Patent Application
20230059921
Despite increasing advancements in gene sequencing, cell surface proteomics, and single-cell genomics, conversion of these data into personalized therapies has remained limited in the realm of cell-specific targeted delivery. Gene therapy and targeted nanomedicine approaches, in particular, have been in great need of improvements to cell-specific delivery technologies. Given the broadly varying expression profiles on various cells, tissues and organs within healthy and diseased physiology, there is a need for “diagnostically-responsive” medicine that can target a given cell/tissue/organ and present a precise set of instructions to that cell/tissue/organ.
One major hurdle for the successful treatment of cancer is that cancer manifests in many forms across all organ systems with each exhibiting diverse physiology. As such, response to treatment can be variable, and the effectiveness of some therapeutics is limited to specific phenotypes. Furthermore, genetic diseases and other degenerative conditions associated with aging morbidity pose a need for cell-specific targeting of genetic engineering tools.
Drug delivery to cancerous tissue can be accomplished via passive targeting due to leaky and irregular tumor vasculature with enhanced permeability and retention, which promotes the accumulation of macromolecules and nanoscale materials. However, this phenomena may not be consistent across patient populations. Furthermore, this phenomenon is not sufficient for achieving specific targeting of a given cell, tissue or organ type. Compositions and methods for efficiently targeting disease are provided in this disclosure, as well as for creating a diagnostically-responsive infrastructure for targeting a given cell/tissue/organ and delivering arbitrary gene editing or gene expressing instructions to those targets.
One difficulty in cancer immunotherapy stems from the fact that vaccination against cancers must bypass two forms of tolerance: central and peripheral. Central tolerance involves auto-reactive T cells being deleted whereas peripheral tolerance involves suppression of mature T cells through regulatory mechanisms and immune checkpoints. Such checkpoints can include the high expression of CTLA-4 or PD-1 receptors on tumor infiltrating lymphocytes. Recently, identifying and targeting tumor-specific antigens (neoantigens) which are only expressed in tumor cells has been of high interest as it can bypass central tolerance. However, the neoantigens can be patient specific and generally require either predictive modeling or patient genome sequencing. Thus, patient specific cancer vaccines are subject to significant time and cost. Efficient compositions and methods for patient-specific (diagnostically-responsive) treatments are provided in this disclosure, whereby a cancerous cell/tissue/organ (or another cell/tissue/organ being treated for disease) can be targeted for its specific receptor profile via an iterative nanoparticle development approach. The nanoparticles can furthermore deliver specific genetic instructions and be designed from bioresponsive materials that allow for additional cell-specific behaviors.
Oncolytic viruses (OVs) have been extensively studied as a cancer therapeutic as they selectively replicate and kill cancer cells without harming normal tissue. As an immunotherapy, OVs are used to tag, alert, and direct lymphocytes towards the tumors. Additionally, they have been used to transfect environment regulating cytokines such as GM-CSF into cancer cells to modulate the TME. However, the efficacy of these OVs to promote an immune response toward tumor cells is largely overshadowed by the immune response toward the OVs. Non-viral compositions and methods for efficiently targeting disease are provided in this disclosure.
Diagnostically-responsive medicine described herein can utilize a holistic nanoscale architecture coupled to a variety of cell-affinity-generating approaches for creating bioresponsive materials with many layers of precision in delivering a transient or permanent change in gene activity to a precisely-targeted cell, tissue or organ. Furthermore, an integrated robotics+software platform allows for rapid peptide synthesis, nanoparticle synthesis, and screening of formulations as part of a recursive machine learning approach for nanoparticle formulation optimization.
This approach goes beyond antibody-drug conjugates and traditional ligand-targeted medicine to create an end-to-end “diagnostically-responsive” medicine infrastructure featuring design, simulation, and synthesis suites driven by robotics, machine learning, biological characterization, nanomaterials characterization, and real-time data processing surrounding top-performing nanomedicine candidates as part of the detailed iterative improvement methodologies. Not only do these approaches offer combinatorial screening capabilities surrounding a comprehensive set of programmable matter, but each component of the nanomedicine/cell-targeting platform is designed to enhance specificity and afford patient-personalized therapeutic effect. These ligand-targeted solutions are readily manufacturing at cGMP grade through synthetic and/or recombinant means, to bolster industry adoption of cell-specific targeting technologies that are “user-specified” based on diagnostically-responsive traits and the payloads (e.g. CRISPR, DNA, mRNA, etc.) that are being delivered. Numerous formulations, embodiments, simulation and computation approaches, screening and synthesis approaches, methods, uses and variations thereof are detailed in the disclosure herein.
Using existing databases of cell, tissue and organ surface marker expression profiles, we show a novel approach for creating cell/tissue/organ-specific targeting technologies whereby a targeting ligand or array of targeting ligands designed to have specificity for a given surface marker profile are capable of shuttling a variety of payloads (e.g. gene therapies, RNPs, small molecules) to cells/tissues/organs bearing those surface markers. An integrative omics approach combines with novel nanomaterials and gene therapy/gene editing modalities such as CRISPR, DNA, and mRNA to allow for predictive targeting and amelioration of disease states, or synthetic biology characteristics (e.g. inserting chimeric antigen receptors into a particular immune subpopulation, or creating cell-specifically-expressed transmembrane motifs for subsequent affinity for an immunotherapy or gene therapy, and the like), in either healthy or diseased cell populations within specific cells/tissues/organs.
Design of targeted nanomedicine can allow for targeting specific cell types, including cancer neoantigens and known receptor profiles of target cells. Prior to this disclosure a diagnostically-responsive technology has not yet been deployed for rapidly tailoring cell-specific targeting technologies to a given patient's needs. Such a technology, as described in this disclosure, facilitates a future where patients see personalized medicine that is either permanent (e.g. CRISPR) or transient (e.g. mRNA), whereby targeted cells/tissues/organs are conferred disease resistance, genetic modifications, or immunomodulatory instructions.
Provided are methods and compositions for the heterologous expression of a payload (e.g., DNA, RNA, protein) of interest in a target cell (e.g., cancer cell, disease-causing cell/tissue/organ). In some cases payload delivery results in expression of a secreted protein, e.g., an immune signal such as a cytokine (e.g., by a cancer cell in vivo). In some cases payload delivery results in expression of a plasma membrane-tethered affinity marker (e.g., by cancer cells in vivo—thus resulting in an induced immune response). In some cases payload delivery results in expression of a cytotoxic protein such as an apoptosis inducer (e.g., by a cancer cell in vivo). In other cases, unknown cell types or cell types with known or acquired genomics/mRNA/proteomics data may be targeted “diagnostically-responsively” via a tailored cell targeting approach. In further cases, a combination of tumor surface marker engineering that is cell/tissue/organ-specific (e.g. under cancer-specific or cell-specific promoters) coupled to an immune engineering approach (e.g. causing antigen-presenting cells, γδ T cells, or other immune cells to hone in on the aforementioned cancer beacons).
Payloads are delivered with a delivery vehicle and in some cases the delivery vehicle is a nanoparticle. In some cases a subject nanoparticle for delivering payloads such as those discussed above includes a targeting ligand for targeted delivery to a specific cell type/tissue type (e.g., a cancerous tissue/cell).
In some embodiments, payload delivery and design of ligand-targeted, cell-specific nanomedicine is “personalized” in the sense that the delivery vehicle and/or payload can be designed based on patient-specific information—such embodiments are referred to herein as “personalized” or “diagnostically-responsive” methods. These diagnostically-responsive methods are facilitated by a nanomedicine infrastructure whereby design of optimal nanoparticles for a given payload, an appropriate cell-specific targeting strategy, and ultimately a cell-specific payload (e.g. promoter-driven expression, cell-specific Cas9 activity) are facilitated by a robotic, computationally-driven synthesis, screening and iteration approach. As such, in some cases a subject method involves diagnostically-responsive payload delivery (i.e., personalized payload delivery)—in such cases the delivery vehicle and/or the payload can be considered “personalized” where the “personalized” aspect relates to the ability to 1) identify ligand-receptor interactions based on native protein sequences (described herein) or alternative means (e.g. phage display, SELEX, etc.), 2) rapidly synthesize a cell-specific targeting ligand or combination of heteromultivalent cell-specific targeting ligands (e.g. through customized, ultra-high-speed robotic peptide synthesis described herein, or through other library generation techniques), 3) tethering these targeting ligands to a variety of nanoparticle chemistries (including electrostatic, lipidic and other embodiments), either through direct ligand condensation into a nanoparticle or upon the surface of a nanoparticle (or an alternative ligand-drug conjugate), 4) assaying for nanomaterials properties and biological effects (through a workflow described herein), 5) identifying top hit formulations via the properties of (4), and 6) iterating through the formulations, combinations of ligands and combinations/ratios of nanoparticle constituents (where applicable) through a software-driven approach (“recursive automation/machine learning”). The combination of this infrastructure with diagnostics data (e.g. receptor profiles, disease state of targeted cell, cell-specific promoter identification, target genes for expression/suppression/editing) and an underlying nanomaterials platform disclosed herein allows for customized, cell-specific targeting technologies to be developed in days or weeks vs. current industry approaches which take several months to years.
Such delivery systems offer flexibility and tailorability towards targeting patient-specific surface proteins and/or using selected promoters to drive expression of introduced sequences. For example, a promoter can be selected based on patient expression profiles. Thus, compositions and methods of this disclosure can be designed in a diagnostically responsive manner such that the composition/method can be tailored specifically for each patient. For example, once a tumor's unique characteristics are identified, a patient-specific and diagnostically-responsive nanomedicine (e.g., delivery vehicle that includes a payload) may be administered to the patient with or without the need for an autologous/allogeneic immunotherapy.
When compared to alternative delivery methods such as viruses, nanoparticles offer several key advantages. First, a lesser degree of immunogenicity may be achieved, and stealth properties may be incorporated in the design to prevent immune response, complement activation and subsequent clearance by the reticuloendothelial system. This immunogenicity may be further reduced by protein fragments (e.g. synthetic peptide sequences per the diagnostically-responsive workflow identified herein) being derived from native proteins when designing ligand-receptor pairings. Additionally, nanoparticles offer greater flexibility in the variety of payloads that may be encapsulated, as well as the potential for co-delivery of multiple payloads.
Further, nanoparticles composed of synthetic biopolymers such as peptides and nucleic acids may be easily tailored for different applications. This is particularly relevant to diagnostically responsive medicine.
The embodiments disclosed herein have broad application to drug delivery, immunotherapy, and oncology. Additionally, the embodiments herein present a universal approach for engineering cancer cells in a diagnostically responsive manner—e.g., to express markers that lead to adaptive immune learning, creating a novel cancer treatment that my augment autologous or allogeneic cell transplantation and engineered cell lines. The embodiments described herein can allow for improved tumor chemotaxis and prolonged adaptive immune learning.
This application contains at least one drawing executed in color. Copies of this application with color drawing(s) will be provided by the Office upon request and payment of the necessary fees.
The invention is best understood from the following detailed description when read in conjunction with the accompanying drawings. It is emphasized that, according to common practice, the various features of the drawings are not to-scale. On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity. Included in the drawings are the following figures.
FIG. 11D1 depicts an algorithmic comparison of top uniquely expressed in human naive CD8+ T cells. This particular dataset compares the top-expressed genes vs. the top uniquely expressed genes in the naive CD8+ T cell example, and compares to other immunological and blood cells. The y-axis of each graph shows transcripts per million.
FIG. 11D2 depicts an algorithmic comparison of top expressed genes in human naive CD8+ T cells. This particular dataset compares the top-expressed genes vs. the top uniquely expressed genes in the naive CD8+ T cell example, and compares to other immunological and blood cells. The y-axis of each graph shows transcripts per million.
FIG. 11S1 depicts a machine learning based approach for determining unique surface markers in a mixed cell population, allowing for improved classification of cell specificity indices. In this example, hematopoietic stem cells and their progenitors are shown. tSNE, principle component analysis (PCA) and similar unsupervised learning techniques may be used to determine initial sets of surface markers corresponding to a particular cell population subtype.
FIG. 11S2 depicts an enlarged view of the top nine plots of FIG. 11S1.
FIG. 11S3 depicts an enlarged view of the bottom six plots of FIG. 11S1.
FIG. 15A1 depicts synthesis results of bulk mixing histone-derived, cysteine-substituted amino acid sequences in various pH conditions and with variable crosslinking time, which yielded an optimal condensation profile with cores made in 30 mM pH 5.5 HEPES. These nanoparticles were used to deliver CRISPR Cas9 RNPs. Inclusion of serum in these particle formulations led to enhanced particle condensation as assessed via SYBR inclusion assay. RNP (5 ng/uL) control fluorescent values (+ and −serum) are shown for baseline SYBR assay values prior to nanoparticle condensation.
FIG. 15A2 depicts the particle sizes corresponding to the FIG. 15A1 embodiment.
FIG. 15A3 depicts the particle sizes distribution corresponding to the FIG. 15A1 embodiment.
FIG. 15B1 depicts orders of addition studies of poly(glutamic acid) and cysteine-modified histone fragments with CRISPR Cas9 RNPs, whereby particle size and formation behaviors were not shown to be different between the two orders of addition when the synthesis was performed via microfluidic devices, and microfluidic mixing led to enhanced particle sizes with uniform size peaks versus bulk synthesis approaches (FIG. 15A1-3). Adding PLE before H2B or H2B before PLE in the microfluidic approach did not impact core particle formation. Inclusion of serum in these particle formulations led to enhanced particle condensation as assessed via SYBR inclusion assay.
FIG. 15B2 depicts the particle sizes corresponding to the FIG. 15B1 embodiment.
FIG. 15B3 depicts the particle sizes distribution corresponding to the FIG. 15B1 embodiment.
FIG. 15C1 depicts nanoparticle cores prepared in FIG. 15B1-3 were subsequently patterned in a variety of electrostatic surface ligands, and the SYBR inclusion/exclusion assay values were measured for each formulation with and without serum inclusion. Particles synthesized with a 1 h crosslinking time demonstrated less stability than particles that had ligands immediately added to them prior to crosslinking, as inferred by the increase in SYBR fluorescence values in the 1 h crosslinked cores. This is perhaps due to serum dissociating the ligands and destabilizing the particles with 1 h of crosslinking, which led to a less stable colloid. Alternatively, ligand inclusion at an earlier stage may form a more stable suspension. Each ligand coating in these examples where a 0 h crosslinking time was utilized prior to ligand decoration demonstrated excellent SYBR fluorescence values with serum inclusion, and particle sizes remained stable with the RNP-H2B; RNP-H2B-PLE; Core—CD28(80), CD28(86), CD3e, IL2R; Core—CD28(80), CD28(86), CD3e, IL2R; and other heteromultivalent variants. Particle sizes were also demonstrably uniform for a variety of surface coats. See
FIG. 15C2 depicts the particle sizes corresponding to the FIG. 15C1 embodiment.
FIG. 15C3 depicts the particle sizes distribution corresponding to the FIG. 15C1 embodiment.
FIG. 15D1 depicts expanded datasets for FIG. 15C1-3 for particle size following microfluidic core particle synthesis and subsequent layering with ligands. The size and zeta potential for each formulation, with cores that were crosslinked for either 0 h or 1 h, is shown. Size and zeta potential is compared with and without serum.
FIG. 15D2 depicts the zeta potential corresponding to the FIG. 15D1 embodiment.
FIG. 15E1 depicts extended SYBR fluorescence assays (24 h) without serum a for CRISPR RNP formulations in FIGS. 15A1-15D3.
FIG. 15E2 depicts the data corresponding to the FIG. 15E1 embodiment with serum.
FIG. 16B1 depicts an untreated control for Cas9 uptake in T cells and PBMC. Negative Control+/−1%=noise Used as the basis to set gates for positive Cas9 signal.
FIG. 16B2 depicts the T cell data corresponding to FIG. 16B1.
FIG. 16B3 depicts the PBMC data corresponding to FIG. 16B1.
FIG. 16C1 depicts core nanoparticle only Cas9 uptake in T cells and PBMC. Does not contain targeting moieties.
FIG. 16C2 depicts the T cell data corresponding to FIG. 16C1.
FIG. 16C3 depicts the PBMC data corresponding to FIG. 16C1.
FIG. 16D1 depicts core nanoparticle+PLR10 cell penetrating peptide Cas9 uptake in T cells and PBMC. General cell surface proteoglycan targeting. Does not confer cell specificity
FIG. 16D2 depicts the T cell data corresponding to FIG. 16D1.
FIG. 16D3 depicts the PBMC data corresponding to FIG. 16D1.
FIG. 16E1 depicts core nanoparticle+CD3epsilon ligand Cas9 uptake in T cells and PBMC.
Monovalent surface targeting CD3. Broad T cell/Thymocyte specificity.
FIG. 16E2 depicts the T cell data corresponding to FIG. 16E1.
FIG. 16E3 depicts the PBMC data corresponding to FIG. 16E1.
FIG. 16F1 depicts core nanoparticle+CD8 ligand Cas9 uptake in T cells and PBMC. Monovalent surface targeting CD8. Results in significant uptake in T-cells and PBMCs.
FIG. 16F2 depicts the T cell data corresponding to FIG. 16F1.
FIG. 16F3 depicts the PBMC data corresponding to FIG. 16F1.
FIG. 16G1 depicts core nanoparticle only+CD80-derived CD28-targeting ligand Cas9 uptake in T cells and PBMC. Targets CD28, a T-cell marker. Ligand mimics CD80 on antigen-presenting cells. Modest uptake in T-cells.
FIG. 16G2 depicts the T cell data corresponding to FIG. 16G1.
FIG. 16G3 depicts the PBMC data corresponding to FIG. 16G1.
FIG. 16H1 depicts core nanoparticle+CD86-derived CD28-targeting ligand Cas9 uptake in T cells and PBMC. Targets CD28, a T-cell marker. Ligand mimics CD86 on antigen-presenting cells. No uptake in T-cells.
FIG. 16H2 depicts the T cell data corresponding to FIG. 16H1.
FIG. 16H3 depicts the PBMC data corresponding to FIG. 16H1.
FIG. 16J1 depicts core nanoparticle+CD3epsilon-targeting ligand+CD8-targeting ligand Cas9 uptake in T cells and PBMC. Heterodivalent combination of ligands targeting CD3 and CD8.
FIG. 16J2 depicts the T cell data corresponding to FIG. 16J1.
FIG. 16J3 depicts the PBMC data corresponding to FIG. 16J1.
FIG. 16K1 depicts core nanoparticle+CD3epsilon ligand+CD80-derived CD28-targeting ligand Cas9 uptake in T cells and PBMC. Heterodivalent combination of ligands targeting CD3 and CD28 (derived from CD80).
FIG. 16K2 depicts the T cell data corresponding to FIG. 16K1.
FIG. 16K3 depicts the PBMC data corresponding to FIG. 16K1.
FIG. 16L1 depicts core nanoparticle+CD3epsilon ligand+CD86-derived CD28-targeting ligand Cas9 uptake in T cells and PBMC. Heterodivalent combination of ligands targeting CD3 and CD28 (derived from CD86).
FIG. 16L2 depicts the T cell data corresponding to FIG. 16L1.
FIG. 16L3 depicts the PBMC data corresponding to FIG. 16L1.
FIG. 16M1 depicts core nanoparticle+CD3epsilon ligand+IL2-derived IL2R-targeting ligand Cas9 uptake in T cells and PBMC. Heterodivalent combination of ligands targeting CD3 and IL2R.
FIG. 16M2 depicts the T cell data corresponding to FIG. 16M1.
FIG. 16M3 depicts the PBMC data corresponding to FIG. 16M1.
