Throughout this application various publications are referred to in brackets. Full citations for these references may be found at the end of the specification. The disclosures of these publications, and of all patents, patent application publications and books referred to herein, are hereby incorporated by reference in their entirety into the subject application to more fully describe the art to which the subject invention pertains.
The discovery of a functional RNAi pathway in mammals has provided a powerful tool for reverse genetics as a method for identifying gene function. The potential of RNAi to silence any gene has also made it an attractive therapeutic modality [1]. However, the main obstacle to RNAi as a clinical agent is delivery. A delivery vehicle must transport its cargo through the body and, upon encountering cells must cross the plasma membrane and gain access to the cytosolic compartment, where the RNAi machinery resides. Furthermore, to be useful as a clinical reagent, ease of formulation and administration to the patient, overall cost and any associated toxicities must be considered.
Due to their ability to knockdown expression of any gene, siRNAs have been heralded as ideal candidates for treating a wide variety of diseases including “undruggable” targets. However, the therapeutic potential of siRNAs is severely limited by a lack of effective delivery vehicles. Recently, lipid nanoparticles (LNPs), containing ionizable cationic lipids, such as DLinDMA have been used to deliver siRNAs to the liver.
Currently, lipid nanoparticles (LNPs) are one of the most advanced delivery platforms being developed for systemic RNAi delivery [2]. LNPs contain ionizable cationic lipids that bind nucleic acids via electrostatic interactions, resulting in efficient siRNA encapsulation and a uniform LNP population of ˜100 nM diameter. Studies have also shown that following cellular uptake of the LNP, the ionizable lipid promotes siRNA escape from the endosome to the cytosol [3]. These properties make LNPs an attractive platform for siRNA delivery in vivo. LNPs are also associated with minimal toxicity, including little induction of pro-inflammatory cytokines following administration of physiologically relevant doses [4]. LNPs containing the ionizable lipid DLinDMA have been used for systemic delivery of siRNAs in mice, non-human primates (NHPs) and are in clinical trials [5]. Biodistribution studies show that systemic injection of LNPs results in accumulation mainly in the liver and spleen, and these LNPs are being evaluated in the clinic for conditions that require hepatic gene silencing [5,6]. The ED50 (the dose required to observe 50% gene silencing) in liver-expressed genes following DLinDMA-formulated LNPs is ˜1 mg/kg3. Modifications to DLinDMA have resulted in identification of more potent lipids. Injection of murine clotting factor VII-specific siRNAs encapsulated in LNPs formulated with the lipid 1,2-dilinoleyl-4-(2-dimethylaminoethyl)[1,3]-dioxolane (DLinKC2-DMA), resulted in gene-specific silencing with an ED50 of 0.1 mg/kg3. Additional modifications to this lipid identified the dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA) with a potency of 0.01 mg/kg when incorporated into siRNA-containing LNPs [2].
These LNPs provide a well-characterized, potent, minimally toxic system for siRNA delivery to cells in the liver and spleen. Splenic delivery makes these LNPs attractive candidates for silencing in immune cell types that are well represented in the spleen such as T cells, B cells, macrophages (MOs) and dendritic cells (DCs), thereby opening up the possibility of using LNPs for delivery of immune-modulatory siRNAs. Recently, Cullis and colleagues used a range of LNPs, including those containing DLinDMA and DLin-KC2-DMA, to determine efficacy of RNAi-mediated gene silencing in MOs and DCs in vitro and in vivo. Systemic injection of DLin-KC2-DMA-containing LNPs encapsulating an siRNA specific for GAPDH resulted in uptake by MOs and DCs isolated from the peritoneum and spleen. LNP uptake was shown to induce RNAi-mediated gene-specific silencing [7]. This study, together with a complementary study [8], indicates that gene silencing in immune cells using the LNP technology is feasible. However, the main target for systemically administered LNPs is the liver. Using such a non-targeted approach suffers from potential drawbacks including the possibility of toxicity if genes are silenced in the liver as well as in the desired splenic immune cell population(s). Furthermore, these studies showed a requirement for injection of relatively high doses of siRNA to achieve effective gene silencing in splenic DC and/or MO populations (3-5 mg/kg [7]).
The present invention provides a facile method of adapting LNPs for specifically targeted siRNA delivery.
The present invention provides improved vehicles and methods for delivering siRNAs or other small cargoes.
