, the neurodegenerative disease is selected from Alzheimer's disease, Parkinson's disease, or Huntington disease.
In some embodiments, the subject comprises a human. In some embodiments, the human has or is suspected of having a cancer.
The following examples are set forth below to illustrate the compounds, systems, methods, and results according to the disclosed subject matter. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations of the present invention which are apparent to one skilled in the art.
The data disclosed herein show that adoptive transfer of macrophages loaded with antimicrobial peptides/cathepsin B in the lysosomes (MACs) provides the immunocompromised sepsis host to boost innate immunity, prevent bacterial immune evasion, and eliminate multidrug resistant (MDR) bacteria. As illustrated in
Through the initial screening and two rounds of characterization, the formulation of vitamin C lipid nanoparticles (VcLNPs) allowed AMP-CatB mRNA to be effectively delivered into macrophages. The mRNA is translated to functional proteins in the cytoplasm and the proteins are further translocated into lysosomes. In the lysosomes, the CatB sensitive linker is cleaved by CatB proteins and thus the APM-IB367 is released. When phagosomes encapsulating bacteria fuse with lysosomes, the ingested bacteria are exposed to both pre-stored AMP-IB367 and lysosomal antimicrobial components. Although immune evasion strategies may protect MDR bacteria from phagolysosomal killing mechanisms, the AMP-IB367 is able to kill these bacteria because of its high antibacterial activity against MDR bacteria in animal models and in humans. Consequently, the adoptive transfer of MACs rescues MDR bacteria induced sepsis with immunosuppression by restoring the innate immunity, overcoming bacterial immune evasion, and eradicating the infection.
Five vitamins were first selected: vitamin B3, vitamin C, vitamin D, vitamin E, and vitamin H (also called vitamin B7), and then incorporated lipid tails using the methods reported previously. The amino lipids were installed on these vitamins through an ester bond or amide bond (
Next, these vitamin-derived lipids were formulated into vitamin-lipid nanoparticles (VLNPs) accordingly. Particle size of VLNPs ranged from 127±1 to 174±1 nm with polydispersity index (PDI)<0.3 except VHLNPs (
Using a fluorescent probe, Alexa-Fluor 647 labelled RNA, 99.2% Alexa-Fluor 647 positive cells was observed in the group of VcLNPs, 21.5% in the group of Lipofectamine 3000, and 2.4% in the group of electroporation (
To test whether the cathepsin B (CatB) was able to transport the payload into the lysosomes, the eGFP-CatB mRNA was constructed and delivered into the RAW264.7 cells using VcLNPs. The confocal microscopy of the live cells exhibited that eGFP-CatB co-localized with the LysoTracker® Red DND-99 in the lysosomes, with a Pearson's correlation coefficient of 0.91±0.15 (
Given the potent in vitro bactericidal activity of MAC-RAWs, testing was performed assessing the therapeutic effects in MDRSA induced sepsis mice with immunosuppression. After 3 consecutive days of cyclophosphamide (CY) treatment, the decrease of body weights (BWs), white blood cells (WBCs), and lymphocytes (LYMs) mimicked an immunocompromised state in the sepsis patient. After infected by MDRSA, the mice were treated with PBS, PBS-RAWs, or MAC-RAWs. PBS-RAWs were injected both intraperitoneally (i.p.) and intravenously (i.v.) to treat the local and blood bacteria. For the MAC-RAWs, three administration methods were conducted with the same total cell number: i.p. injection alone, i.v. injection alone, and i.p.+i.v. injections. Because the lethality in immunosuppressed sepsis is associated with ineradicable pathogens, the bacterial colony forming units (CFUs) in the mouse blood were measured after 24 h of cell transfer. Similar to PBS treatment, PBS-RAWs did not reduce bacterial burden in the blood, while MAC-RAWs administrated via i.p. injection alone (MAC-RAWs (i.p.)) and MAC-RAWs administrated via both i.p. and i.v. injection (MAC-RAWs (i.p.+i.v.)) significantly reduced the bacterial CFUs in the blood (P<0.01 and P<0.001, respectively) (
In the MAC-RAW (i.p.+i.v.) group, bacteria in the blood were not detectable in 3 out of the 7 survived mice at the 480 h (