FIG. 16N1 depicts core nanoparticle+CD3epsilon ligand+PLR10 cell penetrating peptide Cas9 uptake in T cells and PBMC. Poly(L-Arginine) coating along with CD3 ligand greatly reduces efficacy from 26%.
FIG. 16N2 depicts the T cell data corresponding to FIG. 16N1.
FIG. 16N3 depicts the PBMC data corresponding to FIG. 16N1.
FIG. 16P1 depicts core nanoparticle+CD3epsilon ligand+CD86-derived CD28-targeting ligand+CD8-targeting ligand Cas9 uptake in T cells and PBMC. Heterotrivalent surface targeting CD3, CD28 and CD. Slight bias of CD8+ T-cell targeting.
FIG. 16P2 depicts the T cell data corresponding to FIG. 16P1.
FIG. 16P3 depicts the PBMC data corresponding to FIG. 16P1.
FIG. 16Q1 depicts core nanoparticle+CD3epsilon ligand+CD8-targeting ligand+IL2-derived IL2R-targeting ligand Cas9 uptake in T cells and PBMC. Heterotrivalent surface targeting CD3, CD8, and IL2R. Slight bias of CD8+ T-cell targeting. ˜44.4% efficient CD8+ T Cell targeting.
FIG. 16Q2 depicts the T cell data corresponding to FIG. 16Q1.
FIG. 16Q3 depicts the PBMC data corresponding to FIG. 16Q1.
FIG. 16R1 depicts core nanoparticle+CD3epsilon ligand+CD80-derived CD28-targeting ligand+CD8-targeting ligand Cas9 uptake in T cells and PBMC. Heterotrivalent surface targeting CD3, CD28, and CD8. ˜5% bias in targeting CD8+vs. CD4+ T-cells. ˜43.9% efficient CD8+ T-cell targeting.
FIG. 16R2 depicts the T cell data corresponding to FIG. 16R1.
FIG. 16R3 depicts the PBMC data corresponding to FIG. 16R1.
FIG. 16S1 depicts core nanoparticle+CD3epsilon ligand+CD86-derived CD28-targeting ligand+CD80-derived CD28-targeting ligand Cas9 uptake in T cells and PBMC.Heterotrivalent surface targeting CD3 and CD28 (mimicking CD80 and CD86 co-presentation). Reduction in uptake vs. CD8-containing heterotrivalent surface without CD28(86). ˜4% bias in targeting CD8+vs. CD4+ T-cells.
FIG. 16S2 depicts the T cell data corresponding to FIG. 16S1.
FIG. 16S3 depicts the PBMC data corresponding to FIG. 16S1.
FIG. 16T1 depicts core nanoparticle+CD8-targeting ligand+CD80-derived CD28-targeting ligand+CD86-derived CD28-targeting ligand Cas9 uptake in T cells and PBMC. Heterotrivalent surface targeting CD8 and CD28 (mimicking CD80 and CD86 co-presentation). Efficient CD8+ T-cell targeting. ˜6% bias in targeting CD8+vs. CD4+ T-cells.
FIG. 16T2 depicts the T cell data corresponding to FIG. 16T1.
FIG. 16T3 depicts the PBMC data corresponding to FIG. 16T1.
FIG. 16U1 depicts core nanoparticle+CD8-targeting ligand+CD80-derived CD28-targeting ligand+IL2-derived IL2R-targeting ligand Cas9 uptake in T cells and PBMC. Heterotrivalent surface targeting CD8, CD28(80) and IL2R. Efficient CD8+ T-cell targeting. ˜6% bias in targeting CD8+vs. CD4+ T-cells.
FIG. 16U2 depicts the T cell data corresponding to FIG. 16U1.
FIG. 16U3 depicts the PBMC data corresponding to FIG. 16U1.
FIG. 16V1 depicts core nanoparticle+CD8-targeting ligand+CD86-derived CD28-targeting ligand+IL2-derived IL2R-targeting ligand Cas9 uptake in T cells and PBMC. Heterotrivalent surface targeting CD8, CD28(86) and IL2R. Efficient CD8+ T-cell targeting. ˜6% bias in targeting CD8+vs. CD4+ T-cells.
FIG. 16V2 depicts the T cell data corresponding to FIG. 16V1.
FIG. 16V3 depicts the PBMC data corresponding to FIG. 16V1.
FIG. 18J1 depicts high homology of coils 1A, 1B, and 2 between keratin, type I cuticular Hal (top) and keratin, type I cytoskeletal 14 (bottom).
FIG. 18J2 depicts an enlarged version of the top diagram of FIG. 18J1.
FIG. 18J3 depicts an enlarged version of the bottom diagram of FIG. 18J1.
FIG. 18K1 depicts human SCF in complex with an extracellular domain of Kit (green) vs. mouse SCF (blue) prior to sequence alignment.
FIG. 18K2 depicts an enlarged version of a section of FIG. 18K1.
FIG. 18L1 depicts human SCF in complex with an extracellular domain of Kit (green) vs. mouse SCF (blue) following sequence alignment. The c-Kit receptor and SCF have high sequence homology between species, allowing higher translatability of murine to human experiments when performing SCF studies targeting ltHSC, stHSC, and/or CD34+ hematopoietic stem cells. Both mouse and human variants exhibit identical lengths for the signal peptide vs. Kit ligand domains, and high degrees of sequence alignment.
FIG. 18L2 depicts an enlarged version of a section of FIG. 18L1.
FIG. 19F1 depicts computer-assisted formulation design. The table's values represent volume (IL) of the respective solution, whereby a robotic fluid handling system executes the instructions from left to right. Subsequent physicochemical and biological studies examined dsDNA condensation with various ratios of poly(L-glutamic acid) and poly(D-glutamic acid) (PLE20 and PDE20) and applied to a Cas9 ribonucleoprotein (RNP) condensation experiment with either NLS-Cas9-2NLS with a LL236 gRNA (targeting TRAC locus), or NLS-Cas9-EGFP with a LL224 gRNA (targeting TRAC locus). The associated physicochemical and biological properties of nanoparticles are to assess performance of each formulation. Shown are particles condensed with various charge ratios (CR) of 9R-PEG-CD8 ligand or mPEG5K-PLK30. CRX-Y indicates the charge ratio of cationic polypeptides (X) vs. the respective formulation breakdown on the right (Y=1-4).
FIG. 19F2 depicts representative associated formulations corresponding to the embodiment of FIG. 19F1.
Before the present methods and compositions are described, it is to be understood that this invention is not limited to the particular methods or compositions described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. It is understood that the present disclosure supersedes any disclosure of an incorporated publication to the extent there is a contradiction.
As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.
It must be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” includes a plurality of such cells and reference to “the nanoparticle” includes reference to one or more nanoparticles and equivalents thereof, known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any element, e.g., any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.
The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.
As noted above, provided are methods and compositions for the heterologous expression of a payload (e.g., DNA, RNA, protein) of interest in a target cell (e.g., cancer cell). In some cases payload delivery results in expression of a secreted protein, e.g., an immune signal such as a cytokine (e.g., by a cancer cell in vivo). In some cases payload delivery results in expression of a plasma membrane-tethered affinity marker (e.g., by cancer cells in vivo—thus resulting in an induced immune response). In some cases payload delivery results in expression of a cytotoxic protein such as an apoptosis inducer (e.g., by a cancer cell in vivo). Payloads are delivered with a delivery vehicle and in some cases the delivery vehicle is a nanoparticle. In some cases a subject nanoparticle for delivering payloads such as those discussed above includes a targeting ligand for targeted delivery to a specific cell type/tissue type (e.g., a cancerous tissue/cell).
In some embodiments, payload delivery is “personalized” in the sense that the delivery vehicle and/or payload is designed based on patient-specific information—such embodiments are referred to herein as “personalized” or “diagnostically-responsive” methods. As such, in some cases a subject method involves diagnostically-responsive payload delivery (i.e., personalized payload delivery)—in such cases the delivery vehicle and/or the payload can be considered “personalized.” In some embodiments, the “personalized” or “diagnostically-responsive” designation is due to the fact that one or more targeting ligands were identified/selected/designed/screened—for based on an individual's molecular data (e.g., sequencing data, array data, expression data, proteomics data, and the like). In some embodiments, the “personalized” or “diagnostically-responsive” designation is due to the fact that the payload was selected based on an individual's molecular data (e.g., sequencing data, array data, expression data, proteomics data, and the like).
Below is a general description of suitable “delivery vehicles” such as nanoparticles and their components, including an initial general description of payloads. This is followed by a description of ways in which such delivery vehicles and/or payloads can be ‘personalized’ in a diagnostically responsive way. Various payloads of interest (e.g., secreted proteins or nucleic acids encoding them, cytotoxic proteins or nucleic acids encoding them, and affinity markers or nucleic acids encoding them) are also described.
In some embodiments, one or more of the steps of the disclosed methods may be performed in a automated way—for example by a processor executing instructions, e.g., a non-transitory recording medium comprising instructions which, when executed by a processor of the system, cause the processor to perform any one or more of a variety of tasks, which can include but are not limited to: evaluating expression data, identifying one or more cell surface targets for targeting a cell, tissue, or organ of interest, generating a list of candidate targeting ligands (e.g., by evaluating crystal structures of the one or more cell surface targets to derive protein-ligand or protein-protein interaction information for the one or more cell surface targets), designing candidate targeting ligands, producing candidate targeting ligands (e.g., by actuating a robotic devise such as a liquid handling robot), producing a library of candidate delivery vehicles such as a library of nanoparticle formulations (e.g., by actuating a robotic devise such as a liquid handling robot), contacting surface targets (e.g., targets on the surface of cells) with candidate delivery vehicles such as candidate nanoparticle formulations, evaluating effectiveness of candidate targeting ligands and/or candidate delivery vehicles (e.g., via calculating measures of success based on a list of evaluation parameters), selecting the top-performing targeting ligands and/or delivery vehicle formulations, performing any of the above as part of a recursive screen (e.g., for targeting ligand and/or delivery vehicle optimization), and the like.
Delivery Vehicles
A delivery vehicle is a vehicle for delivering a payload (e.g., nucleic acid and/or protein payload) to a cell. Delivery vehicles can include, but are not limited to, non-viral vehicles, viral vehicles, nanoparticles (e.g., a nanoparticle that includes a targeting ligand and/or a core comprising an anionic polymer composition, a cationic polymer composition, and a cationic polypeptide composition), liposomes, micelles, water-oil-water emulsion particles, oil-water emulsion micellar particles, multilamellar water-oil-water emulsion particles, a targeting ligand (e.g., peptide targeting ligand) conjugated to a charged polymer polypeptide domain (where the targeting ligand provides for targeted binding to a cell surface protein, and the charged polymer polypeptide domain is condensed with a nucleic acid payload and/or is interacting electrostatically with a protein payload), a targeting ligand (e.g., peptide targeting ligand) conjugated to payload (where the targeting ligand provides for targeted binding to a cell surface protein). In some cases payloads are introduced into the cell as a deoxyribonucleoprotein complex or a ribo-deoxyribonucleoprotein complex.
In some cases, a delivery vehicle is a water-oil-water emulsion particle. In some cases, a delivery vehicle is an oil-water emulsion micellar particle. In some cases, a delivery vehicle is a multilamellar water-oil-water emulsion particle. In some cases, a delivery vehicle is a multilayered particle. In some cases, a delivery vehicle is a DNA origami nanobot. For any of the above a payload (nucleic acid and/or protein) can be inside of the particle, either covalently, bound as nucleic acid complementary pairs, or within a water phase of a particle. In some cases a delivery vehicle includes a targeting ligand, e.g., in some cases a targeting ligand (described in more detail elsewhere herein) coated upon a water-oil-water emulsion particle, upon an oil-water emulsion micellar particle, upon a multilamellar water-oil-water emulsion particle, upon a multilayered particle, or upon a DNA origami nanobot. In some cases a delivery vehicle has a solid core particle (e.g., metal particle core, quantum dot core, and the like)—in which case the payload can be conjugated to (covalently bound to) the core.
Payloads
Delivery vehicles (e.g., nanoparticles) of the disclosure include a payload (they are used to deliver a payload). A payload can be any compound one wishes to deliver to a cell. For example, in some cases a payload is a nucleic acid and/or protein. In some cases, a subject nanoparticle (e.g., a nanoparticle that includes a targeting ligand and/or a core comprising an anionic polymer composition, a cationic polymer composition, and a cationic polypeptide composition) is used to deliver a nucleic acid payload (e.g., a DNA and/or RNA). In some cases a subject nanoparticle (e.g., a nanoparticle that includes a targeting ligand and/or a core comprising an anionic polymer composition, a cationic polymer composition, and a cationic polypeptide composition) is used to deliver a protein payload. In some cases a subject nanoparticle (e.g., a nanoparticle that includes a targeting ligand and/or a core comprising an anionic polymer composition, a cationic polymer composition, and a cationic polypeptide composition) is used to deliver a payload of protein and nucleic acid, e.g., a ribonucleic acid protein complex (an RNP). A payload can be any desired compound. For example, in some cases a payload is a small molecule drug (e.g., which can be delivered via liposomes, nanoparticles as described herein such as PLGA particles, via direct conjugation to a targeting ligand, etc). For example in some cases a targeting ligand is used to direct the delivery of a small molecule drug via any convenient delivery vehicle (e.g., any of the delivery vehicles described herein can be used to deliver a small molecule drug payload).
A nucleic acid payload can be any nucleic acid of interest, e.g., the nucleic acid payload can be linear or circular, and can be a plasmid, a viral genome, an RNA (e.g., a coding RNA such as an mRNA or a non-coding RNA such as a guide RNA, a short interfering RNA (siRNA), a short hairpin RNA (shRNA), a microRNA (miRNA), and the like), a DNA, etc. In some cases, the nucleic payload is an RNAi agent (e.g., an shRNA, an siRNA, a miRNA, etc.) or a DNA template encoding an RNAi agent. In some cases, the nucleic acid payload is an siRNA molecule (e.g., one that targets an mRNA, one that targets a miRNA). In some cases, the nucleic acid payload is an LNA molecule (e.g., one that targets a miRNA). In some cases, the nucleic acid payload is a miRNA. In some cases the nucleic acid payload includes an mRNA that encodes a protein of interest (e.g., one or more reprograming and/or transdifferentiation factors such as Oct4, Sox2, Klf4, c-Myc, Nanog, and Lin28, e.g., alone or in any desired combination such as (i) Oct4, Sox2, Klf4, and c-Myc; (ii) Oct4, Sox2, Nanog, and Lin28; and the like; a gene editing endonuclease; a therapeutic protein; and the like). In some cases the nucleic acid payload includes a non-coding RNA (e.g., an RNAi agent, a CRISPR/Cas guide RNA, etc.) and/or a DNA molecule encoding the non-coding RNA. In some embodiments a nucleic acid payload includes a nucleic acid (DNA and/or mRNA) that encodes IL2Ra and IL12R7 (e.g., to modulate the behavior or survival of a target cell), and in some cases the payload is released intracellularly from a subject nanoparticle over the course of from 7-90 days (e.g., from 7-80, 7-60, 7-50, 7-40, 7-35, or 7-30 days). In some cases the nucleic acid payload includes a self-replicating RNA.
In some embodiments a nucleic acid payload includes a nucleic acid (DNA and/or mRNA) that encodes BCL-XL (e.g., to prevent apoptosis of a target cell due to engagement of Fas or TNFα receptors). In some embodiments a nucleic acid payload includes a nucleic acid (DNA and/or mRNA) that encodes Foxp3 (e.g., to promote an immune effector phenotype in targeted T-cells). In some embodiments a nucleic acid payload includes a nucleic acid (DNA and/or mRNA) that encodes SCF. In some embodiments a nucleic acid payload includes a nucleic acid (DNA and/or mRNA) that encodes HoxB4. In some embodiments a nucleic acid payload includes a nucleic acid (DNA and/or mRNA) that encodes SIRT6. In some embodiments a nucleic acid payload includes a nucleic acid molecule (e.g., an siRNA, an LNA, etc.) that targets (reduces expression of) a microRNA such as miR-155 (see, e.g., MiR Base accession: MI0000681 and MI0000177). In some embodiments a nucleic acid payload includes an siRNA that targets ku70 and/or an siRNA that targets ku80.
The term “nucleic acid payload” encompasses modified nucleic acids. Likewise, the terms “RNAi agent” and “siRNA” encompass modified nucleic acids. For example, the nucleic acid molecule can be a mimetic, can include a modified sugar backbone, one or more modified internucleoside linkages (e.g., one or more phosphorothioate and/or heteroatom internucleoside linkages), one or more modified bases, and the like. In some embodiments, a subject payload includes triplex-forming peptide nucleic acids (PNAs) (see, e.g., McNeer et al., Gene Ther. 2013 June; 20(6):658-69). Thus, in some cases a subject core includes PNAs. In some cases a subject core includes PNAs and DNAs.
A subject nucleic acid payload (e.g., an siRNA) can have a morpholino backbone structure. In some case, a subject nucleic acid payload (e.g., an siRNA) can have one or more locked nucleic acids (LNAs). Suitable sugar substituent groups include methoxy (—O—CH3), aminopropoxy (—O CH2 CH2 CH2NH2), allyl (—CH2—CH═CH2), —O-allyl (—O—CH2—CH═CH2) and fluoro (F). 2′-sugar substituent groups may be in the arabino (up) position or ribo (down) position. Suitable base modifications include synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (—C═C—CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine(1H-pyrimido(5,4-b)(1,4)benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido(5,4-(b) (1,4)benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido(4,5-b)indol-2-one), pyridoindole cytidine (H-pyrido(3′,2′:4,5)pyrrolo(2,3-d)pyrimidin-2-one).
In some cases, a nucleic acid payload can include a conjugate moiety (e.g., one that enhances the activity, stability, cellular distribution or cellular uptake of the nucleic acid payload). These moieties or conjugates can include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups. Conjugate groups include, but are not limited to, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Suitable conjugate groups include, but are not limited to, cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties include groups that improve uptake, enhance resistance to degradation, and/or strengthen sequence-specific hybridization with the target nucleic acid. Groups that enhance the pharmacokinetic properties include groups that improve uptake, distribution, metabolism or excretion of a subject nucleic acid.
Any convenient polynucleotide can be used as a subject nucleic acid payload. Examples include but are not limited to: species of RNA and DNA including mRNA, m1A modified mRNA (monomethylation at position 1 of Adenosine), siRNA, miRNA, aptamers, shRNA, AAV-derived nucleic acids and scaffolds, morpholino RNA, peptoid and peptide nucleic acids, cDNA, DNA origami, DNA and RNA with synthetic nucleotides, DNA and RNA with predefined secondary structures, multimers and oligomers of the aforementioned, and payloads whose sequence may encode other products such as any protein or polypeptide whose expression is desired.
In some cases a payload of a subject delivery vehicle (e.g., nanoparticle) includes a protein. Examples of protein payloads include, but are not limited to: programmable gene editing proteins (e.g., transcription activator-like (TAL) effectors (TALEs), TALE nucleases (TALENs), zinc-finger proteins (ZFPs), zinc-finger nucleases (ZFNs), DNA-guided polypeptides such as Natronobacterium gregoryi Argonaute (NgAgo), CRISPR/Cas RNA-guided polypeptide (Class 2 CRISPR/Cas effector protein) (e.g., Cas9, CasX, CasY, Cpf1, Cas13, MAD7, and the like); transposons (e.g., a Class I or Class II transposon—e.g., piggybac, sleeping beauty, Tc1/mariner, To12, PIF/harbinger, hAT, mutator, merlin, transib, helitron, maverick, frog prince, minos, Himarl and the like); meganucleases (e.g., I-SceI, I-CeuI, I-CreI, I-DmoI, I-ChuI, I-DirI, I-FlmuI, I-FlmuII, I-Anil, I-SceIV, I-CsmI, I-PanI, I-PanII, I-PanMI, I-SceII, I-PpoI, I-SceIII, I-LtrI, I-GpiI, I-GZeI, I-OnuI, I-HjeMI, I-Msol, I-Teel, I-TevII, I-TevIII, PI-MleI, PI-MtuI, PI-PspI, PI-Tli I, PI-Tli II, PI-SceV, and the like); megaTALs (see, e.g., Boissel et al., Nucleic Acids Res. 2014 February; 42(4): 2591-2601); SCF; BCL-XL; Foxp3; HoxB4; and SiRT6. For any of the above proteins, a payload of a subject delivery vehicle (e.g., nanoparticle) can include a nucleic acid (DNA and/or mRNA) encoding the protein, and/or can include the actual protein.