This invention provides a composition comprising a lipid nanoparticle which comprises a lipid bi-layer, and a lipid having a single-chain variable fragment (scFv) attached thereto via a hydrophilic polymer wherein the scFv is directed against an antigen present on the surface of a cell.
Also provided is a method of delivering a small molecule, a nucleic acid, an antigen or a vaccine to a cell expressing a cell-surface antigen, comprising contacting the cell with any of the compositions described herein, wherein the cell surface antigen is the antigen to which the scFv is directed, in an amount effective to deliver a small molecule, a nucleic acid, an antigen or a vaccine to a cell expressing a cell-surface antigen.
Also provided is a method of increasing the efficacy of a predetermined dose of a nucleic acid, such as an RNAi nucleic acid, administered to a subject comprising delivering the nucleic acid to a cell expressing a cell-surface antigen, comprising contacting the cell with any of the compositions described herein, wherein the cell surface antigen is the antigen to which the scFv is directed, in an amount effective to deliver the nucleic acid to a cell expressing a cell-surface antigen and thereby increase the efficacy of the predetermined dose of a nucleic acid.
Also provided is a method of treating an immune system disorder in a subject comprising administering to the subject an amount of any of the compositions described herein, wherein the siRNA is an immunomodulatory siRNA or the small organic molecule is an immunomodulatory small organic molecule. In embodiment, the subject is a human.
The present invention provides improved vehicles and methods for delivering siRNAs or other small cargoes.
This invention provides a composition comprising a lipid nanoparticle which comprises a lipid bi-layer, and a lipid having a single-chain variable fragment (scFv) attached thereto via a hydrophilic polymer wherein the scFv is directed against an antigen present on the surface of a cell.
Single-chain variable fragments are known in the art as a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, connected with a short linker peptide of ten to about 25 amino acids. The linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the VH with the C-terminus of the VL, or vice versa. In an embodiment, the scFv is monovalent. In an embodiment, the scFv is bivalent, directed against two different cell-surface antigens (e.g. two of CD80, CD86 and CD40).
In an embodiment, the composition comprises an ionizable cationic lipid. In an embodiment, the composition comprises distearoylphosphatidylcholine (DSPC). In an embodiment, the composition comprises the ionizable cationic lipid comprises 1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA).
In an embodiment, the composition comprises one or more of 1,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3] -dioxolane (DLinKC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl phosphatidyl ethanolamine, 16-O-dimethyl phosphatidyl ethanolamine, 18-1-trans phosphatidyl ethanolamine, 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), and N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), and 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLendMA).
In an embodiment, the lipid having the hydrophilic polymer attached comprises distearoyl-phosphatidyl-ethanolamine-polyethylene glycol (DSPE-PEG). In an embodiment, the lipid nanoparticle comprises at least one interior lumen. In an embodiment, the lipid bi-layer encapsulates at least one interior lumen.
In an embodiment, (i) a nucleic acid molecule, (ii) a small organic molecule, (iii) a vaccine or (iv) an antigen is present in the lipid nanoparticle. In an embodiment, (i) a nucleic acid molecule, (ii) a small organic molecule, (iii) a vaccine or (iv) an antigen is present in an interior lumen of the lipid nanoparticle.
In an embodiment, the nucleic acid molecule is present and is a siRNA, shRNA, plasmid, mRNA, miRNA or ncRNA. In embodiment, the siRNA or shRNA is not 2′-fluoro modified.
In embodiment, the siRNA or shRNA is 2′-OMe modified.
In an embodiment, the scFv is an anti-DEC-205 scFv, anti-Cd11c scFv, anti-33d1 scFv or anti-Clec9A (DNGR1) scFv In an embodiment, the lipid nanoparticle comprises DSPC, DLinDMA, DSPE-PEG-malemide and cholesterol.
In an embodiment, the composition comprises 15% DSPC, 40% DLinDMA, 5% DSPE-PEG-malemide and 40% cholesterol.
In an embodiment, the interior lumen is aqueous.
In an embodiment, the hydrophilic polymer of the lipid having the hydrophilic polymer attached is PEG, and the scFv is attached to the PEGylated lipid via a covalent bond to a maleimide bonded to the PEG. In an embodiment, the bond is a —C—S— bond. In an embodiment, the scFv comprises a C-terminal cysteine, a Vl and a Vh, and the C-terminal cysteine is attached to the Vl or the Vh of the scFv. In an embodiment, the bond is between the maleimide attached to the PEG and a C-terminal cysteine of the scFv.