The bactericidal activity using primary bone marrow derived macrophages (BMDMs) was evaluated next, which are translatable for clinical applications. BMDMs were generated from the mouse bone marrow as reported in the literature and confirmed ˜83.5% cells were F4/80 positive. (
After validating the results in vitro, MAC-BMDMs were applied to mice with immunosuppression to treat MDRSA induced sepsis. Based on the data from the RAW264.7 cells, MAC-BMDMs were administrated to mice via both i.p. and i.v. injections. Analysis of the blood at 24 h showed that MAC-BMDMs were more effective in eliminating bacteria than PBS-BMDMs (P<0.001,
Bacteria are distributed into multiple organs within a few hours of infections. To profile the biodistribution of these macrophages, mRNA encoding firefly luciferase was delivered into the BMDMs (FLuc-BMDMs). Six hours post i.p.+i.v. injections of the FLuc-BMDMs, the luminescence intensity was measured in the following major organs usually infected by bacteria in this mouse model: peritoneal cavity, spleen, liver, lung, kidney, heart, and blood. The results showed a similar biodistribution in healthy and sepsis mice except that a higher luminescence intensity was detected in the lung of sepsis mice than that in healthy mice (
Because sepsis hosts are usually exposed to mixed bacterial infections, a formidable challenge for curing sepsis, a mouse sepsis model was established with infections by both MDRSA and MDR E. coli. Because the mixed infections lead to more severe symptoms compared to single bacterial infection, mice were infected with a total 2×108 CUFs of bacteria, 2.5 fold less of the bacteria dose than that in the single infection model. Treatment with MAC-BMDMs significantly reduced 43% (P<0.01) and 39% (P<0.05) bacterial burden in the blood compared to the treatment with PBS and PBS-BMDMs, respectively (
What is shown herein are macrophages loaded with antimicrobial peptides/cathepsin B in the lysosomes (MACs) by delivery of mRNA encapsulated in the vitamin C lipid nanoparticles (VcLNPs). The data show that adoptive transfer of MACs beneficially reduced the bacterial burden and improved survival in multidrug resistant (MDR) bacteria induced sepsis mice with immunosuppression by restoring innate immune defense, preventing bacterial immune evasion, and killing MDR bacteria. MACs were efficacious against sepsis induced by mixed strains of MDR bacteria. The superior therapeutic effects of the MACs administrated via i.p.+i.v. injections than i.p. or i.v. injection alone can be due to the infection process of sepsis. In the sepsis mice, bacteria are transported by the lymphatic system to the blood within a short period of time and then distributed to other organs via the blood circulation. The administration of i.v.+i.p. contributes to eliminating the bacteria invaded into the blood or colonized in the peritoneal cavity, leading to reduced bacterial CFUs and improved survival. When sepsis is diagnosed in an early stage, autologous macrophages can be prepared and engineered in approximately seven days. Next, these autologous MACs can be transferred back to patients with immunosuppression, which are the majority of sepsis patients under current treatment guidelines. Additionally, with the advance of induced pluripotent stem cells (iPSCs) technologies, allogeneic “universal” macrophages can be manufactured, enabling the iPSCs-derived MACs to become an off-the-shelf therapy for broad clinical applications including sepsis. Overall, adoptive transfer of MACs provides a curable strategy for patients with sepsis and MDR bacterial infection in the future.
Chemicals and reagents. The following agents were purchased from Sigma-Aldrich, including cholesterol, gentamicin, cathepsin B (CatB) inhibitor II, cyclophosphamide (CY), and accutase cell detachment solution. The following agents were obtained from Thermo Fisher Scientific, including F4/80 monoclonal antibody, recombinant murine macrophage colony-stimulating factor (M-CSF), and LysoTracker® DND-99. 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) was purchased from Avanti Polar Lipids. Bright-Glo luciferase substrate was purchased from Promega.