Gene Editing Tools (as Payloads)
In some cases, a nucleic acid payload includes or encodes a gene editing tool (i.e., a component of a gene editing system, e.g., a site specific gene editing system such as a programmable gene editing system). For example, a nucleic acid payload can include one or more of: (i) a CRISPR/Cas guide RNA, (ii) a DNA encoding a CRISPR/Cas guide RNA, (iii) a DNA and/or RNA encoding a programmable gene editing protein such as a zinc finger protein (ZFP) (e.g., a zinc finger nuclease—ZFN), a transcription activator-like effector (TALE) protein (e.g., fused to a nuclease—TALEN), a DNA-guided polypeptide such as Natronobacterium gregoryi Argonaute (NgAgo), and/or a CRISPR/Cas RNA-guided polypeptide (Class 2 CRISPR/Cas effector protein) (e.g., Cas9, CasX, CasY, Cpf1, Cas13, MAD7, and the like); (iv) a DNA donor template; (v) a nucleic acid molecule (DNA, RNA) encoding a site-specific recombinase (e.g., Cre recombinase, Dre recombinase, Flp recombinase, KD recombinase, B2 recombinase, B3 recombinase, R recombinase, Hin recombinase, Tre recombinase, PhiC31 integrase, Bxb 1 integrase, R4 integrase, lambda integrase, HK022 integrase, HP1 integrase, and the like); (vi) a DNA encoding a resolvase and/or invertase (e.g., Gin, Hin, γδ3, Tn3, Sin, Beta, and the like); and (vii) a transposon and/or a DNA derived from a transposon (e.g., bacterial transposons such as Tn3, Tn5, Tn7, Tn9, Tn10, Tn903, Tn1681, and the like; eukaryotic transposons such as Tc1/mariner super family transposons, PiggyBac superfamily transposons, hAT superfamily transposons, PiggyBac, Sleeping Beauty, Frog Prince, Minos, Himarl, and the like). In some cases a subject delivery vehicle (e.g., nanoparticle) is used to deliver a protein payload, e.g., a gene editing protein such as a ZFP (e.g., ZFN), a TALE (e.g., TALEN), a DNA-guided polypeptide such as Natronobacterium gregoryi Argonaute (NgAgo), a CRISPR/Cas RNA-guided polypeptide (Class 2 CRISPR/Cas effector protein) (e.g., Cas9, CasX, CasY, Cpf1, Cas13, MAD7, and the like), a site-specific recombinase (e.g., Cre recombinase, Dre recombinase, Flp recombinase, KD recombinase, B2 recombinase, B3 recombinase, R recombinase, Hin recombinase, Tre recombinase, PhiC31 integrase, Bxb 1 integrase, R4 integrase, lambda integrase, HK022 integrase, HP1 integrase, and the like), a resolvase/invertase (e.g., Gin, Hin, γδ3, Tn3, Sin, Beta, and the like); and/or a transposase (e.g., a transposase related to transposons such as bacterial transposons such as Tn3, Tn5, Tn7, Tn9, Tn10, Tn903, Tn1681, and the like; or eukaryotic transposons such as Tc1/mariner super family transposons, PiggyBac superfamily transposons, hAT superfamily transposons, PiggyBac, Sleeping Beauty, Frog Prince, Minos, Himarl, and the like). In some cases, the delivery vehicle (e.g., nanoparticle) is used to deliver a nucleic acid payload and a protein payload, and in some such cases the payload includes a ribonucleoprotein complex (RNP).
Depending on the nature of the system and the desired outcome, a gene editing system (e.g. a site specific gene editing system such as a a programmable gene editing system) can include a single component (e.g., a ZFP, a ZFN, a TALE, a TALEN, a site-specific recombinase, a resolvase/integrase, a transpose, a transposon, and the like) or can include multiple components. In some cases a gene editing system includes at least two components. For example, in some cases a gene editing system (e.g. a programmable gene editing system) includes (i) a donor template nucleic acid; and (ii) a gene editing protein (e.g., a programmable gene editing protein such as a ZFP, a ZFN, a TALE, a TALEN, a DNA-guided polypeptide such as Natronobacterium gregoryi Argonaute (NgAgo), a CRISPR/Cas RNA-guided polypeptide (Class 2 CRISPR/Cas effector protein) (e.g., Cas9, CasX, CasY, Cpf1, Cas13, MAD7, and the like), or a nucleic acid molecule encoding the gene editing protein (e.g., DNA or RNA such as a plasmid or mRNA). As another example, in some cases a gene editing system (e.g. a programmable gene editing system) includes (i) a CRISPR/Cas guide RNA, or a DNA encoding the CRISPR/Cas guide RNA; and (ii) a CRISPR/Cas RNA-guided polypeptide (Class 2 CRISPR/Cas effector protein) (e.g., Cas9, CasX, CasY, Cpf1, Cas13, MAD7, and the like), or a nucleic acid molecule encoding the RNA-guided polypeptide (e.g., DNA or RNA such as a plasmid or mRNA). As another example, in some cases a gene editing system (e.g. a programmable gene editing system) includes (i) an NgAgo-like guide DNA; and (ii) a DNA-guided polypeptide (e.g., NgAgo), or a nucleic acid molecule encoding the DNA-guided polypeptide (e.g., DNA or RNA such as a plasmid or mRNA). In some cases a gene editing system (e.g. a programmable gene editing system) includes at least three components: (i) a donor DNA template; (ii) a CRISPR/Cas guide RNA, or a DNA encoding the CRISPR/Cas guide RNA; and (iii) a CRISPR/Cas RNA-guided polypeptide (Class 2 CRISPR/Cas effector protein) (e.g., Cas9, CasX, CasY, Cpf1, Cas13, MAD7, and the like), or a nucleic acid molecule encoding the RNA-guided polypeptide (e.g., DNA or RNA such as a plasmid or mRNA). In some cases a gene editing system (e.g. a programmable gene editing system) includes at least three components: (i) a donor DNA template; (ii) an NgAgo-like guide DNA, or a DNA encoding the NgAgo-like guide DNA; and (iii) a DNA-guided polypeptide (e.g., NgAgo), or a nucleic acid molecule encoding the DNA-guided polypeptide (e.g., DNA or RNA such as a plasmid or mRNA).
In some embodiments, a subject delivery vehicle (e.g., nanoparticle) is used to deliver a gene editing tool. In other words in some cases the payload includes one or more gene editing tools. The term “gene editing tool” is used herein to refer to one or more components of a gene editing system. Thus, in some cases the payload includes a gene editing system and in some cases the payload includes one or more components of a gene editing system (i.e., one or more gene editing tools). For example, a target cell might already include one of the components of a gene editing system and the user need only add the remaining components. In such a case the payload of a subject delivery vehicle (e.g., nanoparticle) does not necessarily include all of the components of a given gene editing system. As such, in some cases a payload includes one or more gene editing tools.
As an illustrative example, a target cell might already include a gene editing protein (e.g., a ZFP, a TALE, a DNA-guided polypeptide (e.g., NgAgo), a CRISPR/Cas RNA-guided polypeptide (Class 2 CRISPR/Cas effector protein) (e.g., Cas9, CasX, CasY, Cpf1, Cas13, MAD7, and the like, a site-specific recombinase such as Cre recombinase, Dre recombinase, Flp recombinase, KD recombinase, B2 recombinase, B3 recombinase, R recombinase, Hin recombinase, Tre recombinase, PhiC31 integrase, Bxb 1 integrase, R4 integrase, lambda integrase, HK022 integrase, HP1 integrase, and the like, a resolvase/invertase such as Gin, Hin, γδ3, Tn3, Sin, Beta, and the like, a transposase, etc.) and/or a DNA or RNA encoding the protein, and therefore the payload can include one or more of: (i) a donor template; and (ii) a CRISPR/Cas guide RNA, or a DNA encoding the CRISPR/Cas guide RNA; or an NgAgo-like guide DNA. Likewise, the target cell may already include a CRISPR/Cas guide RNA and/or a DNA encoding the guide RNA or an NgAgo-like guide DNA, and the payload can include one or more of: (i) a donor template; and (ii) a CRISPR/Cas RNA-guided polypeptide (Class 2 CRISPR/Cas effector protein) (e.g., Cas9, CasX, CasY, Cpf1, Cas13, MAD7, and the like), or a nucleic acid molecule encoding the RNA-guided polypeptide (e.g., DNA or RNA such as a plasmid or mRNA); or a DNA-guided polypeptide (e.g., NgAgo), or a nucleic acid molecule encoding the DNA-guided polypeptide.
As would be understood by one of ordinary skill in the art, a gene editing system need not be a system that ‘edits’ a nucleic acid. For example, it is well recognized that a gene editing system can be used to modify target nucleic acids (e.g., DNA and/or RNA) in a variety of ways without creating a double strand break (DSB) in the target DNA. For example, in some cases a double stranded target DNA is nicked (one strand is cleaved), and in some cases (e.g., in some cases where the gene editing protein is devoid of nuclease activity, e.g., a CRISPR/Cas RNA-guided polypeptide may harbor mutations in the catalytic nuclease domains), the target nucleic acid is not cleaved at all. For example, in some cases a CRISPR/Cas protein (e.g., Cas9, CasX, CasY, Cpf1) with or without nuclease activity, is fused to a heterologous protein domain. The heterologous protein domain can provide an activity to the fusion protein such as (i) a DNA-modifying activity (e.g., nuclease activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer forming activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity or glycosylase activity), (ii) a transcription modulation activity (e.g., fusion to a transcriptional repressor or activator), or (iii) an activity that modifies a protein (e.g., a histone) that is associated with target DNA (e.g., methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity or demyristoylation activity). As such, a gene editing system can be used in applications that modify a target nucleic acid in way that do not cleave the target nucleic acid, and can also be used in applications that modulate transcription from a target DNA.
For additional information related to programmable gene editing tools (e.g., CRISPR/Cas RNa-guided proteins such as Cas9, CasX, CasY, Cpf1, Cas13, MAD7, and the like, Zinc finger proteins such as Zinc finger nucleases, TALE proteins such as TALENs, CRISPR/Cas guide RNAs, and the like) refer to, for example, Dreier, et al., (2001) J Biol Chem 276:29466-78; Dreier, et al., (2000) J Mol Biol 303:489-502; Liu, et al., (2002) J Biol Chem 277:3850-6); Dreier, et al., (2005) J Biol Chem 280:35588-97; Jamieson, et al., (2003) Nature Rev Drug Discov 2:361-8; Durai, et al., (2005) Nucleic Acids Res 33:5978-90; Segal, (2002) Methods 26:76-83; Porteus and Carroll, (2005) Nat Biotechnol 23:967-73; Pabo, et al., (2001) Ann Rev Biochem 70:313-40; Wolfe, et al., (2000) Ann Rev Biophys Biomol Struct 29:183-212; Segal and Barbas, (2001) Curr Opin Biotechnol 12:632-7; Segal, et al., (2003) Biochemistry 42:2137-48; Beerli and Barbas, (2002) Nat Biotechnol 20:135-41; Carroll, et al., (2006) Nature Protocols 1:1329; Ordiz, et al., (2002) Proc Natl Acad Sci USA 99:13290-5; Guan, et al., (2002) Proc Natl Acad Sci USA 99:13296-301; Sanjana et al., Nature Protocols, 7:171-192 (2012); Zetsche et al, Cell. 2015 Oct. 22; 163(3):759-71; Makarova et al, Nat Rev Microbiol. 2015 November; 13(11):722-36; Shmakov et al., Mol Cell. 2015 Nov. 5; 60(3):385-97; Jinek et al., Science. 2012 Aug. 17; 337(6096):816-21; Chylinski et al., RNA Biol. 2013 May; 10(5):726-37; Ma et al., Biomed Res Int. 2013; 2013:270805; Hou et al., Proc Natl Acad Sci USA. 2013 Sep. 24; 110(39):15644-9; Jinek et al., Elife. 2013; 2:e00471; Pattanayak et al., Nat Biotechnol. 2013 September; 31(9):839-43; Qi et al, Cell. 2013 Feb. 28; 152(5):1173-83; Wang et al., Cell. 2013 May 9; 153(4):910-8; Auer et. al., Genome Res. 2013 Oct. 31; Chen et. al., Nucleic Acids Res. 2013 Nov. 1; 41(20):e19; Cheng et. al., Cell Res. 2013 October; 23(10):1163-71; Cho et. al., Genetics. 2013 November; 195(3):1177-80; DiCarlo et al., Nucleic Acids Res. 2013 April; 41(7):4336-43; Dickinson et. al., Nat Methods. 2013 October; 10(10):1028-34; Ebina et. al., Sci Rep. 2013; 3:2510; Fujii et. al, Nucleic Acids Res. 2013 Nov. 1; 41(20):e187; Hu et. al., Cell Res. 2013 November; 23(11):1322-5; Jiang et. al., Nucleic Acids Res. 2013 Nov. 1; 41(20):e188; Larson et. al., Nat Protoc. 2013 November; 8(11):2180-96; Mali et. al., Nat Methods. 2013 October; 10(10):957-63; Nakayama et. al., Genesis. 2013 December; 51(12):835-43; Ran et. al., Nat Protoc. 2013 November; 8(11):2281-308; Ran et. al., Cell. 2013 Sep. 12; 154(6):1380-9; Upadhyay et. al., G3 (Bethesda). 2013 Dec. 9; 3(12):2233-8; Walsh et. al., Proc Natl Acad Sci USA. 2013 Sep. 24; 110(39):15514-5; Xie et. al., Mol Plant. 2013 Oct. 9; Yang et. al., Cell. 2013 Sep. 12; 154(6):1370-9; Briner et al., Mol Cell. 2014 Oct. 23; 56(2):333-9; Burstein et al., Nature. 2016 Dec. 22—Epub ahead of print; Gao et al., Nat Biotechnol. 2016 Jul. 34(7):768-73; as well as international patent application publication Nos. WO2002099084; WO00/42219; WO02/42459; WO2003062455; WO03/080809; WO05/014791; WO05/084190; WO08/021207; WO09/042186; WO09/054985; and WO10/065123; U.S. patent application publication Nos. 20030059767, 20030108880, 20140068797; 20140170753; 20140179006; 20140179770; 20140186843; 20140186919; 20140186958; 20140189896; 20140227787; 20140234972; 20140242664; 20140242699; 20140242700; 20140242702; 20140248702; 20140256046; 20140273037; 20140273226; 20140273230; 20140273231; 20140273232; 20140273233; 20140273234; 20140273235; 20140287938; 20140295556; 20140295557; 20140298547; 20140304853; 20140309487; 20140310828; 20140310830; 20140315985; 20140335063; 20140335620; 20140342456; 20140342457; 20140342458; 20140349400; 20140349405; 20140356867; 20140356956; 20140356958; 20140356959; 20140357523; 20140357530; 20140364333; 20140377868; 20150166983; and 20160208243; and U.S. Pat. Nos. 6,140,466; 6,511,808; 6,453,242 8,685,737; 8,906,616; 8,895,308; 8,889,418; 8,889,356; 8,871,445; 8,865,406; 8,795,965; 8,771,945; and 8,697,359; all of which are hereby incorporated by reference in their entirety.
In some cases an inserted nucleotide sequence (e.g., of a donor DNA) encodes a receptor whereby the target that is targeted (bound) by the receptor is specific to an individual's disease (e.g., cancer/tumor). In some cases an inserted nucleotide sequence (e.g., of a donor DNA) encodes a heteromultivalent receptor, whereby the combination of targets that are targeted by the heteromultivalent receptor are specific to an individual's disease (e.g., cancer/tumor). As one illustrative example, an individual's cancer (e.g., tumor, e.g., via biopsy) can be sequenced (nucleic acid sequence, proteomics, metabolomics etc.) to identify antigens of diseased cells that can be targets (such as antigens that are overexpressed by or are unique to a tumor relative to control cells of the individual), and a nucleotide sequence encoding a receptor (e.g., heteromultivalent receptor) that binds to one or more of those targets (e.g., 2 or more, 3 or more, 5 or more, 10 or more, 15 or more, or about 20 of those targets) can be inserted into an immune cell (e.g., an NK cell, a B-Cell, a T-Cell, e.g., using a CAR or TCR) so that the immune cell specifically targets the individual's disease cells (e.g., tumor cells). As such, an inserted nucleotide sequence (e.g., of a donor DNA) can be designed to be diagnostically responsive—in the sense that the encoded receptor(s) (e.g., heteromultivalent receptor(s)) can be designed after receiving unique insights related to a patient's proteomics, genomics or metabolomics (e.g., through sequencing etc.)—thus generating an avid and specific immune system response. In this way, immune cells (such as NK cells, B cell, T cells, and the like) can be genome edited to express receptors such as CAR and/or TCR proteins (e.g., heteromultivalent versions) that are designed to be effective against an individual's own disease (e.g., cancer). In some cases, regulatory T cells can be given similar avidity for tissues affected by autoimmunity following diagnostically-responsive medicine. In some cases, antigen presenting cells (such as Macrophages, Dendritic cells, B cells, and the like) can be edited to more effectively present or recognize antigens based on a diagnostically-responsive process.
In some cases the nucleotide sequence, of a donor DNA that is inserted into a cell's genome includes a protein-coding nucleotide sequence that does not have introns. In some cases the nucleotide sequence that does not have introns encodes all or a portion of a TCR protein.
In some embodiments more than one delivery vehicle is introduced into a target cell. For example, in some cases a subject method includes introducing a first and a second of said delivery vehicles into the cell, where a nucleotide sequence of a donor DNA of the first delivery vehicle, that is inserted into the cell's genome, encodes a T cell receptor (TCR) Alpha or Delta subunit, and the nucleotide sequence of the donor DNA of the second delivery vehicle, that is inserted into the cell's genome, encodes a TCR Beta or Gamma subunit. In some cases a subject method includes introducing a first and a second of said delivery vehicles into the cell, where the nucleotide sequence of the donor DNA of the first delivery vehicle, that is inserted into the cell's genome, encodes a T cell receptor (TCR) Alpha or Delta subunit constant region, and the nucleotide sequence of the donor DNA of the second delivery vehicle, that is inserted into the cell's genome, encodes a TCR Beta or Gamma subunit constant region.