In an embodiment, the scFv is attached to the hydrophilic polymer of the lipid via a azide/alkyne covalent bond or an amide bond.
In an embodiment, the cell is a mammalian cell. In an embodiment, the mammalian cell is human. In an embodiment, the cell is a dendritic cell.
Also provided is a method of delivering a small molecule, a nucleic acid, an antigen or a vaccine to a cell expressing a cell-surface antigen, comprising contacting the cell with any of the compositions described herein, wherein the cell surface antigen is the antigen to which the scFv is directed, in an amount effective to deliver a small molecule, a nucleic acid, an antigen or a vaccine to a cell expressing a cell-surface antigen.
A method of increasing the efficacy of a predetermined dose of a nucleic acid administered to a subject, such as an RNAi nucleic acid, comprising delivering the nucleic acid to a cell expressing a cell-surface antigen, comprising contacting the cell with any of the compositions described herein, wherein the cell surface antigen is the antigen to which the scFv is directed, in an amount effective to deliver a nucleic acid to a cell expressing a cell-surface antigen and thereby increase the efficacy of the predetermined dose of a nucleic acid. In an embodiment, the nucleic acid is a siRNA or shRNA.
Also provided is a method of treating an immune system disorder in a subject comprising administering to the subject an amount of any of the compositions described herein, wherein the siRNA is an immunomodulatory siRNA or the small organic molecule is an immunomodulatory small organic molecule. In embodiment, the subject is a human.
In an embodiment, the immune system disorder is an autoimmune disease. Autoimmune diseases include acute disseminated encephalomyelitis (ADEM), alopecia areata, antiphospholipid syndrome, autoimmune cardiomyopathy, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome, autoimmune peripheral neuropathy, autoimmune pancreatitis, autoimmune polyendocrine syndrome, autoimmune progesterone dermatitis, autoimmune thrombocytopenic purpura, autoimmune urticarial, autoimmune uveitis, Behcet's disease, celiac disease, Chagas disease, cold agglutinin disease, Crohn's disease, dermatomyositis, diabetes mellitus type 1, eosinophilic fasciitis, gastrointestinal pemphigoid, Goodpasture's syndrome, Grave's syndrome, Guillain-Barré syndrome, Hashimoto's encephalopathy, Hashimoto's thyroiditis, lupus erythematosus, Miller-Fisher syndrome, mixed connective tissue disease, myasthenia gravis, pemphigus vulgaris, pernicious anaemia, polymyositis, psoriasis, psoriatic arthritis, relapsing polychondritis, rheumatoid arthritis, rheumatic fever, Sjogren's syndrome, temporal arteritis, transverse myelitis, ulcerative colitis, undifferentiated connective tissue disease, vasculitis, and Wegener's granulomatosis.
In an embodiment of the methods, the cell is a dendritic cell. In an embodiment of the methods, the nucleic acid is delivered.
In embodiment of the methods or compositions, the nucleic acid is an RNAi nucleic acid. In an embodiment of the methods, the nucleic acid is a siRNA, shRNA, plasmid, mRNA, miRNA or ncRNA. In an embodiment, the siRNA or shRNA is not 2′-fluoro modified. In an embodiment, the siRNA or shRNA is 2′-fluoro modified. In embodiment, the siRNA or shRNA is 2′-OMe modified. In embodiment, the siRNA or shRNA is not 2′-OMe modified. In embodiment, the nucleic acid is an immune-modulatory siRNA. In embodiment of the methods or compositions, the RNAi nucleic acid is directed against a nucleic acid encoding CD80 (for example, encoding NCBI Reference Sequence: NP—005182.1), CD40 (for example, encoding NCBI Reference Sequence: NP—001241.1), CD86 (for example, encoding NCBI Reference Sequence: NP—001193853.1), MyD88, TRIF, TRAF6, IRAK, RIG-1, MDA-5, p38, ERK, jnk, PDL1, or IRAK-M. In a preferred embodiment, said RNAi nucleic acid is an siRNA. In embodiment, the nucleic acid is a gene. In embodiment, said gene is a human gene.
In an embodiment, the siRNA or shRNA is directed against a mammalian CD80. In an embodiment, the siRNA or shRNA is directed against a mammalian CD86. In an embodiment, the siRNA or shRNA is directed against a mammalian CD40.