Cells and bacteria. The RAW264.7 cell line was obtained from the American Type Culture Collection (ATCC) and cultured in Dulbecco's modified Eagle's medium (DMEM, ATCC) with 10% fetal bovine serum (Gibco, Invitrogen). Murine bone marrow-derived macrophages (BMDMs) were obtained by adaptation of previous procedures, cultured in DMEM containing 10% fetal bovine serum and 100 ng/ml M-CSF. MDR Staphylococcus aureus (MDRSA, ATCC BAA-44) were grown in trypticase soy agar or broth at 37° C. with aeration. According to the data from ATCC, the MDRSA are resistant to the following antibiotic: amoxicillin/clavulanic acid, penicillin, ciprofloxacin, cephalothin, doxycycline, gentamicin, erythromycin, imipenem, methicillin, tetracycline, oxacillin, azithromycin, clindamycin, ceftriaxone, rifampin, amikacin, and tobramycin. Multidrug resistant Escherichia coli (MDR E. coli, ATCC BAA-2340) was grown in nutrient agar or nutrient broth (BD Biosciences) at 37° C. with aeration. According to the data from ATCC, the MDR E. coli are resistant to the following: amoxicillin/clavulanic acid, ticarcillin, piperacillin, ampicillin/sulbactam, cefalotin, cefuroxime, cefotetan, cefpodoxime, cefotaxime, ceftizoxime, cefazolin, cefoxitin, ceftazidime, ceftriaxone, cefepime, doripenem, meropenem, ertapenem, imipenem, nalidixic acid, moxifloxacin, norfloxacin, ciprofloxacin, levofloxacin, tobramycin, aztreonam, and trimethoprim/sulfamethoxazole.
Synthesis of vitamin-derived lipids. Compound 1, vitamin B3 derivative, vitamin C derivative, and vitamin H (also called vitamin B7) derivative, were synthesized according to the methods reported previously.
Preparation and characterization of VLNP. mRNAs used herein were constructed by the mRNA platform based on the reported method. The preparation of mRNA LNPs was reported previously. Briefly, newly synthesized vitamin-derived lipids were formulated with DOPE, Cholesterol as well as firefly luciferase (FLuc) mRNA by pipetting for in vitro screenings or by a microfluidic mixing device for ex vivo studies. The NanoZS Zetasizer (Malvern) was used to measure the size and zeta potential. The entrapment efficiency was measured by the Ribogreen assay. The morphology of VcLNPs was examined on Thermo Scientific™ Glacios™ CryoTEM using the methods described before. In order to effectively deliver mRNA into macrophages, an initial screening was performed using the five vitamin-lipid nanoparticles (VLNPs), Lipofectamine 3000, and electroporation. Newly synthesized vitamin-derived lipids were formulated with DOPE, Cholesterol (Lipid:DOPE:Cholesterol=20:30:40, mole ratio) as well as FLuc mRNA (Lipid:mRNA=10:1, mass ratio) in the initial screening. Delivery efficiency of mRNA was determined by the luciferase expression assay. Next, the dynamics of FLuc expression followed by two rounds of characterization were carried out after the initial screening. Briefly, VcLNPs from A-1 to A-16 were prepared based on the orthogonal array design table L16 (4)4 and the top formulation was predicted by the FLuc expression data. After the top formulation was validated, the second round of characterization focused on fine tuning the mass ratio of Vc-Lipid:mRNA. Electroporation for macrophages was performed using Nucleofector kit (Lonza) and the suggested protocols by the Nucleofector 2b Device.
Cellular uptake and endosomal escape. RAW264.7 cells were seeded in a 6-well plate at 105 cells/well and cultured for 24 h. Then, cells were treated by FLuc mRNA and Alexa-Fluor 647-labeled RNA (1:1, weight ratio) using Lipofectamine 3000, VcLNPs, or electroporation. After 3 h incubation, cellular uptake was quantified by a flow cytometer (LSRII, BD). To study endocytosis pathways of VcLNPs, cellular uptake assay was performed in the presence of different endocytotic inhibitors, including 5-(N-methyl-N-isopropyl)amiloride (EIPA), methyl-beta-cyclodextrin (MPCD), and chlorpromazine hydrochloride (CPZ). For the endosomal escape assay, 2×104 cells were plated in an imaging dish (ibidi) for 24 h, and then 150 μg/mL of calcein was added to the cells with or without VcLNPs containing Alexa-Fluor 647-labeled RNA for 6 h at 37° C. After washing with PBS to remove extracellular calcein and nanoparticles, cells were lively imaged under the Nikon A1R Live Cell Imaging Confocal Microscope via the 487 nm and 647 nm lasers.
Analysis of lysosome co-localization. To test whether AMP-CatB can specifically accumulate in the lysosomes, the eGFP-CatB mRNA were prepared and delivered into the RAW264.7 cells using VcLNPs. Then, lysosomes were stained with LysoTracker® Red DND-99, a well-established lysosome probe. The co-localization of eGFP-CatB and LysoTracker® Red DND-99 was analyzed under the Nikon A1R Live Cell Imaging Confocal Microscope via the 487 nm and 561 nm lasers.