In some cases a subject method includes introducing a first and a second of said delivery vehicles into the cell, wherein the nucleotide sequence of a donor DNA of the first delivery vehicle is inserted within a nucleotide sequence that functions as a T cell receptor (TCR) Alpha or Delta subunit promoter, and the nucleotide sequence of a donor DNA of the second delivery vehicle is inserted within a nucleotide sequence that functions as a TCR Beta or Gamma subunit promoter. For more information related to TCR proteins and CDRs, see, e.g., Dash et al., Nature. 2017 Jul. 6; 547(7661):89-93. Epub 2017 Jun. 21; and Glanville et al., Nature. 2017 Jul. 6; 547(7661):94-98. Epub 2017 Jun. 21. In some cases, a 147 bp TCRbeta promoter can drive high cell-specific gene expression in T cells, and may include the sequence:
Agtcacccaagtgtggtctaatataaatcctgtgttcctgaggtcatgcagattgagagaggaagtgatgtcactgtgggaacttccgtgtaagga cggggcgtccctcctcctctgctcctgctcacagtgatcctgatctggtaa (SEQ ID NO: xx)
In some cases a subject method includes introducing a first and a second of said delivery vehicles into the cell, where the nucleotide sequence of a donor DNA of the first delivery vehicle, that is inserted into the cell's genome, encodes a T cell receptor (TCR) Alpha or Gamma subunit, and the nucleotide sequence of a donor DNA of the second delivery vehicle, that is inserted into the cell's genome, encodes a TCR Beta or Delta subunit. In some cases a subject method includes introducing a first and a second of said delivery vehicles into the cell, where the nucleotide sequence of the donor DNA of the first delivery vehicle, that is inserted into the cell's genome, encodes a T cell receptor (TCR) Alpha or Delta subunit constant region, and the nucleotide sequence of the donor DNA of the second delivery vehicle, that is inserted into the cell's genome, encodes a TCR Beta or Gamma subunit constant region. In some cases a subject method includes introducing a first and a second of said delivery vehicles into the cell, wherein the nucleotide sequence of the donor DNA of the first delivery vehicle is inserted within a nucleotide sequence that functions as a T cell receptor (TCR) Alpha or Gamma subunit promoter, and the nucleotide sequence of the donor DNA of the second delivery vehicle is inserted within a nucleotide sequence that functions as a TCR Beta or Delta subunit promoter. For more information related to TCR proteins and CDRs, see, e.g., Dash et al., Nature. 2017 Jul. 6; 547(7661):89-93. Epub 2017 Jun. 21; and Glanville et al., Nature. 2017 Jul. 6; 547(7661):94-98. Epub 2017 Jun. 21.
Payloads for Co-Delivery
In some embodiments, more than one payload is delivered as part of the same package (e.g., nanoparticle), e.g., in some cases different payloads are part of different cores. One advantage of delivering multiple payloads as part of the same package (e.g., nanoparticle) is that the efficiency of each payload is not diluted. As an illustrative example, if payload A and payload B are delivered in two separate packages (package A and package B, respectively), then the efficiencies are multiplicative, e.g., if package A and package B each have a 1% transfection efficiency, the chance of delivering payload A and payload B to the same cell is 0.01% (1%×1%). However, if payload A and payload B are both delivered as part of the same package (e.g., part of the same nanoparticle—package A), then the chance of delivering payload A and payload B to the same cell is 1%, a 100-fold improvement over 0.01%.
Likewise, in a scenario where package A and package B each have a 0.1% transfection efficiency, the chance of delivering payload A and payload B to the same cell is 0.0001% (0.1%×0.1%). However, if payload A and payload B are both delivered as part of the same package (e.g., part of the same nanoparticle—package A) in this scenario, then the chance of delivering payload A and payload B to the same cell is 0.1%, a 1000-fold improvement over 0.0001%.
As such, in some embodiments, one or more gene editing tools (e.g., as described above) is delivered in combination with (e.g., as part of the same nanoparticle) a protein (and/or a DNA or mRNA encoding same) and/or a non-coding RNA that increases genomic editing efficiency. In some cases, one or more gene editing tools (e.g., as described above) is delivered in combination with (e.g., as part of the same nanoparticle) a protein (and/or a DNA or mRNA encoding same) and/or a non-coding RNA that controls cell division and/or differentiation. In some cases, one or more gene editing tools (e.g., as described above) is delivered in combination with (e.g., as part of the same nanoparticle) a protein (and/or a DNA or mRNA encoding same) and/or a non-coding RNA that biases the cell DNA repair machinery toward non-homologous end joining (NHEJ) or homology directed repair (HDR).
As non-limiting examples of the above, in some embodiments one or more gene editing tools can be delivered in combination with one or more of: SCF (and/or a DNA or mRNA encoding SCF), HoxB4 (and/or a DNA or mRNA encoding HoxB4), BCL-XL (and/or a DNA or mRNA encoding BCL-XL), SIRT6 (and/or a DNA or mRNA encoding SIRT6), a nucleic acid molecule (e.g., an siRNA and/or an LNA) that suppresses miR-155, a nucleic acid molecule (e.g., an siRNA, an shRNA, a microRNA) that reduces ku70 expression, and a nucleic acid molecule (e.g., an siRNA, an shRNA, a microRNA) that reduces ku80 expression.
For examples of microRNAs that can be delivered in combination with a gene editing tool, see
For examples of signaling proteins (e.g., extracellular signaling proteins) that can be delivered (e.g., as protein or as DNA or RNA encoding the protein) in combination with a gene editing tool, see
Examples of proteins that can be delivered (e.g., as protein and/or a nucleic acid such as DNA or RNA encoding the protein) in combination with a gene editing tool include but are not limited to: SOX17, HEX, OSKM (Oct4/Sox2/K1f4/c-myc), and/or bFGF (e.g., to drive differentiation toward hepatic stem cell lineage); HNF4a (e.g., to drive differentiation toward hepatocyte fate); Poly (I:C), BMP-4, bFGF, and/or 8-Br-cAMP (e.g., to drive differentiation toward endothelial stem cell/progenitor lineage); VEGF (e.g., to drive differentiation toward arterial endothelium fate); Sox-2, Brn4, Mytl1, Neurod2, Ascl1 (e.g., to drive differentiation toward neural stem cell/progenitor lineage); and BDNF, FCS, Forskolin, and/or SHH (e.g., to drive differentiation neuron, astrocyte, and/or oligodendrocyte fate).
Examples of signaling proteins (e.g., extracellular signaling proteins) that can be delivered (e.g., as protein and/or a nucleic acid such as DNA or RNA encoding the protein) in combination with a gene editing tool include but are not limited to: cytokines (e.g., IL-2 and/or IL-15, e.g., for activating CD8+ T-cells); ligands and or signaling proteins that modulate one or more of the Notch, Wnt, and/or Smad signaling pathways; SCF; stem cell differentiating factors (e.g. Sox2, Oct3/4, Nanog, Klf4, c-Myc, and the like); and temporary surface marker “tags” and/or fluorescent reporters for subsequent isolation/purification/concentration. For example, a fibroblast may be converted into a neural stem cell via delivery of Sox2, while it will turn into a cardiomyocyte in the presence of Oct3/4 and small molecule “epigenetic resetting factors.” In a patient with Huntington's disease or a CXCR4 mutation, these fibroblasts may respectively encode diseased phenotypic traits associated with neurons and cardiac cells. By delivering gene editing corrections and these factors in a single package, the risk of deleterious effects due to one or more, but not all of the factors/payloads being introduced can be significantly reduced.
Because the timing and/or location of payload release can be controlled (described in more detail elsewhere in this disclosure), the packaging of multiple payloads in the same package (e.g., same nanoparticle) does not preclude one from achieving different release times and/or locations for different payloads. For example the release of the above proteins (and/or a DNAs or mRNAs encoding same) and/or non-coding RNAs can be controlled separately from the release of the one or more gene editing tools that are part of the same package. For example, proteins and/or nucleic acids (e.g., DNAs, mRNAs, non-coding RNAs, miRNAs) that control cell proliferation and/or differentiation, or that control bias toward NHEJ or HDR, can be released earlier than the one or more gene editing tools or can be released later than the one or more gene editing tools. This can be achieved, e.g., by using more than one sheddable layer and/or by using more than one core (e.g., where one core has a different release profile than the other, e.g., uses a different D- to L-isomer ratio, uses a different ESP:ENP:EPP profile, and the like).
Applications include in vivo approaches wherein a cell death cue may be conditional upon a gene edit not being successful, and cell differentiation/proliferation/activation is tied to a tissue/organ-specific promoter and/or exogenous factor. A diseased cell receiving a gene edit may activate and proliferate, but due to the presence of another promoter-driven expression cassette (e.g. one tied to the absence of tumor suppressor such as p21 or p53), those cells will subsequently be eliminated. The cells expressing desired characteristics, on the other hand, may be triggered to further differentiate into the desired downstream lineages.
In some cases, a subject nucleic acid payload includes a morpholino backbone structure. In some case, a subject nucleic acid payload can have one or more locked nucleic acids (LNAs). Suitable sugar substituent groups include methoxy (—O—CH3), aminopropoxy (—O CH2 CH2 CH2NH2), allyl (—CH2—CH═CH2), —O-allyl (—O— CH2—CH═CH2) and fluoro (F). 2′-sugar substituent groups may be in the arabino (up) position or ribo (down) position. Suitable base modifications include synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (—C═C—CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine(1H-pyrimido(5,4-b)(1,4)benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido(5,4-(b) (1,4)benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido(4,5-b)indol-2-one), pyridoindole cytidine (H-pyrido(3′,2′:4,5)pyrrolo(2,3-d)pyrimidin-2-one).
In some cases, a nucleic acid payload can include a conjugate moiety (e.g., one that enhances the activity, stability, cellular distribution or cellular uptake of the nucleic acid payload). These moieties or conjugates can include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups. Conjugate groups include, but are not limited to, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Suitable conjugate groups include, but are not limited to, cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties include groups that improve uptake, enhance resistance to degradation, and/or strengthen sequence-specific hybridization with the target nucleic acid. Groups that enhance the pharmacokinetic properties include groups that improve uptake, distribution, metabolism or excretion of a subject nucleic acid.
Any convenient polynucleotide can be used as a subject nucleic acid payload. Examples include but are not limited to: species of RNA and DNA including mRNA, m1A modified mRNA (monomethylation at position 1 of Adenosine), morpholino RNA, peptoid and peptide nucleic acids, cDNA, DNA origami, DNA and RNA with synthetic nucleotides, DNA and RNA with predefined secondary structures, and multimers and oligomers of the aforementioned.
Because the timing and/or location of payload release can be controlled (described in more detail elsewhere in this disclosure), the packaging of multiple payloads in the same package (e.g., same nanoparticle) does not preclude one from achieving different release times/rates and/or locations for different payloads. For example, the release of the above proteins (and/or a DNAs or mRNAs encoding same) and/or non-coding RNAs can be controlled separately from the release of the one or more gene editing tools that are part of the same package. For example, proteins and/or nucleic acids (e.g., DNAs, mRNAs, non-coding RNAs, miRNAs) that control cell proliferation and/or differentiation can be released earlier than the one or more gene editing tools or can be released later than the one or more gene editing tools. This can be achieved, e.g., by using more than one sheddable layer and/or by using more than one core (e.g., where one core has a different release profile than the other, e.g., uses a different D- to L-isomer ratio, uses a different ESP:ENP:EPP profile, and the like). In this way, a donor and nuclease may be released in a stepwise manner that allows for optimal editing and insertion efficiencies.
Nanoparticles of the disclosure include a payload, which can be made of nucleic acid and/or protein. For example, in some cases a subject nanoparticle is used to deliver a nucleic acid payload (e.g., a DNA and/or RNA). The payloads function to influence cellular phenotype, or result in the expression of proteins to be secreted or presented on the cell surface. In some cases the core of the nanoparticle includes the payload(s). In some such cases a nanoparticle core can also include an anionic polymer composition, a cationic polymer composition, and a cationic polypeptide composition. In some cases the nanoparticle has a metallic core and the payload associates with (in some cases is conjugated to, e.g., the outside of) the core. In some embodiments, the payload is part of the nanoparticle core. Thus the core of a subject nanoparticle can include nucleic acid, DNA, RNA, and/or protein. Thus, in some cases a subject nanoparticle includes nucleic acid (DNA and/or RNA) and protein. In some cases a subject nanoparticle core includes a ribonucleoprotein (RNA and protein) complex. In some cases a subject nanoparticle core includes a deoxyribonucleoprotein (DNA and protein, e.g., donor DNA and ZFN, TALEN, or CRISPR/Cas effector protein) complex. In some cases a subject nanoparticle core includes a ribo-deoxyribonucleoprotein (RNA and DNA and protein, e.g., a guide RNA, a donor DNA and a CRISPR/Cas effector protein) complex. In some cases a subject nanoparticle core includes PNAs. In some cases a subject core includes PNAs and DNAs.
Nanoparticles as described herein are modular and can be tailored for various scenarios: for example, each component (e.g., payload, core, coat, targeting ligand, etc.) can be selected based on the desired outcome, e.g., as part of a set of degrees of freedom across the entire nanoparticle platform.
Nanoparticle Core
The core of a subject nanoparticle can include an anionic polymer composition (e.g., poly(glutamic acid)), a cationic polymer composition (e.g., poly(arginine), a cationic polypeptide composition (e.g., a histone tail peptide), and a payload (e.g., nucleic acid and/or protein payload). In some cases the core is generated by condensation of a cationic amino acid polymer and payload in the presence of an anionic amino acid polymer (and in some cases in the presence of a cationic polypeptide of a cationic polypeptide composition). In some embodiments, condensation of the components that make up the core can mediate increased transfection efficiency compared to conjugates of cationic polymers with a payload. Inclusion of an anionic polymer in a nanoparticle core may prolong the duration of intracellular residence of the nanoparticle and release of payload.
Other nanoparticle cores may include proteins as substrates, whereas a molecule such as Cas9 has its surface modified by subsequent electrostatic or covalent layers encoding cell-specific targeting, subcellular trafficking characteristics, or tethering together multiple payloads (e.g. Cas9 protein and RNP forms with DNA covalently attached).
For the cationic and anionic polymer compositions of the core, ratios of D-isomer polymers to L-isomer polymers can be controlled in order to control the timed release of payload, where increased ratio of D-isomer polymers to L-isomer polymers leads to increased stability (reduced payload release rate), which for example can enable longer lasting gene expression from a payload delivered by a subject nanoparticle. In some cases modifying the ratio of D-to-L isomer polypeptides within the nanoparticle core can cause gene expression profiles (e.g., expression of a protein encoded by a payload molecule) to be on the order of from 1-90 days (e.g. from 1-80, 1-70, 1-60, 1-50, 1-40, 1-30, 1-25, 1-20, 1-15, 1-10, 3-90, 3-80, 3-70, 3-60, 3-50, 3-40, 3-30, 3-25, 3-20, 3-15, 3-10, 5-90, 5-80, 5-70, 5-60, 5-50, 5-40, 5-30, 5-25, 5-20, 5-15, or 5-10 days). The control of payload release (e.g., when delivering a gene editing tool), can be particularly effective for performing genomic edits e.g., in some cases where homology-directed repair is desired.
In some embodiments, a nanoparticle includes a core and a sheddable layer encapsulating the core, where the core includes: (a) an anionic polymer composition; (b) a cationic polymer composition; (c) a cationic polypeptide composition; and (d) a nucleic acid and/or protein payload, where one of (a) and (b) includes a D-isomer polymer of an amino acid, and the other of (a) and (b) includes an L-isomer polymer of an amino acid, and where the ratio of the D-isomer polymer to the L-isomer polymer is in a range of from 10:1 to 1.5:1 (e.g., from 8:1 to 1.5:1, 6:1 to 1.5:1, 5:1 to 1.5:1, 4:1 to 1.5:1, 3:1 to 1.5:1, 2:1 to 1.5:1, 10:1 to 2:1; 8:1 to 2:1, 6:1 to 2:1, 5:1 to 2:1, 10:1 to 3:1; 8:1 to 3:1, 6:1 to 3:1, 5:1 to 3:1, 10:1 to 4:1; 4:1 to 2:1, 6:1 to 4:1, or 10:1 to 5:1), or from 1:1.5 to 1:10 (e.g., from 1:1.5 to 1:8, 1:1.5 to 1:6, 1:1.5 to 1:5, 1:1.5 to 1:4, 1:1.5 to 1:3, 1:1.5 to 1:2, 1:2 to 1:10, 1:2 to 1:8, 1:2 to 1:6, 1:2 to 1:5, 1:2 to 1:4, 1:2 to 1:3, 1:3 to 1:10, 1:3 to 1:8, 1:3 to 1:6, 1:3 to 1:5, 1:4 to 1:10, 1:4 to 1:8, 1:4 to 1:6, or 1:5 to 1:10). In some such cases, the ratio of the D-isomer polymer to the L-isomer polymer is not 1:1. In some such cases, the anionic polymer composition includes an anionic polymer selected from poly(D-glutamic acid) (PDEA) and poly(D-aspartic acid) (PDDA), where (optionally) the cationic polymer composition can include a cationic polymer selected from poly(L-arginine), poly(L-lysine), poly(L-histidine), poly(L-ornithine), and poly(L-citrulline). In some cases the cationic polymer composition comprises a cationic polymer selected from poly(D-arginine), poly(D-lysine), poly(D-histidine), poly(D-ornithine), and poly(D-citrulline), where (optionally) the anionic polymer composition can include an anionic polymer selected from poly(L-glutamic acid) (PLEA) and poly(L-aspartic acid) (PLDA).
In some embodiments, a nanoparticle includes a core and a sheddable layer encapsulating the core, where the core includes: (i) an anionic polymer composition; (ii) a cationic polymer composition; (iii) a cationic polypeptide composition; and (iv) a nucleic acid and/or protein payload, wherein (a) said anionic polymer composition includes polymers of D-isomers of an anionic amino acid and polymers of L-isomers of an anionic amino acid; and/or (b) said cationic polymer composition includes polymers of D-isomers of a cationic amino acid and polymers of L-isomers of a cationic amino acid. In some such cases, the anionic polymer composition comprises a first anionic polymer selected from poly(D-glutamic acid) (PDEA) and poly(D-aspartic acid) (PDDA); and comprises a second anionic polymer selected from poly(L-glutamic acid) (PLEA) and poly(L-aspartic acid) (PLDA). In some cases, the cationic polymer composition comprises a first cationic polymer selected from poly(D-arginine), poly(D-lysine), poly(D-histidine), poly(D-ornithine), and poly(D-citrulline); and comprises a second cationic polymer selected from poly(L-arginine), poly(L-lysine), poly(L-histidine), poly(L-ornithine), and poly(L-citrulline). In some cases, the polymers of D-isomers of an anionic amino acid are present at a ratio, relative to said polymers of L-isomers of an anionic amino acid, in a range of from 10:1 to 1:10. In some cases, the polymers of D-isomers of a cationic amino acid are present at a ratio, relative to said polymers of L-isomers of a cationic amino acid, in a range of from 10:1 to 1:10.
Nanoparticle Components (Delayed and/or Extended Payload Release)
In some embodiments, timing of payload release can be controlled by selecting particular types of proteins, e.g., as part of the core (e.g., part of a cationic polypeptide composition, part of a cationic polymer composition, and/or part of an anionic polymer composition). For example, it may be desirable to delay payload release for a particular range of time, or until the payload is present at a particular cellular location (e.g., cytosol, nucleus, lysosome, endosome) or under a particular condition (e.g., low pH, high pH, etc.). As such, in some cases a protein is used (e.g., as part of the core) that is susceptible to a specific protein activity (e.g., enzymatic activity), e.g., is a substrate for a specific protein activity (e.g., enzymatic activity), and this is in contrast to being susceptible to general ubiquitous cellular machinery, e.g., general degradation machinery. A protein that is susceptible to a specific protein activity is referred to herein as an ‘enzymatically susceptible protein’ (ESP). Illustrative examples of ESPs include but are not limited to: (i) proteins that are substrates for matrix metalloproteinase (MMP) activity (an example of an extracellular activity), e.g., a protein that includes a motif recognized by an MMP; (ii) proteins that are substrates for cathepsin activity (an example of an intracellular endosomal activity), e.g., a protein that includes a motif recognized by a cathepsin; and (iii) proteins such as histone tails peptides (HTPs) that are substrates for methyltransferase and/or acetyltransferase activity (an example of an intracellular nuclear activity), e.g., a protein that includes a motif that can be enzymatically methylated/de-methylated and/or a motif that can be enzymatically acetylated/de-acetylated. For example, in some cases a nucleic acid payload is condensed with a protein (such as a histone tails peptide) that is a substrate for acetyltransferase activity, and acetylation of the protein causes the protein to release the payload—as such, one can exercise control over payload release by choosing to use a protein that is more or less susceptible to acetylation.