In an embodiment, the siRNA (small interfering RNA) as used in the methods or compositions described herein comprises a portion which is complementary to an mRNA sequence encoded by NCBI Reference Sequence for the stated genes/proteins. In an embodiment, the siRNA comprises a double-stranded portion (duplex). In an embodiment, the siRNA is 20-25 nucleotides in length. In an embodiment the siRNA comprises a 19-21 core RNA duplex with a one or 2 nucleotide 3′ overhang on, independently, either one or both strands. In an embodiment, the overhang is UU. The siRNA can be 5′ phosphorylated or not and may be modified with any of the known modifications in the art to improve efficacy and/or resistance to nuclease degradation. In a non-limiting embodiment, the siRNA can be administered such that it is transfected into one or more cells.
In one embodiment, a siRNA of the invention comprises a double-stranded RNA comprising a first and second strand, wherein one strand of the RNA is 80, 85, 90, 95 or 100% complementary to a portion of an RNA transcript of a gene (e.g. encoding human CD80, CD40, or CD86). Thus, in an embodiment, the invention encompasses an siRNA comprising a 19, 20 or 21 nucleotide first RNA strand which is 80, 85, 90, 95 or 100% complementary to a 19, 20 or 21 nucleotide portion, respectively, of an RNA transcript of a gene (e.g. encoding human CD80, CD40, or CD86). In embodiment, the second RNA strand of the double-stranded RNA is also 19, 20 or 21 nucleotides, respectively, a 100% complementary to the first strand. In another embodiment, a siRNA of the invention comprises a double-stranded RNA wherein one strand of the RNA comprises a portion having a sequence the same as a portion of 18-25 consecutive nucleotides of an RNA transcript of a gene (e.g. encoding human CD80, CD40, or CD86). In yet another embodiment, a siRNA of the invention comprises a double-stranded RNA wherein both strands of RNA are connected by a non-nucleotide linker. Alternately, a siRNA of the invention comprises a double-stranded RNA wherein both strands of RNA are connected by a nucleotide linker, such as a loop or stem loop structure.
In one embodiment, a single strand component of a siRNA of the invention is from 14 to 50 nucleotides in length. In another embodiment, a single strand component of a siRNA of the invention is 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides in length. In yet another embodiment, a single strand component of a siRNA of the invention is 21 nucleotides in length. In yet another embodiment, a single strand component of a siRNA of the invention is 22 nucleotides in length. In yet another embodiment, a single strand component of a siRNA of the invention is 23 nucleotides in length. In one embodiment, a siRNA of the invention is from 28 to 56 nucleotides in length.
In an embodiment, an siRNA of the invention comprises at least one 2′-sugar modification. In an embodiment, an siRNA of the invention comprises at least one 2′-O-fluorine modification. In an embodiment, an siRNA of the invention comprises at least one 2′-O-methyl modification. In an embodiment, an siRNA of the invention comprises at least one nucleic acid base modification. In an embodiment, an siRNA of the invention comprises at least one phosphate backbone modification.
In one embodiment, RNAi inhibition of the relevant gene is effected by a short hairpin RNA (“shRNA”). The shRNA is introduced into the cell by transduction with a vector inside the liposome. In an embodiment, the vector is a lentiviral vector. In an embodiment, the vector comprises a promoter. In an embodiment, the promoter is a U6 or H1 promoter. In an embodiment the shRNA encoded by the vector is a first nucleotide sequence ranging from 19-29 nucleotides complementary to the target gene, in the present case relevant gene. In an embodiment the shRNA encoded by the vector also comprises a short spacer of 4-15 nucleotides (a loop, which does not hybridize) and a 19-29 nucleotide sequence that is a reverse complement of the first nucleotide sequence. In an embodiment the siRNA resulting from intracellular processing of the shRNA has overhangs of 1 or 2 nucleotides. In an embodiment the siRNA resulting from intracellular processing of the shRNA overhangs has two 3′ overhangs. In an embodiment the overhangs are UU. The shRNA may comprise one or more 2′-O-fluorine modification and/or one or more 2′-O-methyl modification.