Cytotoxicity of vitamin C lipid nanoparticles in BMDMs. Cytotoxicity of VcLNPs in BMDMs was examined by an MTT assay. 2×104 BMDMs were seeded into each well of 96-well plates in 100 μL of growth medium. After 12 h of incubation with PBS, free AMP-CatB mRNA, empty VcLNPs, AMP-CatB mRNA VcLNPs/CatB inhibitor II, and AMP-CatB mRNA VcLNPs, MTT solution was added. After additional 4 h incubation, 100 uL of 10% SDS-HCl was added into each well. The purple formazan was dissolved overnight and the absorbance was measured at 570 nm by a plate reader.
In vitro antimicrobial assay. The intracellular antimicrobial assay was conducted according to the reported method. Briefly, after treated by PBS, free AMP-CatB mRNA, empty VcLNPs, AMP-CatB mRNA VcLNPs/CatB inhibitor II, and AMP-CatB mRNA VcLNPs, the RAW264.7 cells or BMDMs were incubated with MDRSA or MDR E. coli at multiplicity of infection (MOI) of 25 for 120 min. After washing by PBS, medium containing gentamicin (100 ρg/mL) was supplemented and the cells were incubated for additional 1 h to clear extracellular bacteria. At different time points, cells were washed by PBS and lysed with 0.1% Triton-X 100. Lastly, the lysates were cultured on the nutrient agar or trypticase soy agar for counting bacterial colony forming units (CFUs).
In vivo treatment of MDR induced sepsis mice with immunosuppression. All mouse experiments were performed under the protocols approved by the Institutional Animal Care and Use Committee (IACUC) of the Ohio State University. C57BL/6 mice (6-7 weeks) were purchased from the Jackson Lab. The immunosuppressed model of sepsis was conducted as the method in the literature. Briefly, C57BL/6 mice were intraperitoneally injected cyclophosphamide (CY) at a dosage of 100 mg/kg for 3 consecutive days before bacterial infection. Their immunocompromised states were assessed by monitoring the body weights (BWs), counting white blood cells (WBCs) by a hemocytometer, and counting lymphocytes (LYMs) by the Kwik-Diff stain. Mice were then intraperitoneally inoculated with 0.1 ml of bacterial suspension (5×108 CFUs/mouse for MDRSA infections and 2×108 CFUs/mouse for mixed MDRSA and MDR E. coli infections). Afterwards, the mice were administrated by 0.2 mL PBS or 0.2 mL cell suspension (total 2 million cells/mouse). After 24 h infection, the blood was collected from the facial vein to quantify bacterial CFUs. The survival of mice was assessed every 12 h within the first 6 days and then every 24 h for the following 24 days based on 20% loss of BW and the early removal criteria. After 30 days, blood was collected from all survived mice and the number of WBC and LYM were counted. After that, the mice were euthanized and major organs (heart, liver, spleen, lung, and kidneys) were aseptically homogenized to quantify the bacterial CFUs.
Biodistribution of macrophages and bacteria. The biodistribution of macrophages was performed on both healthy and sepsis C57BL/6 mice (6-7 weeks). In this experiment, mRNA encoding firefly luciferase was first delivered into the BMDMs (FLuc-BMDMs) for 12 h. Next, each mouse was administrated 0.2 mL PBS or 0.2 mL cell suspension (total 4 million cells) via the intraperitoneal (i.p.)+intravenous (i.v.) injection. After 6 h, mice were i.p. injected with 150 L of the D-luciferin substrate (30 mg/mL) and then euthanized by CO2 after 8 min of injection. Bioluminescence signals in the blood, peritoneal fluid, and major organs were immediately measured using a Xenogen IVIS imaging system (Caliper, Alameda, Calif.). The bacterial CFUs in the blood, peritoneal fluid, and major organs of sepsis mice were quantified after 6 h of infection.
Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.
Those skilled in the art will appreciate that numerous changes and modifications can be made to the preferred embodiments of the invention and that such changes and modifications can be made without departing from the spirit of the invention. It is, therefore, intended that the appended claims cover all such equivalent variations as fall within the true spirit and scope of the invention.
This application claims the benefit of U.S. Provisional Patent Application Ser. No. 62/898,846 filed Sep. 11, 2019, which is expressly incorporated herein by reference.
This invention was made with government support under grant number R35GM119679 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2020/050366 | 9/10/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62898846 | Sep 2019 | US |