In some cases, a core of a subject nanoparticle includes an enzymatically neutral polypeptide (ENP), which is a polypeptide homopolymer (i.e., a protein having a repeat sequence) where the polypeptide does not have a particular activity and is neutral. For example, unlike NLS sequences and HTPs, both of which have a particular activity, ENPs do not.
In some cases, a core of a subject nanoparticle includes an enzymatically protected polypeptide (EPP), which is a protein that is resistant to enzymatic activity. Examples of PPs include but are not limited to: (i) polypeptides that include D-isomer amino acids (e.g., D-isomer polymers), which can resist proteolytic degradation; and (ii) self-sheltering domains such as a polyglutamine repeat domains (e.g., QQQQQQQQQQ) (SEQ ID NO: 170).
By controlling the relative amounts of susceptible proteins (ESPs), neutral proteins (ENPs), and protected proteins (EPPs), that are part of a subject nanoparticle (e.g., part of the nanoparticle core), one can control the release of payload. For example, use of more ESPs can in general lead to quicker release of payload than use of more EPPs. In addition, use of more ESPs can in general lead to release of payload that depends upon a particular set of conditions/circumstances, e.g., conditions/circumstances that lead to activity of proteins (e.g., enzymes) to which the ESP is susceptible.
In some cases, ratios of carrier molecules relative to one another are modulating while designing delivery vehicle (e.g., nanoparticle) formulations. Term “carrier molecules” refers to components of the delivery vehicle that are not the payload or targeting ligand—for example: anionic polymer, cationic polymer, cationic polypeptide (e.g., HTP), a lipid, and the like.
Anionic Polymer Composition (e.g., of a Nanoparticle)
An anionic polymer composition can include one or more anionic amino acid polymers. For example, in some cases a subject anionic polymer composition includes a polymer selected from: poly(glutamic acid)(PEA), poly(aspartic acid)(PDA), and a combination thereof. In some cases a given anionic amino acid polymer can include a mix of aspartic and glutamic acid residues. Each polymer can be present in the composition as a polymer of L-isomers or D-isomers, where D-isomers are more stable in a target cell because they take longer to degrade. Thus, inclusion of D-isomer poly(amino acids) in the nanoparticle core delays degradation of the core and subsequent payload release. A suitable ratio of D to L isomer polypeptides can be determined by performing a robotic screen utilizing a formulator app, such as shown in
In some cases an anionic polymer composition of a subject nanoparticle includes polymers of D-isomers and polymers of L-isomers of an anionic amino acid polymer (e.g., poly(glutamic acid)(PEA) and poly(aspartic acid)(PDA)). In some cases the D- to L-isomer ratio is in a range of from 10:1-1:10 (e.g., from 8:1-1:10, 6:1-1:10, 4:1-1:10, 3:1-1:10, 2:1-1:10, 1:1-1:10, 10:1-1:8, 8:1-1:8, 6:1-1:8, 4:1-1:8, 3:1-1:8, 2:1-1:8, 1:1-1:8, 10:1-1:6, 8:1-1:6, 6:1-1:6, 4:1-1:6, 3:1-1:6, 2:1-1:6, 1:1-1:6, 10:1-1:4, 8:1-1:4, 6:1-1:4, 4:1-1:4, 3:1-1:4, 2:1-1:4, 1:1-1:4, 10:1-1:3, 8:1-1:3, 6:1-1:3, 4:1-1:3, 3:1-1:3, 2:1-1:3, 1:1-1:3, 10:1-1:2, 8:1-1:2, 6:1-1:2, 4:1-1:2, 3:1-1:2, 2:1-1:2, 1:1-1:2, 10:1-1:1, 8:1-1:1, 6:1-1:1, 4:1-1:1, 3:1-1:1, or 2:1-1:1).
Thus, in some cases an anionic polymer composition includes a first anionic polymer (e.g., amino acid polymer) that is a polymer of D-isomers (e.g., selected from poly(D-glutamic acid) (PDEA) and poly(D-aspartic acid) (PDDA)); and includes a second anionic polymer (e.g., amino acid polymer) that is a polymer of L-isomers (e.g., selected from poly(L-glutamic acid) (PLEA) and poly(L-aspartic acid) (PLDA)). In some cases the ratio of the first anionic polymer (D-isomers) to the second anionic polymer (L-isomers) is in a range of from 10:1-1:10 (e.g., from 8:1-1:10, 6:1-1:10, 4:1-1:10, 3:1-1:10, 2:1-1:10, 1:1-1:10, 10:1-1:8, 8:1-1:8, 6:1-1:8, 4:1-1:8, 3:1-1:8, 2:1-1:8, 1:1-1:8, 10:1-1:6, 8:1-1:6, 6:1-1:6, 4:1-1:6, 3:1-1:6, 2:1-1:6, 1:1-1:6, 10:1-1:4, 8:1-1:4, 6:1-1:4, 4:1-1:4, 3:1-1:4, 2:1-1:4, 1:1-1:4, 10:1-1:3, 8:1-1:3, 6:1-1:3, 4:1-1:3, 3:1-1:3, 2:1-1:3, 1:1-1:3, 10:1-1:2, 8:1-1:2, 6:1-1:2, 4:1-1:2, 3:1-1:2, 2:1-1:2, 1:1-1:2, 10:1-1:1, 8:1-1:1, 6:1-1:1, 4:1-1:1, 3:1-1:1, or 2:1-1:1).
In some embodiments, an anionic polymer composition of a core of a subject nanoparticle includes (e.g., in addition to or in place of any of the foregoing examples of anionic polymers) a glycosaminoglycan, a glycoprotein, a polysaccharide, poly(mannuronic acid), poly(guluronic acid), heparin, heparin sulfate, chondroitin, chondroitin sulfate, keratan, keratan sulfate, aggrecan, poly(glucosamine), or an anionic polymer that comprises any combination thereof.
In some embodiments, an anionic polymer within the core can have a molecular weight in a range of from 1-200 kDa (e.g., from 1-150, 1-100, 1-50, 5-200, 5-150, 5-100, 5-50, 10-200, 10-150, 10-100, 10-50, 15-200, 15-150, 15-100, or 15-50 kDa). As an example, in some cases an anionic polymer includes poly(glutamic acid) with a molecular weight of approximately 15 kDa.
In some cases, an anionic amino acid polymer includes a cysteine residue, which can facilitate conjugation, e.g., to a linker, an NLS, and/or a cationic polypeptide (e.g., a histone or HTP). For example, a cysteine residue can be used for crosslinking (conjugation) via sulfhydryl chemistry (e.g., a disulfide bond) and/or amine-reactive chemistry. Thus, in some embodiments an anionic amino acid polymer (e.g., poly(glutamic acid) (PEA), poly(aspartic acid) (PDA), poly(D-glutamic acid) (PDEA), poly(D-aspartic acid) (PDDA), poly(L-glutamic acid) (PLEA), poly(L-aspartic acid) (PLDA)) of an anionic polymer composition includes a cysteine residue. In some cases the anionic amino acid polymer includes cysteine residue on the N- and/or C-terminus. In some cases the anionic amino acid polymer includes an internal cysteine residue.
In some cases, an anionic amino acid polymer includes (and/or is conjugated to) a nuclear localization signal (NLS) (described in more detail below). Thus, in some embodiments an anionic amino acid polymer (e.g., poly(glutamic acid) (PEA), poly(aspartic acid) (PDA), poly(D-glutamic acid) (PDEA), poly(D-aspartic acid) (PDDA), poly(L-glutamic acid) (PLEA), poly(L-aspartic acid) (PLDA)) of an anionic polymer composition includes (and/or is conjugated to) one or more (e.g., two or more, three or more, or four or more) NLSs. In some cases the anionic amino acid polymer includes an NLS on the N- and/or C-terminus. In some cases the anionic amino acid polymer includes an internal NLS.
In some cases, an anionic polymer is added prior to a cationic polymer when generating a subject nanoparticle core. In some cases, the matrix output of a robotic synthesis of various D:L isomer ratios of constituent polypeptides in a given nanoparticle screen can be used as an input variable for subsequent machine learning and recursive optimization approaches of additional degrees of freedom of the nanoparticle platform as shown in
Cationic Polymer Composition (e.g., of a Nanoparticle)
A cationic polymer composition can include one or more cationic amino acid polymers. For example, in some cases a subject cationic polymer composition includes a polymer selected from: poly(arginine)(PR), poly(lysine)(PK), poly(histidine)(PH), poly(ornithine), poly(citrulline), and a combination thereof. In some cases a given cationic amino acid polymer can include a mix of arginine, lysine, histidine, ornithine, and citrulline residues (in any convenient combination). Each polymer can be present in the composition as a polymer of L-isomers or D-isomers, where D-isomers are more stable in a target cell because they take longer to degrade. Thus, inclusion of D-isomer poly(amino acids) in the nanoparticle core delays degradation of the core and subsequent payload release. The payload release rate can therefore be controlled and is proportional to the ratio of polymers of D-isomers to polymers of L-isomers, where a higher ratio of D-isomer to L-isomer increases duration of payload release (i.e., decreases release rate). In other words, the relative amounts of D- and L-isomers can modulate the nanoparticle core's timed release kinetics and enzymatic susceptibility to degradation and payload release.
In some cases a cationic polymer composition of a subject nanoparticle includes polymers of D-isomers and polymers of L-isomers of an cationic amino acid polymer (e.g., poly(arginine)(PR), poly(lysine)(PK), poly(histidine)(PH), poly(ornithine), poly(citrulline)). In some cases the D- to L-isomer ratio is in a range of from 10:1-1:10 (e.g., from 8:1-1:10, 6:1-1:10, 4:1-1:10, 3:1-1:10, 2:1-1:10, 1:1-1:10, 10:1-1:8, 8:1-1:8, 6:1-1:8, 4:1-1:8, 3:1-1:8, 2:1-1:8, 1:1-1:8, 10:1-1:6, 8:1-1:6, 6:1-1:6, 4:1-1:6, 3:1-1:6, 2:1-1:6, 1:1-1:6, 10:1-1:4, 8:1-1:4, 6:1-1:4, 4:1-1:4, 3:1-1:4, 2:1-1:4, 1:1-1:4, 10:1-1:3, 8:1-1:3, 6:1-1:3, 4:1-1:3, 3:1-1:3, 2:1-1:3, 1:1-1:3, 10:1-1:2, 8:1-1:2, 6:1-1:2, 4:1-1:2, 3:1-1:2, 2:1-1:2, 1:1-1:2, 10:1-1:1, 8:1-1:1, 6:1-1:1, 4:1-1:1, 3:1-1:1, or 2:1-1:1).
Thus, in some cases a cationic polymer composition includes a first cationic polymer (e.g., amino acid polymer) that is a polymer of D-isomers (e.g., selected from poly(D-arginine), poly(D-lysine), poly(D-histidine), poly(D-ornithine), and poly(D-citrulline)); and includes a second cationic polymer (e.g., amino acid polymer) that is a polymer of L-isomers (e.g., selected from poly(L-arginine), poly(L-lysine), poly(L-histidine), poly(L-ornithine), and poly(L-citrulline)). In some cases the ratio of the first cationic polymer (D-isomers) to the second cationic polymer (L-isomers) is in a range of from 10:1-1:10 (e.g., from 8:1-1:10, 6:1-1:10, 4:1-1:10, 3:1-1:10, 2:1-1:10, 1:1-1:10, 10:1-1:8, 8:1-1:8, 6:1-1:8, 4:1-1:8, 3:1-1:8, 2:1-1:8, 1:1-1:8, 10:1-1:6, 8:1-1:6, 6:1-1:6, 4:1-1:6, 3:1-1:6, 2:1-1:6, 1:1-1:6, 10:1-1:4, 8:1-1:4, 6:1-1:4, 4:1-1:4, 3:1-1:4, 2:1-1:4, 1:1-1:4, 10:1-1:3, 8:1-1:3, 6:1-1:3, 4:1-1:3, 3:1-1:3, 2:1-1:3, 1:1-1:3, 10:1-1:2, 8:1-1:2, 6:1-1:2, 4:1-1:2, 3:1-1:2, 2:1-1:2, 1:1-1:2, 10:1-1:1, 8:1-1:1, 6:1-1:1, 4:1-1:1, 3:1-1:1, or 2:1-1:1)
In some embodiments, a cationic polymer composition of a core of a subject nanoparticle includes (e.g., in addition to or in place of any of the foregoing examples of cationic polymers) poly(ethylenimine), poly(amidoamine) (PAMAM), poly(aspartamide), polypeptoids (e.g., for forming “spiderweb”-like branches for core condensation), a charge-functionalized polyester, a cationic polysaccharide, an acetylated amino sugar, chitosan, or a cationic polymer that comprises any combination thereof (e.g., in linear or branched forms).
In some embodiments, a cationic polymer within the core can have a molecular weight in a range of from 1-200 kDa (e.g., from 1-150, 1-100, 1-50, 5-200, 5-150, 5-100, 5-50, 10-200, 10-150, 10-100, 10-50, 15-200, 15-150, 15-100, or 15-50 kDa). As an example, in some cases a cationic polymer includes poly(L-arginine), e.g., with a molecular weight of approximately 29 kDa. As another example, in some cases a cationic polymer includes linear poly(ethylenimine) with a molecular weight of approximately 25 kDa (PEI). As another example, in some cases a cationic polymer includes branched poly(ethylenimine) with a molecular weight of approximately 10 kDa. As another example, in some cases a cationic polymer includes branched poly(ethylenimine) with a molecular weight of approximately 70 kDa. In some cases a cationic polymer includes PAMAM.
In some cases, a cationic amino acid polymer includes a cysteine residue, which can facilitate conjugation, e.g., to a linker, an NLS, and/or a cationic polypeptide (e.g., a histone or HTP). For example, a cysteine residue can be used for crosslinking (conjugation) via sulfhydryl chemistry (e.g., a disulfide bond) and/or amine-reactive chemistry. Thus, in some embodiments a cationic amino acid polymer (e.g., poly(arginine)(PR), poly(lysine)(PK), poly(histidine)(PH), poly(ornithine), and poly(citrulline), poly(D-arginine)(PDR), poly(D-lysine)(PDK), poly(D-histidine)(PDH), poly(D-ornithine), and poly(D-citrulline), poly(L-arginine)(PLR), poly(L-lysine)(PLK), poly(L-histidine)(PLH), poly(L-ornithine), and poly(L-citrulline)) of a cationic polymer composition includes a cysteine residue. In some cases the cationic amino acid polymer includes cysteine residue on the N- and/or C-terminus. In some cases the cationic amino acid polymer includes an internal cysteine residue.
In some cases, a cationic amino acid polymer includes (and/or is conjugated to) a nuclear localization signal (NLS) (described in more detail below). Thus, in some embodiments a cationic amino acid polymer (e.g., poly(arginine)(PR), poly(lysine)(PK), poly(histidine)(PH), poly(ornithine), and poly(citrulline), poly(D-arginine)(PDR), poly(D-lysine)(PDK), poly(D-histidine)(PDH), poly(D-ornithine), and poly(D-citrulline), poly(L-arginine)(PLR), poly(L-lysine)(PLK), poly(L-histidine)(PLH), poly(L-ornithine), and poly(L-citrulline)) of a cationic polymer composition includes (and/or is conjugated to) one or more (e.g., two or more, three or more, or four or more) NLSs. In some cases the cationic amino acid polymer includes an NLS on the N- and/or C-terminus. In some cases the cationic amino acid polymer includes an internal NLS.
Cationic Polypeptide Composition (e.g., of a Nanoparticle)
In some embodiments the cationic polypeptide composition of a nanoparticle can mediate stability, subcellular compartmentalization, and/or payload release. As one example, fragments of the N-terminus of histone proteins, referred to generally as histone tail peptides, within a subject nanoparticle core are in some case not only capable of being deprotonated by various histone modifications, such as in the case of histone acetyltransferase-mediated acetylation, but may also mediate effective nuclear-specific unpackaging of components (e.g., a payload) of a nanoparticle core. In some cases a cationic polypeptide composition includes a histone and/or histone tail peptide (e.g., a cationic polypeptide can be a histone and/or histone tail peptide). In some cases a cationic polypeptide composition includes an NLS-containing peptide (e.g., a cationic polypeptide can be an NLS-containing peptide). In some cases, a cationic polypeptide composition includes one or more NLS-containing peptides separated by cysteine residues to facilitate crosslinking. In some cases a cationic polypeptide composition includes a peptide that includes a mitochondrial localization signal (e.g., a cationic polypeptide can be a peptide that includes a mitochondrial localization signal).
Histone Tail Peptide (HTPs)
In some embodiments a cationic polypeptide composition (e.g., of a subject nanoparticle) includes a histone peptide or a fragment of a histone peptide, such as an N-terminal histone tail (e.g., a histone tail of an H1, H2 (e.g., H2A, H2AX, H2B), H3, or H4 histone protein). A tail fragment of a histone protein is referred to herein as a histone tail peptide (HTP). Because such a protein (a histone and/or HTP) can condense with a nucleic acid payload as part of the core of a subject nanoparticle, a core that includes one or more histones or HTPs (e.g., as part of the cationic polypeptide composition) is sometimes referred to herein as a nucleosome-mimetic core. Histones and/or HTPs can be included as monomers, and in some cases form dimers, trimers, tetramers and/or octamers when condensing a nucleic acid payload into a nanoparticle core. In some cases HTPs are not only capable of being deprotonated by various histone modifications, such as in the case of histone acetyltransferase-mediated acetylation, but may also mediate effective nuclear-specific unpackaging of components of the core (e.g., release of a payload). Trafficking of a core that includes a histone and/or HTP may be reliant on alternative endocytotic pathways utilizing retrograde transport through the Golgi and endoplasmic reticulum. Furthermore, some histones include an innate nuclear localization sequence and inclusion of an NLS in the core can direct the core (including the payload) to the nucleus of a target cell.
In some embodiments a subject cationic polypeptide composition includes a protein having an amino acid sequence of an H2A, H2AX, H2B, H3, or H4 protein. In some cases a subject cationic polypeptide composition includes a protein having an amino acid sequence that corresponds to the N-terminal region of a histone protein. For example, the fragment (an HTP) can include the first 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 N-terminal amino acids of a histone protein. In some cases, a subject HTP includes from 5-50 amino acids (e.g., from 5-45, 5-40, 5-35, 5-30, 5-25, 5-20, 8-50, 8-45, 8-40, 8-35, 8-30, 10-50, 10-45, 10-40, 10-35, or 10-30 amino acids) from the N-terminal region of a histone protein. In some cases a subject a cationic polypeptide includes from 5-150 amino acids (e.g., from 5-100, 5-50, 5-35, 5-30, 5-25, 5-20, 8-150, 8-100, 8-50, 8-40, 8-35, 8-30, 10-150, 10-100, 10-50, 10-40, 10-35, or 10-30 amino acids).