In an embodiment, the delivery of the small molecule or nucleic acid is selective to a cell expressing the cell-surface antigen for which the scFv is directed. In an embodiment, the delivery of the small molecule or nucleic acid is selective to a cell expressing the cell-surface antigen for which the scFv is directed if the small molecule or RNAi molecule is delivered to a cell expressing the cell-surface antigen at a rate 5-fold or greater than it is to a cell not expressing the cell-surface antigen under otherwise the same conditions. In an embodiment, the delivery of the small molecule or nucleic acid is selective to a cell expressing the cell-surface antigen for which the scFv is directed if the small molecule or RNAi molecule is delivered to a cell expressing the cell-surface antigen at a rate 10-fold or greater than it is to a cell not expressing the cell-surface antigen under otherwise the same conditions. In an embodiment, the delivery of the small molecule or nucleic acid is selective to a cell expressing the cell-surface antigen for which the scFv is directed if the small molecule or RNAi molecule is delivered to a cell expressing the cell-surface antigen at a rate 20-fold or greater than it is to a cell not expressing the cell-surface antigen under otherwise the same conditions. In an embodiment, the delivery of the small molecule or nucleic acid is selective to a cell expressing the cell-surface antigen for which the scFv is directed if the small molecule or RNAi molecule is delivered to a cell expressing the cell-surface antigen at a rate 35-fold or greater than it is to a cell not expressing the cell-surface antigen under otherwise the same conditions.
In an embodiment, the lipid nanoparticles of the invention comprise:
In a preferred embodiment t, the lipid nanoparticles of the invention comprise:
Preferred PEG sizes are PEG MW 2000. In an embodiment the PEG is MW 500-5000. Alternatively, PEG can be replaced in the compositions and methods described herein, mutatis mutandis, by other hydrophilic polymers.
Malemide linkages as described in the compositions herein can, alternatively, be substituted with amides, Click chemistry such as azide/alkyne, Staudinger chemistry and reactive animation.
All combinations of the various elements described herein are within the scope of the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
This invention will be better understood from the Experimental Details, which follow. However, one skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the invention as described more fully in the claims that follow thereafter.
Injection of siRNAs encapsulated in DLinDMA-based LNPs, decorated with a single chain antibody (scFv) specific for DEC-205 targeting moiety, resulted in delivery of the siRNA cargo specifically to DEC-205-positive cells. Uptake of siRNA led to gene-specific knockdown via the RNAi pathway. The results suggest that targeting the siRNA to a specific cell subset can reduce the effective dose by at least one log when compared with non-targeted counterparts. Furthermore, restricting siRNA uptake to distinct cell subsets provides a means of limiting undesirable effects in bystander cells.
To achieve DC-directed gene silencing LNPs were modified by attaching a single chain antibody (scFv) via the PEG linker. The scFv specific for the DEC205 receptor was chosen for various reasons. First, DEC205 is expressed at high levels on DCs [9]. Second, uptake of an antigen coupled to anti-DEC205 antibody mediates effective cross-presentation, suggestive that cargoes taken up through the DEC205 pathway gain access to the cytosolic compartment [10]. As much of the RNA-induced silencing complex (RISC) resides in the cytosol this is an important consideration for choosing an appropriate receptor for shuttling siRNAs into a cell.
Systemic injection of scFv DEC205-decorated LNPs resulted in uptake that was limited to DEC205+ cells. Furthermore, delivery of siRNAs specific for CD80, a costimulatory molecule expressed by activated DCs, resulted in a reduction of expression by at least 50%.
The observed gene knockdown is likely mediated via the RNAi pathway, and this can be confirmed using 5′ RACE (to detect appropriately sized mRNA cleavage products). Anti-CD80 transfection of siRNA resulted in protein knockdown via RNAi in vitro.
This system can be extended to include additional gene targets of interest. Here, DC costimulatory molecules are targeted as a method to treat autoimmune diseases. This study has also identified siRNAs that effectively reduce CD86 and CD40 expression in vitro (data not shown). These sequences can be delivered by the LNPs disclosed herein in vivo.
Targeting DCs is significant because of the immune-modulatory capacity of this cell type. Knocking down inhibitory genes in DCs is useful for potentiating immune responses e.g. for use as a prophylactic or therapeutic vaccine. Conversely, silencing stimulatory genes is useful for treatment of autoimmune diseases.