In some cases a cationic polypeptide (e.g., a histone or HTP, e.g., H1, H2, H2A, H2AX, H2B, H3, or H4) of a cationic polypeptide composition includes a post-translational modification (e.g., in some cases on one or more histidine, lysine, arginine, or other complementary residues). For example, in some cases the cationic polypeptide is methylated (and/or susceptible to methylation/demethylation), acetylated (and/or susceptible to acetylation/deacetylation), crotonylated (and/or susceptible to crotonylation/decrotonylation), ubiquitinylated (and/or susceptible to ubiquitinylation/deubiquitinylation), phosphorylated (and/or susceptible to phosphorylation/dephosphorylation), SUMOylated (and/or susceptible to SUMOylation/deSUMOylation), farnesylated (and/or susceptible to farnesylation/defarnesylation), sulfated (and/or susceptible to sulfation/desulfation) or otherwise post-translationally modified. In some cases a cationic polypeptide (e.g., a histone or HTP, e.g., H1, H2, H2A, H2AX, H2B, H3, or H4) of a cationic polypeptide composition is p300/CBP substrate (e.g., see example HTPs below, e.g., SEQ ID NOs: 129-130). In some cases a cationic polypeptide (e.g., a histone or HTP, e.g., H1, H2, H2A, H2AX, H2B, H3, or H4) of a cationic polypeptide composition includes one or more thiol residues (e.g., can include a cysteine and/or methionine residue) that is sulfated or susceptible to sulfation (e.g., as a thiosulfate sulfurtransferase substrate). In some cases a cationic polypeptide (e.g., a histone or HTP, e.g., H1, H2, H2A, H2AX, H2B, H3, or H4) of a cationic polypeptide is amidated on the C-terminus. Histones H2A, H2B, H3, and H4 (and/or HTPs) may be monomethylated, dimethylated, or trimethylated at any of their lysines to promote or suppress transcriptional activity and alter nuclear-specific release kinetics.
A cationic polypeptide can be synthesized with a desired modification or can be modified in an in vitro reaction. Alternatively, a cationic polypeptide (e.g., a histone or HTP) can be expressed in a cell population and the desired modified protein can be isolated/purified. In some cases the cationic polypeptide composition of a subject nanoparticle includes a methylated HTP, e.g., includes the HTP sequence of H3K4(Me3)—includes the amino acid sequence set forth as SEQ ID NO: 75 or 88). In some cases a cationic polypeptide (e.g., a histone or HTP, e.g., H1, H2, H2A, H2AX, H2B, H3, or H4) of a cationic polypeptide composition includes a C-terminal amide.
Examples of Histones and HTPs
Examples include but are not limited to the following sequences:
As such, a cationic polypeptide of a subject cationic polypeptide composition can include an amino acid sequence having the amino acid sequence set forth in any of SEQ ID NOs: 62-139. In some cases a cationic polypeptide of subject a cationic polypeptide composition includes an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, or 100% sequence identity) with the amino acid sequence set forth in any of SEQ ID NOs: 62-139. In some cases a cationic polypeptide of subject a cationic polypeptide composition includes an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 98% or more, 99% or more, or 100% sequence identity) with the amino acid sequence set forth in any of SEQ ID NOs: 62-139. The cationic polypeptide can include any convenient modification, and a number of such contemplated modifications are discussed above, e.g., methylated, acetylated, crotonylated, ubiquitinylated, phosphorylated, SUMOylated, farnesylated, sulfated, and the like.
In some cases a cationic polypeptide of a cationic polypeptide composition includes an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, or 100% sequence identity) with the amino acid sequence set forth in SEQ ID NO: 94. In some cases a cationic polypeptide of a cationic polypeptide composition includes an amino acid sequence having 95% or more sequence identity (e.g., 98% or more, 99% or more, or 100% sequence identity) with the amino acid sequence set forth in SEQ ID NO: 94. In some cases a cationic polypeptide of a cationic polypeptide composition includes the amino acid sequence set forth in SEQ ID NO: 94. In some cases a cationic polypeptide of a cationic polypeptide composition includes the sequence represented by H3K4(Me3) (SEQ ID NO: 95), which comprises the first 25 amino acids of the human histone 3 protein, and tri-methylated on the lysine 4 (e.g., in some cases amidated on the C-terminus).
In some embodiments a cationic polypeptide (e.g., a histone or HTP, e.g., H1, H2, H2A, H2AX, H2B, H3, or H4) of a cationic polypeptide composition includes a cysteine residue, which can facilitate conjugation to: a cationic (or in some cases anionic) amino acid polymer, a linker, an NLS, and/or other cationic polypeptides (e.g., in some cases to form a branched histone structure). For example, a cysteine residue can be used for crosslinking (conjugation) via sulfhydryl chemistry (e.g., a disulfide bond) and/or amine-reactive chemistry. In some cases the cysteine residue is internal. In some cases the cysteine residue is positioned at the N-terminus and/or C-terminus. In some cases, a cationic polypeptide (e.g., a histone or HTP, e.g., H1, H2, H2A, H2AX, H2B, H3, or H4) of a cationic polypeptide composition includes a mutation (e.g., insertion or substitution) that adds a cysteine residue. Examples of HTPs that include a cysteine include but are not limited to:
In some embodiments a cationic polypeptide (e.g., a histone or HTP, e.g., H1, H2, H2A, H2AX, H2B, H3, or H4) of a cationic polypeptide composition is conjugated to a cationic (and/or anionic) amino acid polymer of the core of a subject nanoparticle. As an example, a histone or HTP can be conjugated to a cationic amino acid polymer (e.g., one that includes poly(lysine)), via a cysteine residue, e.g., where the pyridyl disulfide group(s) of lysine(s) of the polymer are substituted with a disulfide bond to the cysteine of a histone or HTP.
Modified/Branching Structure
In some embodiments a cationic polypeptide of a subject a cationic polypeptide composition has a linear structure. In some embodiments a cationic polypeptide of a subject a cationic polypeptide composition has a branched structure.
For example, in some cases, a cationic polypeptide (e.g., HTPs, e.g., HTPs with a cysteine residue) is conjugated (e.g., at its C-terminus) to the end of a cationic polymer (e.g., poly(L-arginine), poly(D-lysine), poly(L-lysine), poly(D-lysine)), thus forming an extended linear polypeptide. In some cases, one or more (two or more, three or more, etc.) cationic polypeptides (e.g., HTPs, e.g., HTPs with a cysteine residue) are conjugated (e.g., at their C-termini) to the end(s) of a cationic polymer (e.g., poly(L-arginine), poly(D-lysine), poly(L-lysine), poly(D-lysine)), thus forming an extended linear polypeptide. In some cases the cationic polymer has a molecular weight in a range of from 4,500-150,000 Da).
As another example, in some cases, one or more (two or more, three or more, etc.) cationic polypeptides (e.g., HTPs, e.g., HTPs with a cysteine residue) are conjugated (e.g., at their C-termini) to the side-chains of a cationic polymer (e.g., poly(L-arginine), poly(D-lysine), poly(L-lysine), poly(D-lysine)), thus forming a branched structure (branched polypeptide).
Formation of a branched structure by components of the nanoparticle core (e.g., components of a subject cationic polypeptide composition) can in some cases increase the amount of core condensation (e.g., of a nucleic acid payload) that can be achieved. Thus, in some cases it is desirable to used components that form a branched structure. Various types of branches structures are of interest, and examples of branches structures that can be generated (e.g., using subject cationic polypeptides such as HTPs, e.g., HTPs with a cysteine residue; peptoids, polyamides, and the like) include but are not limited to: brush polymers, webs (e.g., spider webs), graft polymers, star-shaped polymers, comb polymers, polymer networks, dendrimers, and the like.
In some cases, a branched structure includes from 2-30 cationic polypeptides (e.g., HTPs) (e.g., from 2-25, 2-20, 2-15, 2-10, 2-5, 4-30, 4-25, 4-20, 4-15, or 4-10 cationic polypeptides), where each can be the same or different than the other cationic polypeptides of the branched structure. In some cases the cationic polymer has a molecular weight in a range of from 4,500-150,000 Da). In some cases, 5% or more (e.g., 10% or more, 20% or more, 25% or more, 30% or more, 40% or more, or 50% or more) of the side-chains of a cationic polymer (e.g., poly(L-arginine), poly(D-lysine), poly(L-lysine), poly(D-lysine)) are conjugated to a subject cationic polypeptide (e.g., HTP, e.g., HTP with a cysteine residue). In some cases, up to 50% (e.g., up to 40%, up to 30%, up to 25%, up to 20%, up to 15%, up to 10%, or up to 5%) of the side-chains of a cationic polymer (e.g., poly(L-arginine), poly(D-lysine), poly(L-lysine), poly(D-lysine)) are conjugated to a subject cationic polypeptide (e.g., HTP, e.g., HTP with a cysteine residue). Thus, an HTP can be branched off of the backbone of a polymer such as a cationic amino acid polymer.
In some cases formation of branched structures can be facilitated using components such as peptoids (polypeptoids), polyamides, dendrimers, and the like. For example, in some cases peptoids (e.g., polypeptoids) are used as a component of a nanoparticle core, e.g., in order to generate a web (e.g., spider web) structure, which can in some cases facilitate condensation of the nanoparticle core.
One or more of the natural or modified polypeptide sequences herein may be modified with terminal or intermittent arginine, lysine, or histidine sequences. In one embodiment, each polypeptide is included in equal amine molarities within a nanoparticle core. In this embodiment, each polypeptide's C-terminus can be modified with 5R (5 arginines). In some embodiments, each polypeptide's C-terminus can be modified with 9R (9 arginines). In some embodiments, each polypeptide's N-terminus can be modified with 5R (5 arginines). In some embodiments, each polypeptide's N-terminus can be modified with 9R (9 arginines). In some cases, an H2A, H2B, H3 and/or H4 histone fragment (e.g., HTP) are each bridged in series with a FKFL Cathepsin B proteolytic cleavage domain or RGFFP Cathepsin D proteolytic cleavage domain. In some cases, an H2A, H2B, H3 and/or H4 histone fragment (e.g., HTP) can be bridged in series by a 5R (5 arginines), 9R (9 arginines), 5K (5 lysines), 9K (9 lysines), 5H (5 histidines), or 9H (9 histidines) cationic spacer domain. In some cases, one or more H2A, H2B, H3 and/or H4 histone fragments (e.g., HTPs) are disulfide-bonded at their N-terminus to protamine.
To illustrate how to generate a branched histone structure, example methods of preparation are provided. One example of such a method includes the following: covalent modification of equimolar ratios of Histone H2AX [134-143], Histone H3 [1-21 Cys], Histone H3 [23-34 Cys], Histone H4 [8-25 WC] and SV40 T-Ag-derived NLS can be performed in a reaction with 10% pyridyl disulfide modified poly(L-Lysine) [MW=5400, 18000, or 45000 Da; n=30, 100, or 250]. In a typical reaction, a 29 μL aqueous solution of 700 μM Cys-modified histone/NLS (20 nmol) can be added to 57 μL of 0.2 M phosphate buffer (pH 8.0). Second, 14 μL of 100 μM pyridyl disulfide protected poly(lysine) solution can then be added to the histone solution bringing the final volume to 100 μL with a 1:2 ratio of pyridyl disulfide groups to Cysteine residues. This reaction can be carried out at room temperature for 3 h. The reaction can be repeated four times and degree of conjugation can be determined via absorbance of pyridine-2-thione at 343 nm.
As another example, covalent modification of a 0:1, 1:4, 1:3, 1:2, 1:1, 1:2, 1:3, 1:4, or 1:0 molar ratio of Histone H3 [1-21 Cys] peptide and Histone H3 [23-34 Cys] peptide can be performed in a reaction with 10% pyridyl disulfide modified poly(L-Lysine) or poly(L-Arginine) [MW=5400, 18000, or 45000 Da; n=30, 100, or 250]. In a typical reaction, a 29 μL aqueous solution of 700 μM Cys-modified histone (20 nmol) can be added to 57 μL of 0.2 M phosphate buffer (pH 8.0). Second, 14 μL of 100 μM pyridyl disulfide protected poly(lysine) solution can then be added to the histone solution bringing the final volume to 100 with a 1:2 ratio of pyridyl disulfide groups to Cysteine residues. This reaction can be carried out at room temperature for 3 h. The reaction can be repeated four times and degree of conjugation can be determined via absorbance of pyridine-2-thione at 343 nm.
In some cases, an anionic polymer is conjugated to a targeting ligand.
Nuclear Localization Sequence (NLS)
In some embodiments a cationic polypeptide (e.g., a histone or HTP, e.g., H1, H2, H2A, H2AX, H2B, H3, or H4) of a cationic polypeptide composition includes (and/or is conjugated to) one or more (e.g., two or more, three or more, or four or more) nuclear localization sequences (NLSs). Thus in some cases the cationic polypeptide composition of a subject nanoparticle includes a peptide that includes an NLS. In some cases a histone protein (or an HTP) of a subject nanoparticle includes one or more (e.g., two or more, three or more) natural nuclear localization signals (NLSs). In some cases a histone protein (or an HTP) of a subject nanoparticle includes one or more (e.g., two or more, three or more) NLSs that are heterologous to the histone protein (i.e., NLSs that do not naturally occur as part of the histone/HTP, e.g., an NLS can be added by humans). In some cases the HTP includes an NLS on the N- and/or C-terminus.
In some embodiments a cationic amino acid polymer (e.g., poly(arginine)(PR), poly(lysine)(PK), poly(histidine)(PH), poly(ornithine), poly(citrulline), poly(D-arginine)(PDR), poly(D-lysine)(PDK), poly(D-histidine)(PDH), poly(D-ornithine), poly(D-citrulline), poly(L-arginine)(PLR), poly(L-lysine)(PLK), poly(L-histidine)(PLH), poly(L-ornithine), or poly(L-citrulline)) of a cationic polymer composition includes (and/or is conjugated to) one or more (e.g., two or more, three or more, or four or more) NLSs. In some cases the cationic amino acid polymer includes an NLS on the N- and/or C-terminus. In some cases the cationic amino acid polymer includes an internal NLS.
In some embodiments an anionic amino acid polymer (e.g., poly(glutamic acid) (PEA), poly(aspartic acid) (PDA), poly(D-glutamic acid) (PDEA), poly(D-aspartic acid) (PDDA), poly(L-glutamic acid) (PLEA), or poly(L-aspartic acid) (PLDA)) of an anionic polymer composition includes (and/or is conjugated to) one or more (e.g., two or more, three or more, or four or more) NLSs. In some cases the anionic amino acid polymer includes an NLS on the N- and/or C-terminus. In some cases the anionic amino acid polymer includes an internal NLS.
Any convenient NLS can be used (e.g., conjugated to a histone, an HTP, a cationic amino acid polymer, an anionic amino acid polymer, and the like). Examples include, but are not limited to Class 1 and Class 2 ‘monopartite NLSs’, as well as NLSs of Classes 3-5 (see, e.g.,
In some embodiments a cationic polypeptide of a cationic polypeptide composition includes one more (e.g., two or more, three or more, or four or more) NLSs. In some cases the cationic polypeptide is not a histone protein or histone fragment (e.g., is not an HTP). Thus, in some cases the cationic polypeptide of a cationic polypeptide composition is an NLS-containing peptide.
In some cases, the NLS-containing peptide includes a cysteine residue, which can facilitate conjugation to: a cationic (or in some cases anionic) amino acid polymer, a linker, histone protein for HTP, and/or other cationic polypeptides (e.g., in some cases as part of a branched histone structure). For example, a cysteine residue can be used for crosslinking (conjugation) via sulfhydryl chemistry (e.g., a disulfide bond) and/or amine-reactive chemistry. In some cases the cysteine residue is internal. In some cases the cysteine residue is positioned at the N-terminus and/or C-terminus. In some cases, an NLS-containing peptide of a cationic polypeptide composition includes a mutation (e.g., insertion or substitution) (e.g., relative to a wild type amino acid sequence) that adds a cysteine residue.
Examples of NLSs that can be used as an NLS-containing peptide (or conjugated to any convenient cationic polypeptide such as an HTP or cationic polymer or cationic amino acid polymer or anionic amino acid polymer) include but are not limited to (some of which include a cysteine residue):
For non-limiting examples of NLSs that can be used, see, e.g., Kosugi et al., J Biol Chem. 2009 Jan. 2; 284(1):478-85, e.g., see
Mitochondrial Localization Signal
In some embodiments a cationic polypeptide (e.g., a histone or HTP, e.g., H1, H2, H2A, H2AX, H2B, H3, or H4), an anionic polymer, and/or a cationic polymer of a subject nanoparticle includes (and/or is conjugated to) one or more (e.g., two or more, three or more, or four or more) mitochondrial localization sequences. Any convenient mitochondrial localization sequence can be used. Examples of mitochondrial localization sequences include but are not limited to: PEDEIWLPEPESVDVPAKPISTSSMMMP (SEQ ID NO: 149), a mitochondrial localization sequence of SDHB, mono/di/triphenylphosphonium or other phosphoniums, VAMP 1A, VAMP 1B, the 67 N-terminal amino acids of DGAT2, and the 20 N-terminal amino acids of Bax.
Sheddable Layer (Sheddable Coat)—e.g., of a Nanoparticle
In some embodiments, a subject nanoparticle includes a sheddable layer (also referred to herein as a “transient stabilizing layer”) that surrounds (encapsulates) the core. In some cases a subject sheddable layer can protect the payload before and during initial cellular uptake. For example, without a sheddable layer, much of the payload can be lost during cellular internalization. Once in the cellular environment, a sheddable layer ‘sheds’ (e.g., the layer can be pH- and/or or glutathione-sensitive), exposing the components of the core.
In some cases a subject sheddable layer includes silica. In some cases, when a subject nanoparticle includes a sheddable layer (e.g., of silica), greater intracellular delivery efficiency can be observed despite decreased probability of cellular uptake. Without wishing to be bound by any particular theory, coating a nanoparticle core with a sheddable layer (e.g., silica coating) can seal the core, stabilizing it until shedding of the layer, which leads to release of the payload (e.g., upon processing in the intended subcellular compartment). Following cellular entry through receptor-mediated endocytosis, the nanoparticle sheds its outermost layer, the sheddable layer degrades in the acidifying environment of the endosome or reductive environment of the cytosol, and exposes the core, which in some cases exposes localization signals such as nuclear localization signals (NLSs) and/or mitochondrial localization signals. Moreover, nanoparticle cores encapsulated by a sheddable layer can be stable in serum and can be suitable for administration in vivo.
Any desired sheddable layer can be used, and one of ordinary skill in the art can take into account where in the target cell (e.g., under what conditions, such as low pH) they desire the payload to be released (e.g., endosome, cytosol, nucleus, lysosome, and the like). Different sheddable layers may be more desirable depending on when, where, and/or under what conditions it would be desirable for the sheddable coat to shed (and therefore release the payload). For example, a sheddable layer can be acid labile. In some cases the sheddable layer is an anionic sheddable layer (an anionic coat). In some cases the sheddable layer comprises silica, a peptoid, a polycysteine, and/or a ceramic (e.g., a bioceramic). In some cases the sheddable includes one or more of: calcium, manganese, magnesium, iron (e.g., the sheddable layer can be magnetic, e.g., Fe3MnO2), and lithium. Each of these can include phosphate or sulfate. As such, in some cases the sheddable includes one or more of: calcium phosphate, calcium sulfate, manganese phosphate, manganese sulfate, magnesium phosphate, magnesium sulfate, iron phosphate, iron sulfate, lithium phosphate, and lithium sulfate; each of which can have a particular effect on how and/or under which conditions the sheddable layer will ‘shed.’ Thus, in some cases the sheddable layer includes one or more of: silica, a peptoid, a polycysteine, a ceramic (e.g., a bioceramic), calcium, calcium phosphate, calcium sulfate, calcium oxide, hydroxyapatite, manganese, manganese phosphate, manganese sulfate, manganese oxide, magnesium, magnesium phosphate, magnesium sulfate, magnesium oxide, iron, iron phosphate, iron sulfate, iron oxide, lithium, lithium phosphate, and lithium sulfate (in any combination thereof) (e.g., the sheddable layer can be a coating of silica, peptoid, polycysteine, a ceramic (e.g., a bioceramic), calcium phosphate, calcium sulfate, manganese phosphate, manganese sulfate, magnesium phosphate, magnesium sulfate, iron phosphate, iron sulfate, lithium phosphate, lithium sulfate, or a combination thereof). In some cases the sheddable layer includes silica (e.g., the sheddable layer can be a silica coat). In some cases the sheddable layer includes an alginate gel. For example a sheddable layer can in some cases be composed of biocompatible ceramic, organic or biopolymer functionalized ceramic, anionic polypeptides, or cationic polypeptides.
A sheddable layer may include peptide domains that promote endosomal escape or organelle localization such as nuclear localization signals. Additionally, Cathepsin-cleavable and MMP-cleavable domains may be included to promote accumulation and subsequent activity within specific cellular and tissue environments.
In some cases different release times for different payloads are desirable. For example, in some cases it is desirable to release a payload early (e.g., within 0.5-7 days of contacting a target cell) and in some cases it is desirable to release a payload late (e.g., within 6 days-30 days of contacting a target cell). For example, in some cases it may be desirable to release a payload (e.g., a gene editing tool such as a CRISPR/Cas guide RNA, a DNA molecule encoding said CRISPR/Cas guide RNA, a CRISPR/Cas RNA-guided polypeptide, and/or a nucleic acid molecule encoding said CRISPR/Cas RNA-guided polypeptide) within 0.5-7 days of contacting a target cell (e.g., within 0.5-5 days, 0.5-3 days, 1-7 days, 1-5 days, or 1-3 days of contacting a target cell). In some cases it may be desirable to release a payload (e.g., a Donor DNA molecule) within 6-40 days of contacting a target cell (e.g., within 6-30, 6-20, 6-15, 7-40, 7-30, 7-20, 7-15, 9-40, 9-30, 9-20, or 9-15 days of contacting a target cell). In some cases release times can be controlled by delivering nanoparticles having different payloads at different times. In some cases release times can be controlled by delivering nanoparticles at the same time (as part of different formulations or as part of the same formulation), where the components of the nanoparticle are designed to achieve the desired release times. For example, one may use a sheddable layer that degrades faster or slower, core components that are more or less resistant to degradation, core components that are more or less susceptible to de-condensation, etc. —and any or all of the components can be selected in any convenient combination to achieve the desired timing.
In some cases it is desirable to delay the release of a payload (e.g., a Donor DNA molecule) relative to another payload (e.g., one or more gene editing tools). As an example, in some cases a first nanoparticle includes a donor DNA molecule as a payload is designed such that the payload is released within 6-40 days of contacting a target cell (e.g., within 6-30, 6-20, 6-15, 7-40, 7-30, 7-20, 7-15, 9-40, 9-30, 9-20, or 9-15 days of contacting a target cell), while a second nanoparticle that includes one or more gene editing tools (e.g., a ZFP or nucleic acid encoding the ZFP, a TALE or a nucleic acid encoding the TALE, a ZFN or nucleic acid encoding the ZFN, a TALEN or a nucleic acid encoding the TALEN, a CRISPR/Cas guide RNA or DNA molecule encoding the CRISPR/Cas guide RNA, a CRISPR/Cas RNA-guided polypeptide or a nucleic acid molecule encoding the CRISPR/Cas RNA-guided polypeptide, and the like) as a payload is designed such that the payload is released within 0.5-7 days of contacting a target cell (e.g., within 0.5-5 days, 0.5-3 days, 1-7 days, 1-5 days, or 1-3 days of contacting a target cell). The second nanoparticle can be part of the same or part of a different formulation as the first nanoparticle.
In some cases, a nanoparticle includes more than one payload, where it is desirable for the payloads to be released at different times. This can be achieved in a number of different ways. For example, a nanoparticle can have more than one core, where one core is made with components that can release the payload early (e.g., within 0.5-7 days of contacting a target cell, e.g., within 0.5-5 days, 0.5-3 days, 1-7 days, 1-5 days, or 1-3 days of contacting a target cell) (e.g., an siRNA, an mRNA, and/or a genome editing tool such as a ZFP or nucleic acid encoding the ZFP, a TALE or a nucleic acid encoding the TALE, a ZFN or nucleic acid encoding the ZFN, a TALEN or a nucleic acid encoding the TALEN, a CRISPR/Cas guide RNA or DNA molecule encoding the CRISPR/Cas guide RNA, a CRISPR/Cas RNA-guided polypeptide or a nucleic acid molecule encoding the CRISPR/Cas RNA-guided polypeptide, and the like) and the other is made with components that can release the payload (e.g., a Donor DNA molecule) later (e.g., within 6-40 days of contacting a target cell, e.g., within 6-30, 6-20, 6-15, 7-40, 7-30, 7-20, 7-15, 9-40, 9-30, 9-20, or 9-15 days of contacting a target cell).
As another example, a nanoparticle can include more than one sheddable layer, where the outer sheddable layer is shed (releasing a payload) prior to an inner sheddable layer being shed (releasing another payload). In some cases, the inner payload is a Donor DNA molecule and the outer payload is one or more gene editing tools (e.g., a ZFN or nucleic acid encoding the ZFN, a TALEN or a nucleic acid encoding the TALEN, a CRISPR/Cas guide RNA or DNA molecule encoding the CRISPR/Cas guide RNA, a CRISPR/Cas RNA-guided polypeptide or a nucleic acid molecule encoding the CRISPR/Cas RNA-guided polypeptide, and the like). The inner and outer payloads can be any desired payload and either or both can include, for example, one or more siRNAs and/or one or more mRNAs. As such, in some cases a nanoparticle can have more than one sheddable layer and can be designed to release one payload early (e.g., within 0.5-7 days of contacting a target cell, e.g., within 0.5-5 days, 0.5-3 days, 1-7 days, 1-5 days, or 1-3 days of contacting a target cell) (e.g., an siRNA, an mRNA, a genome editing tool such as a ZFP or nucleic acid encoding the ZFP, a TALE or a nucleic acid encoding the TALE, a ZFN or nucleic acid encoding the ZFN, a TALEN or a nucleic acid encoding the TALEN, a CRISPR/Cas guide RNA or DNA molecule encoding the CRISPR/Cas guide RNA, a CRISPR/Cas RNA-guided polypeptide or a nucleic acid molecule encoding the CRISPR/Cas RNA-guided polypeptide, and the like), and another payload (e.g., an siRNA, an mRNA, a Donor DNA molecule) later (e.g., within 6-40 days of contacting a target cell, e.g., within 6-30, 6-20, 6-15, 7-40, 7-30, 7-20, 7-15, 9-40, 9-30, 9-20, or 9-15 days of contacting a target cell).
In some embodiments (e.g., in embodiments described above), time of altered gene expression can be used as a proxy for the time of payload release. As an illustrative example, if one desires to determine if a payload has been released by day 12, one can assay for the desired result of nanoparticle delivery on day 12. For example, if the desired result was to reduce the expression of a target gene of the target cell, e.g., by delivering an siRNA, then the expression of the target gene can be assayed/monitored to determine if the siRNA has been released. As another example, if the desired result was to express a protein of interest, e.g., by delivering a DNA or mRNA encoding the protein of interest, then the expression of the protein of interest can be assayed/monitored to determine if the payload has been released. As yet another example, if the desired result was to alter the genome of the target cell, e.g., via cleaving genomic DNA and/or inserting a sequence of a donor DNA molecule, the expression from the targeted locus and/or the presence of genomic alterations can be assayed/monitored to determine if the payload has been released.
As such, in some cases a sheddable layer provides for a staged release of nanoparticle components. For example, in some cases, a nanoparticle has more than one (e.g., two, three, or four) sheddable layers. For example, for a nanoparticle with two sheddable layers, such a nanoparticle can have, from inner-most to outer-most: a core, e.g., with a first payload; a first sheddable layer, an intermediate layer e.g., with a second payload; and a second sheddable layer surrounding the intermediate layer (see, e.g.,
Surface Coat (Outer Shell) of a Nanoparticle
In some cases, the sheddable layer (the coat), is itself coated by an additional layer, referred to herein as an “outer shell,” “outer coat,” or “surface coat.” A surface coat can serve multiple different functions. For example, a surface coat can increase delivery efficiency and/or can target a subject nanoparticle to a particular cell type. The surface coat can include a peptide, a polymer, or a ligand-polymer conjugate. The surface coat can include a targeting ligand. The surface coat may be a layer upon a substrate (e.g. nanoparticle with electrostatic surface) or may contain its own conjugation or electrostatic condensation domains that independently present a ligand on the surface of a nanoparticle (see click chemistry and electrostatic approaches detailed elsewhere). For example, an aqueous solution of one or more targeting ligands (with or without linker domains) can be added to a coated nanoparticle suspension (suspension of nanoparticles coated with a sheddable layer). For example, in some cases the final concentration of protonated anchoring residues (of an anchoring domain) is between 25 and 300 μM. In some cases, the process of adding the surface coat yields a monodispersed suspension of particles with a mean particle size between 50 and 150 nm and a zeta potential between 0 and −10 mV.
In some cases the surface coat includes a targeting ligand (described in more detail elsewhere herein). In some cases the surface coat includes a stealth motif. A stealth motif is a motif that renders an entity (e.g., a pathogen, a nanoparticle, etc.) invisible a host immune system. Examples of stealth motifs include but are not limited to: polysialic acid, sialic acid and/or neuraminic acid functionalized peptides, hyaluronan, other anionic polypeptide/peptoid/polymer sequences, other glycoprotein modifications, brushed glycoproteins and anionic branches, native human-derived peptide sequences or sequences not found in databases of immunogenicity, and polyethylene glycol [see, e.g., Deepagan et al, J Nanosci Nanotechnol. 2013 Nov.; 13(11):7312-8; Sperisen et al., PLoS Comput Biol. 2005 November; 1(6):e6; and Yu et al., J Control Release. 2016 Oct. 28; 240:24-37]
In some cases, the surface coat interacts electrostatically with the outermost sheddable layer. For example, in some cases, a nanoparticle has two sheddable layers (e.g., from inner-most to outer-most: a core, e.g., with a first payload; a first sheddable layer, an intermediate layer e.g., with a second payload; and a second sheddable layer surrounding the intermediate layer), and the outer shell (surface coat) can interact with (e.g., electrostatically) the second sheddable layer. In some cases, a nanoparticle has only one sheddable layer (e.g., an anionic silica layer), and the outer shell can in some cases electrostatically interact with the sheddable layer.
Thus, in cases where the sheddable layer (e.g., outermost sheddable layer) is anionic (e.g., in some cases where the sheddable layer is a silica coat), the surface coat can interact electrostatically with the sheddable layer if the surface coat includes a cationic component. For example, in some cases the surface coat includes a delivery molecule in which a targeting ligand is conjugated to a cationic anchoring domain. The cationic anchoring domain interacts electrostatically with the sheddable layer and anchors the delivery molecule to the nanoparticle. Likewise, in cases where the sheddable layer (e.g., outermost sheddable layer) is cationic, the surface coat can interact electrostatically with the sheddable layer if the surface coat includes an anionic component.
In some embodiments, the surface coat includes a cell penetrating peptide (CPP). In some cases, a polymer of a cationic amino acid can function as a CPP (also referred to as a ‘protein transduction domain’-PTD), which is a term used to refer to a polypeptide, polynucleotide, carbohydrate, or organic or inorganic compound that facilitates traversing a lipid bilayer, micelle, cell membrane, organelle membrane, or vesicle membrane. A PTD attached to another molecule (e.g., embedded in and/or interacting with a sheddable layer of a subject nanoparticle), which can range from a small polar molecule to a large macromolecule and/or a nanoparticle, facilitates the molecule traversing a membrane, for example going from extracellular space to intracellular space, or cytosol to within an organelle (e.g., the nucleus).
Examples of CPPs include but are not limited to a minimal undecapeptide protein transduction domain (corresponding to residues 47-57 of HIV-1 TAT comprising YGRKKRRQRRR (SEQ ID NO: 160); a polyarginine sequence comprising a number of arginines sufficient to direct entry into a cell (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or 10-50 arginines); a VP22 domain (Zender et al. (2002) Cancer Gene Ther. 9(6):489-96); an Drosophila Antennapedia protein transduction domain (Noguchi et al. (2003) Diabetes 52(7):1732-1737); a truncated human calcitonin peptide (Trehin et al. (2004) Pharm. Research 21:1248-1256); polylysine (Wender et al. (2000) Proc. Natl. Acad. Sci. USA 97:13003-13008); RRQRRTSKLMKR (SEQ ID NO: 161); Transportan GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 162); KALAWEAKLAKALAKALAKHLAKALAKALKCEA (SEQ ID NO: 163); and RQIKIWFQNRRMKWKK (SEQ ID NO: 164). Example CPPs include but are not limited to: YGRKKRRQRRR (SEQ ID NO: 160), RKKRRQRRR (SEQ ID NO: 165), an arginine homopolymer of from 3 arginine residues to 50 arginine residues, RKKRRQRR (SEQ ID NO: 166), YARAAARQARA (SEQ ID NO: 167), THRLPRRRRRR (SEQ ID NO: 168), and GGRRARRRRRR (SEQ ID NO: 169). In some embodiments, the CPP is an activatable CPP (ACPP) (Aguilera et al. (2009) Integr Biol (Camb) June; 1(5-6): 371-381). ACPPs comprise a polycationic CPP (e.g., Arg9 or “R9”) connected via a cleavable linker to a matching polyanion (e.g., Glu9 or “E9”), which reduces the net charge to nearly zero and thereby inhibits adhesion and uptake into cells. Upon cleavage of the linker, the polyanion is released, locally unmasking the polyarginine and its inherent adhesiveness, thus “activating” the ACPP to traverse the membrane
In some cases a CPP can be added to the nanoparticle by contacting a coated core (a core that is surrounded by a sheddable layer) with a composition (e.g., solution) that includes the CPP. The CPP can then interact with the sheddable layer (e.g., electrostatically).
In some cases, the surface coat includes a polymer of a cationic amino acid (e.g., a poly(arginine) such as poly(L-arginine) and/or poly(D-arginine), a poly(lysine) such as poly(L-lysine) and/or poly(D-lysine), a poly(histidine) such as poly(L-histidine) and/or poly(D-histidine), a poly(ornithine) such as poly(L-ornithine) and/or poly(D-ornithine), poly(citrulline) such as poly(L-citrulline) and/or poly(D-citrulline), and the like). As such, in some cases the surface coat includes poly(arginine), e.g., poly(L-arginine).
In some embodiments, the surface coat includes a heptapeptide such as selank (TKPRPGP—SEQ ID NO: 147) (e.g., N-acetyl selank) and/or semax (MEHFPGP—SEQ ID NO: 148) (e.g., N-acetyl semax). As such, in some cases the surface coat includes selank (e.g., N-acetyl selank). In some cases the surface coat includes semax (e.g., N-acetyl semax).
In some embodiments the surface coat includes a delivery molecule. A delivery molecule includes a targeting ligand and in some cases the targeting ligand is conjugated to an anchoring domain (e.g. a cationic anchoring domain or anionic anchoring domain). In some cases a targeting ligand is conjugated to an anchoring domain (e.g. a cationic anchoring domain or anionic anchoring domain) via an intervening linker.
In some cases the surface coat includes any one or more of (in any desired combination): (i) one or more of the above described polymers, (ii) one or more targeting ligands, one or more CPPs, and one or more heptapeptides. For example, in some cases a surface coat can include one or more (e.g., two or more, three or more) targeting ligands, but can also include one or more of the above described cationic polymers. In some cases a surface coat can include one or more (e.g., two or more, three or more) targeting ligands, but can also include one or more CPPs. Further, a surface coat may include any combination of glycopeptides to promote stealth functionality, that is, to prevent serum protein adsorption and complement activity. This may be accomplished through Azide-alkyne click chemistry, coupling a peptide containing propargyl modified residues to azide containing derivatives of sialic acid, neuraminic acid, and the like.
In some cases, a surface coat includes a combination of targeting ligands that provides for targeted binding to CD34 and heparin sulfate proteoglycans. For example, poly(L-arginine) can be used as part of a surface coat to provide for targeted binding to heparin sulfate proteoglycans. As such, in some cases, after surface coating a nanoparticle with a cationic polymer (e.g., poly(L-arginine)), the coated nanoparticle is incubated with hyaluronic acid, thereby forming a zwitterionic and multivalent surface.
In some embodiments, the surface coat is multivalent. A multivalent surface coat is one that includes two or more targeting ligands (e.g., two or more delivery molecules that include different ligands). An example of a multimeric (in this case trimeric) surface coat (outer shell) is one that includes the targeting ligands stem cell factor (SCF) (which targets c-Kit receptor, also known as CD117), CD70 (which targets CD27), and SH2 domain-containing protein 1A (SH2D1A) (which targets CD150). For example, in some cases, to target hematopoietic stem cells (HSCs) [KLS (c-Kit+Lin− Sca-1+) and CD27+/IL-7Ra−/CD150+/CD34+], a subject nanoparticle includes a surface coat that includes a combination of the targeting ligands SCF, CD70, and SH2 domain-containing protein 1A (SH2D1A), which target c-Kit, CD27, and CD150, respectively (see, e.g., Table 1). In some cases, such a surface coat can selectively target HSPCs and long-term HSCs (c-Kit+/Lin-/Sca-1+/CD27+/IL-7Ra-/CD150+/CD34-) over other lymphoid and myeloid progenitors. Other HSC lineages may be targeted in human, mouse, or other animal model cell population subsets using transcriptomics and proteomics data through a diagnostically-responsive ligand panel, e.g. ligands corresponding to overexpressed receptors in htt followed by ps followed by //ww follwed by w.ncbi.nlm followed by .nih.go followed by v/pmc/articles/PMC5305050/, and ht followed by tps followed by ://ww followed by w.nature.c followed by om/articles/s41421-018-0038-x. In some example embodiments, all three targeting ligands (SCF, CD70, and SH2D1A) are anchored to the nanoparticle via fusion to a cationic anchoring domain (e.g., a poly-histidine such as 6H, a poly-arginine such as 9R, and the like). For example, (1) the targeting polypeptide SCF (which targets c-Kit receptor) can include XMEGICRNRVTNNVKDVTKLVANLPKDYMITLKYVPGMDVLPSHCWISEMVVQLSDSLTDLLDKF SNISEGLSNYSIIDKLVNIVDDLVECVKENSSKDLKKSFKSPEPRLFTPEEFFRIFNRSIDAFKDFVVAS ETSDCVVSSTLSPEKDSRVSVTKPFMLPPVAX (SEQ ID NO: 194), where the X is a cationic anchoring domain (e.g., a poly-histidine such as 6H, a poly-arginine such as 9R, and the like), e.g., which can in some cases be present at the N- and/or C-terminal end, or can be embedded within the polypeptide sequence; (2) the targeting polypeptide CD70 (which targets CD27) can include XPEEGSGCSVRRRPYGCVLRAALVPLVAGLVICLVVCIQRFAQAQQQLPLESLGWDVAELQLNHTG PQQDPRLYWQGGPALGRSFLHGPELDKGQLRIHRDGIYMVHIQVTLAICSSTTASRHHPTTLAVGIC SPASRSISLLRLSFHQGCTIASQRLTPLARGDTLCTNLTGTLLPSRNTDETFFGVQWVRPX (SEQ ID NO: 195), where the X is a cationic anchoring domain (e.g., a poly-histidine such as 6H, a poly-arginine such as 9R, and the like), e.g., which can in some cases be present at the N- and/or C-terminal end, or can be embedded within the polypeptide sequence; and (3) the targeting polypeptide SH2D1A (which targets CD150) can include XSSGLVPRGSHMDAVAVYHGKISRETGEKLLLATGLDGSYLLRDSESVPGVYCLCVLYHGYIYTYR VSQTETGSWSAETAPGVHKRYFRKIKNLISAFQKPDQGIVIPLQYPVEKKSSARSTQGTTGIREDPDVC LKAP (SEQ ID NO: 196), where the X is a cationic anchoring domain (e.g., a poly-histidine such as 6H, a poly-arginine such as 9R, and the like), e.g., which can in some cases be present at the N- and/or C-terminal end, or can be embedded within the polypeptide sequence (e.g., such as MGSSXSSGLVPRGSHMDAVAVYHGKISRETGEKLLLATGLDGSYLLRDSESVPGVYCLCVLYHGYIY TYRVSQTETGSWSAETAPGVHKRYFRKIKNLISAFQKPDQGIVIPLQYPVEKKSSARSTQGTTGIRED PDVCLKAP (SEQ ID NO: 197)).