LNPs containing siRNAs specific either for CD80 or a control (non-target) sequence were synthesized. The formulation of the LNPs are DSPC:DLinDMA:DSPE-PEG:cholesterol 15:40:5:40. Anti-DEC205 (murine) scFv with a cysteine at its C-terminal end was attached to the lipid, DSPE-PEG by a maleimide group. A schematic is shown in
Following synthesis, LNPs are subject to quality control, to ensure consistency between batches. LNP diameter (dynamic light scattering) and the level of siRNA incorporation and scFv binding to DSPE-PEG were measured. Analysis of a typical set of LNPs is shown in
To show that scFv-LNPs selectively bound to DEC205, scFv-LNPs that encapsulated Cy3-labeled siRNA were cultured with CHO cells that stably expressed DEC205 or with parental CHO cells. As seen in
Having shown that the scFv-LNPs are specifically taken up by DEC205+ cells in vitro (including by DCs, data not shown), one goal was to determine whether this observation extended in vivo. C57L6 mice were injected systemically (intravenous, i.v.), with ˜0.7 mg/kg scFv-LNPs. These LNPs were synthesized with the inclusion of DOPE-rhodamine B sulfonyl (0.1%) to track lipid uptake. After 24 hrs, spleens were removed and uptake and distribution of LNPs was determined by flow cytometry. As seen in
Having demonstrated specific uptake of scFv-LNPs by DCs in vivo, one goal was to show that this uptake was competent for specific gene silencing. LNPs (containing rhodamine B-labeled DOPE for tracking) were complexed with siRNAs specific for CD80 and these were injected into mice at 0.8 mg/kg. After 24 hrs LNP uptake and knockdown of CD80 was determined. As seen in
In further experiments, 2′-OMe modification of siRNAs was found to diminish dendritic cell activation following delivery via scFv DEC205-LNPs (see
Confirming specificity and uptake pathway of DEC205-LNPs: Bone marrow-derived dendritic cells (BMDCs) derived from wild-type (WT) or DEC205-knockout (DEC205−/−) mice were used to show that LNP uptake required the DEC205 receptor. As seen in
Expanding the specific genes that have been targeted to demonstrate generality of the approach: Initially it was shown that injection of DEC205-LNPs that contained siRNAs targeting CD80 resulted in uptake by DEC205+ DCs, leading to reduced expression of CD80 protein. It was further shown that following injection of DEC205-LNPs containing CD80-specific siRNAs, CD80 mRNA is reduced and that this mRNA knockdown occurs via an RNA interference (RNAi)-specific mechanism (
As a use for these targeted LNPs is as a treatment for autoimmune disease, analysis was extended to another costimulatory molecule, CD86. As seen in
Using chemically-modified siRNAs to minimize unwanted immune responses: Injection of mice with DEC205-LNPs containing unmodified siRNAs resulted in DC activation. Therefore, siRNAs were encapsulated that contained either 2′-O-methyl (2′-Me) or 2′-fluorine (2′-F) substitutions on their backbone, into the DEC205-LNPs (13).
Human- and murine-based experimental assays show different sensitivities following exposure to modified and unmodified siRNAs: As it was observed that delivery of unmodified siRNAs, and some modified siRNAs, to murine DCs in situ results in their activation, it was determined whether a simpler in vitro assay tconfimred whether an siRNA may cause non-specific immune activation. Although the ability of some siRNAs to elicit immune activation was abrogated following backbone modification (
It was tested how a co-culture of LNP-encapsulated siRNAs with BMDCs affected DC activation. Both DEC-coated and uncoated LNPs were tested, which are taken up by cells similarly to liposomes used routinely as transfection reagents. No detectable upregulation of CD80, CD86 or CD40 was observed following culture with any of the LNP complexes tested (
As the ultimate goal is to use LNPs as a delivery agent in humans, we switched to analyzing LNPs in human cell systems. Our scFv targets murine DEC205, therefore non-targeted (uncoated) LNPs were used in these assays. First, human peripheral blood mononuclear cells (PBMCs) were co-cultured with LNPs containing either unmodified, 2′-F or 2′-Me modified siRNAs. As seen in
DEC205-LNPs containing siRNAs mediate functional gene-specific knockdown:
This application claims benefit of U.S. Provisional Application No. 61/833,072, filed Jun. 10, 2013, the contents of which are hereby incorporated by reference.
This invention was made with government support under grant numbers R21 AI093539-01 and R01 AI099567-01 awarded by the National Institutes of Health. The government has certain rights in the invention.
Number | Date | Country | |
---|---|---|---|
61833072 | Jun 2013 | US |