As noted above, nanoparticles of the disclosure can include multiple targeting ligands (as part of a surface coat) in order to target a desired cell type, or in order to target a desired combination of cell types. Examples of cells of interest within the mouse and human hematopoietic cell lineages are depicted in
Provided are delivery molecules (a form of delivery vehicle) that include a targeting ligand (a peptide) conjugated to (i) a protein or nucleic acid payload, or (ii) a charged polymer polypeptide domain. The targeting ligand provides for (i) targeted binding to a cell surface protein, and in some cases (ii) engagement of a long endosomal recycling pathway. In some cases when the targeting ligand is conjugated to a charged polymer polypeptide domain, the charged polymer polypeptide domain interacts with (e.g., is condensed with) a nucleic acid payload and/or a protein payload. In some cases the targeting ligand is conjugated via an intervening linker. Refer to
In some cases, the delivery molecules disclosed herein are designed such that a nucleic acid or protein payload reaches its extracellular target (e.g., by providing targeted biding to a cell surface protein) and is preferentially not destroyed within lysosomes or sequestered into ‘short’ endosomal recycling endosomes. Instead, delivery molecules of the disclosure can provide for engagement of the ‘long’ (indirect/slow) endosomal recycling pathway, which can allow for endosomal escape and/or or endosomal fusion with an organelle.
For example, in some cases, β-arrestin is engaged to mediate cleavage of seven-transmembrane GPCRs (McGovern et al., Handb Exp Pharmacol. 2014; 219:341-59; Goodman et al., Nature. 1996 Oct. 3; 383(6599):447-50; Zhang et al., J Biol Chem. 1997 Oct. 24; 272(43):27005-14) and/or single-transmembrane receptor tyrosine kinases (RTKs) from the actin cytoskeleton (e.g., during endocytosis), triggering the desired endosomal sorting pathway. Thus, in some embodiments the targeting ligand of a delivery molecule of the disclosure provides for engagement of β-arrestin upon binding to the cell surface protein (e.g., to provide for signaling bias and to promote internalization via endocytosis following orthosteric binding).
In some cases a targeting ligand (e.g., of a subject delivery molecule) is conjugated to a charged polymer polypeptide domain (an anchoring domain such as a cationic anchoring domain or an anionic anchoring domain) (see e.g.,
A charged polymer polypeptide domain (a cationic anchoring domain, an anionic anchoring domain) can be any convenient charged domain (e.g., cationic charged domain). For example, such a domain can be a histone tail peptide (HTP) (described elsewhere herein in more detail). In some cases a charged polymer polypeptide domain includes a histone and/or histone tail peptide (e.g., a cationic polypeptide can be a histone and/or histone tail peptide). In some cases a charged polymer polypeptide domain includes an NLS-containing peptide (e.g., a cationic polypeptide can be an NLS-containing peptide). In some cases a charged polymer polypeptide domain includes a peptide that includes a mitochondrial localization signal (e.g., a cationic polypeptide can be a peptide that includes a mitochondrial localization signal).
In some cases, a charged polymer polypeptide domain of a subject delivery molecule is used as a way for the delivery molecular to interact with (e.g., interact electrostatically, e.g., for condensation) the payload (e.g., nucleic acid payload and/or protein payload).
In some cases, a charged polymer polypeptide domain of a subject delivery molecule is used as an anchor to coat the surface of a nanoparticle with the delivery molecule, e.g., so that the targeting ligand is used to target the nanoparticle to a desired cell/cell surface protein (see e.g.,
In some cases a charged polymer polypeptide domain (cationic) can include a polymer selected from: poly(arginine)(PR), poly(lysine)(PK), poly(histidine)(PH), poly(ornithine), poly(citrulline), and a combination thereof. In some cases a given cationic amino acid polymer can include a mix of arginine, lysine, histidine, ornithine, and citrulline residues (in any convenient combination). Polymers can be present as a polymer of L-isomers or D-isomers, where D-isomers are more stable in a target cell because they take longer to degrade. Thus, inclusion of D-isomer poly(amino acids) delays degradation (and subsequent payload release). The payload release rate can therefore be controlled and is proportional to the ratio of polymers of D-isomers to polymers of L-isomers, where a higher ratio of D-isomer to L-isomer increases duration of payload release (i.e., decreases release rate). In other words, the relative amounts of D- and L-isomers can modulate the release kinetics and enzymatic susceptibility to degradation and payload release.
In some cases a cationic polymer includes D-isomers and L-isomers of an cationic amino acid polymer (e.g., poly(arginine)(PR), poly(lysine)(PK), poly(histidine)(PH), poly(ornithine), poly(citrulline)). In some cases the D- to L-isomer ratio is in a range of from 10:1-1:10 (e.g., from 8:1-1:10, 6:1-1:10, 4:1-1:10, 3:1-1:10, 2:1-1:10, 1:1-1:10, 10:1-1:8, 8:1-1:8, 6:1-1:8, 4:1-1:8, 3:1-1:8, 2:1-1:8, 1:1-1:8, 10:1-1:6, 8:1-1:6, 6:1-1:6, 4:1-1:6, 3:1-1:6, 2:1-1:6, 1:1-1:6, 10:1-1:4, 8:1-1:4, 6:1-1:4, 4:1-1:4, 3:1-1:4, 2:1-1:4, 1:1-1:4, 10:1-1:3, 8:1-1:3, 6:1-1:3, 4:1-1:3, 3:1-1:3, 2:1-1:3, 1:1-1:3, 10:1-1:2, 8:1-1:2, 6:1-1:2, 4:1-1:2, 3:1-1:2, 2:1-1:2, 1:1-1:2, 10:1-1:1, 8:1-1:1, 6:1-1:1, 4:1-1:1, 3:1-1:1, or 2:1-1:1).
Thus, in some cases a cationic polymer includes a first cationic polymer (e.g., amino acid polymer) that is a polymer of D-isomers (e.g., selected from poly(D-arginine), poly(D-lysine), poly(D-histidine), poly(D-ornithine), and poly(D-citrulline)); and includes a second cationic polymer (e.g., amino acid polymer) that is a polymer of L-isomers (e.g., selected from poly(L-arginine), poly(L-lysine), poly(L-histidine), poly(L-ornithine), and poly(L-citrulline)). In some cases the ratio of the first cationic polymer (D-isomers) to the second cationic polymer (L-isomers) is in a range of from 10:1-1:10 (e.g., from 8:1-1:10, 6:1-1:10, 4:1-1:10, 3:1-1:10, 2:1-1:10, 1:1-1:10, 10:1-1:8, 8:1-1:8, 6:1-1:8, 4:1-1:8, 3:1-1:8, 2:1-1:8, 1:1-1:8, 10:1-1:6, 8:1-1:6, 6:1-1:6, 4:1-1:6, 3:1-1:6, 2:1-1:6, 1:1-1:6, 10:1-1:4, 8:1-1:4, 6:1-1:4, 4:1-1:4, 3:1-1:4, 2:1-1:4, 1:1-1:4, 10:1-1:3, 8:1-1:3, 6:1-1:3, 4:1-1:3, 3:1-1:3, 2:1-1:3, 1:1-1:3, 10:1-1:2, 8:1-1:2, 6:1-1:2, 4:1-1:2, 3:1-1:2, 2:1-1:2, 1:1-1:2, 10:1-1:1, 8:1-1:1, 6:1-1:1, 4:1-1:1, 3:1-1:1, or 2:1-1:1)
In some embodiments, a cationic polymer includes (e.g., in addition to or in place of any of the foregoing examples of cationic polymers) poly(ethylenimine), poly(amidoamine) (PAMAM), poly(aspartamide), polypeptoids (e.g., for forming “spiderweb”-like branches for core condensation), a charge-functionalized polyester, a cationic polysaccharide, an acetylated amino sugar, chitosan, or a cationic polymer that includes any combination thereof (e.g., in linear or branched forms).
In some embodiments, an cationic polymer can have a molecular weight in a range of from 1-200 kDa (e.g., from 1-150, 1-100, 1-50, 5-200, 5-150, 5-100, 5-50, 10-200, 10-150, 10-100, 10-50, 15-200, 15-150, 15-100, or 15-50 kDa). As an example, in some cases a cationic polymer includes poly(L-arginine), e.g., with a molecular weight of approximately 29 kDa. As another example, in some cases a cationic polymer includes linear poly(ethylenimine) with a molecular weight of approximately 25 kDa (PEI). As another example, in some cases a cationic polymer includes branched poly(ethylenimine) with a molecular weight of approximately 10 kDa. As another example, in some cases a cationic polymer includes branched poly(ethylenimine) with a molecular weight of approximately 70 kDa. In some cases a cationic polymer includes PAMAM.
In some cases, a cationic amino acid polymer includes a cysteine residue, which can facilitate conjugation, e.g., to a linker, an NLS, and/or a cationic polypeptide (e.g., a histone or HTP). For example, a cysteine residue can be used for crosslinking (conjugation) via sulfhydryl chemistry (e.g., a disulfide bond) and/or amine-reactive chemistry. Thus, in some embodiments a cationic amino acid polymer (e.g., poly(arginine)(PR), poly(lysine)(PK), poly(histidine)(PH), poly(ornithine), and poly(citrulline), poly(D-arginine)(PDR), poly(D-lysine)(PDK), poly(D-histidine)(PDH), poly(D-ornithine), and poly(D-citrulline), poly(L-arginine)(PLR), poly(L-lysine)(PLK), poly(L-histidine)(PLH), poly(L-ornithine), and poly(L-citrulline)) of a cationic polymer composition includes a cysteine residue. In some cases the cationic amino acid polymer includes cysteine residue on the N- and/or C-terminus. In some cases the cationic amino acid polymer includes an internal cysteine residue.
In some cases, a cationic amino acid polymer includes (and/or is conjugated to) a nuclear localization signal (NLS) (described in more detail below). Thus, in some embodiments a cationic amino acid polymer (e.g., poly(arginine)(PR), poly(lysine)(PK), poly(histidine)(PH), poly(ornithine), and poly(citrulline), poly(D-arginine)(PDR), poly(D-lysine)(PDK), poly(D-histidine)(PDH), poly(D-ornithine), and poly(D-citrulline), poly(L-arginine)(PLR), poly(L-lysine)(PLK), poly(L-histidine)(PLH), poly(L-ornithine), and poly(L-citrulline)) includes one or more (e.g., two or more, three or more, or four or more) NLSs. In some cases the cationic amino acid polymer includes an NLS on the N- and/or C-terminus. In some cases the cationic amino acid polymer includes an internal NLS.
In some cases, the charged polymer polypeptide domain is condensed with a nucleic acid payload and/or a protein payload (see e.g.,
The anionic polymer of an anionic sheddable matrix (i.e., the anionic polymer that interacts with the charged polymer polypeptide domain of a subject delivery molecule) can be any convenient anionic polymer/polymer composition. Examples include, but are not limited to: poly(glutamic acid) (e.g., poly(D-glutamic acid) (PDE), poly(L-glutamic acid) (PLE), both PDE and PLE in various desired ratios, etc.) In some cases, PDE is used as an anionic sheddable matrix. In some cases, PLE is used as an anionic sheddable matrix (anionic polymer). In some cases, PDE is used as an anionic sheddable matrix (anionic polymer). In some cases, PLE and PDE are both used as an anionic sheddable matrix (anionic polymer), e.g., in a 1:1 ratio (50% PDE, 50% PLE).
Anionic Polymer
An anionic polymer can include one or more anionic amino acid polymers. For example, in some cases a subject anionic polymer composition includes a polymer selected from: poly(glutamic acid)(PEA), poly(aspartic acid)(PDA), and a combination thereof. In some cases a given anionic amino acid polymer can include a mix of aspartic and glutamic acid residues. Each polymer can be present in the composition as a polymer of L-isomers or D-isomers, where D-isomers are more stable in a target cell because they take longer to degrade. Thus, inclusion of D-isomer poly(amino acids) can delay degradation and subsequent payload release. The payload release rate can therefore be controlled and is proportional to the ratio of polymers of D-isomers to polymers of L-isomers, where a higher ratio of D-isomer to L-isomer increases duration of payload release (i.e., decreases release rate). In other words, the relative amounts of D- and L-isomers can modulate the nanoparticle core's timed release kinetics and enzymatic susceptibility to degradation and payload release.
In some cases an anionic polymer composition includes polymers of D-isomers and polymers of L-isomers of an anionic amino acid polymer (e.g., poly(glutamic acid)(PEA) and poly(aspartic acid)(PDA)). In some cases the D- to L-isomer ratio is in a range of from 10:1-1:10 (e.g., from 8:1-1:10, 6:1-1:10, 4:1-1:10, 3:1-1:10, 2:1-1:10, 1:1-1:10, 10:1-1:8, 8:1-1:8, 6:1-1:8, 4:1-1:8, 3:1-1:8, 2:1-1:8, 1:1-1:8, 10:1-1:6, 8:1-1:6, 6:1-1:6, 4:1-1:6, 3:1-1:6, 2:1-1:6, 1:1-1:6, 10:1-1:4, 8:1-1:4, 6:1-1:4, 4:1-1:4, 3:1-1:4, 2:1-1:4, 1:1-1:4, 10:1-1:3, 8:1-1:3, 6:1-1:3, 4:1-1:3, 3:1-1:3, 2:1-1:3, 1:1-1:3, 10:1-1:2, 8:1-1:2, 6:1-1:2, 4:1-1:2, 3:1-1:2, 2:1-1:2, 1:1-1:2, 10:1-1:1, 8:1-1:1, 6:1-1:1, 4:1-1:1, 3:1-1:1, or 2:1-1:1).
Thus, in some cases an anionic polymer composition includes a first anionic polymer (e.g., amino acid polymer) that is a polymer of D-isomers (e.g., selected from poly(D-glutamic acid) (PDEA) and poly(D-aspartic acid) (PDDA)); and includes a second anionic polymer (e.g., amino acid polymer) that is a polymer of L-isomers (e.g., selected from poly(L-glutamic acid) (PLEA) and poly(L-aspartic acid) (PLDA)). In some cases the ratio of the first anionic polymer (D-isomers) to the second anionic polymer (L-isomers) is in a range of from 10:1-1:10 (e.g., from 8:1-1:10, 6:1-1:10, 4:1-1:10, 3:1-1:10, 2:1-1:10, 1:1-1:10, 10:1-1:8, 8:1-1:8, 6:1-1:8, 4:1-1:8, 3:1-1:8, 2:1-1:8, 1:1-1:8, 10:1-1:6, 8:1-1:6, 6:1-1:6, 4:1-1:6, 3:1-1:6, 2:1-1:6, 1:1-1:6, 10:1-1:4, 8:1-1:4, 6:1-1:4, 4:1-1:4, 3:1-1:4, 2:1-1:4, 1:1-1:4, 10:1-1:3, 8:1-1:3, 6:1-1:3, 4:1-1:3, 3:1-1:3, 2:1-1:3, 1:1-1:3, 10:1-1:2, 8:1-1:2, 6:1-1:2, 4:1-1:2, 3:1-1:2, 2:1-1:2, 1:1-1:2, 10:1-1:1, 8:1-1:1, 6:1-1:1, 4:1-1:1, 3:1-1:1, or 2:1-1:1)
In some embodiments, an anionic polymer composition includes (e.g., in addition to or in place of any of the foregoing examples of anionic polymers) a glycosaminoglycan, a glycoprotein, a polysaccharide, poly(mannuronic acid), poly(guluronic acid), heparin, heparin sulfate, chondroitin, chondroitin sulfate, keratan, keratan sulfate, aggrecan, poly(glucosamine), or an anionic polymer that comprises any combination thereof.
In some embodiments, an anionic polymer can have a molecular weight in a range of from 1-200 kDa (e.g., from 1-150, 1-100, 1-50, 5-200, 5-150, 5-100, 5-50, 10-200, 10-150, 10-100, 10-50, 15-200, 15-150, 15-100, or 15-50 kDa). As an example, in some cases an anionic polymer includes poly(glutamic acid) with a molecular weight of approximately 15 kDa.
In some cases, an anionic amino acid polymer includes a cysteine residue, which can facilitate conjugation, e.g., to a linker, an NLS, and/or a cationic polypeptide (e.g., a histone or HTP). For example, a cysteine residue can be used for crosslinking (conjugation) via sulfhydryl chemistry (e.g., a disulfide bond) and/or amine-reactive chemistry. Thus, in some embodiments an anionic amino acid polymer (e.g., poly(glutamic acid) (PEA), poly(aspartic acid) (PDA), poly(D-glutamic acid) (PDEA), poly(D-aspartic acid) (PDDA), poly(L-glutamic acid) (PLEA), poly(L-aspartic acid) (PLDA)) of an anionic polymer composition includes a cysteine residue. In some cases the anionic amino acid polymer includes cysteine residue on the N- and/or C-terminus. In some cases the anionic amino acid polymer includes an internal cysteine residue.
In some cases, an anionic amino acid polymer includes (and/or is conjugated to) a nuclear localization signal (NLS) (described in more detail below). Thus, in some embodiments an anionic amino acid polymer (e.g., poly(glutamic acid) (PEA), poly(aspartic acid) (PDA), poly(D-glutamic acid) (PDEA), poly(D-aspartic acid) (PDDA), poly(L-glutamic acid) (PLEA), poly(L-aspartic acid) (PLDA)) of an anionic polymer composition includes (and/or is conjugated to) one or more (e.g., two or more, three or more, or four or more) NLSs. In some cases the anionic amino acid polymer includes an NLS on the N- and/or C-terminus. In some cases the anionic amino acid polymer includes an internal NLS.
In some cases, an anionic polymer is conjugated to a targeting ligand.
Linker In some embodiments a targeting ligand is conjugated to an anchoring domain (e.g., a cationic anchoring domain, an anionic anchoring domain) or to a payload via an intervening linker. The linker can be a protein linker or non-protein linker. A linker can in some cases aid in stability, prevent complement activation, and/or provide flexibility to the ligand relative to the anchoring domain.
Conjugation of a targeting ligand to a linker or a linker to an anchoring domain can be accomplished in a number of different ways. In some cases the conjugation is via sulfhydryl chemistry (e.g., a disulfide bond, e.g., between two cysteine residues). In some cases the conjugation is accomplished using amine-reactive chemistry. In some cases, a targeting ligand includes a cysteine residue and is conjugated to the linker via the cysteine residue; and/or an anchoring domain includes a cysteine residue and is conjugated to the linker via the cysteine residue. In some cases, the linker is a peptide linker and includes a cysteine residue. In some cases, the targeting ligand and a peptide linker are conjugated by virtue of being part of the same polypeptide; and/or the anchoring domain and a peptide linker are conjugated by virtue of being part of the same polypeptide.
In some cases, a subject linker is a polypeptide and can be referred to as a polypeptide linker. It is to be understood that while polypeptide linkers are contemplated, non-polypeptide linkers (chemical linkers) are used in some cases. For example, in some embodiments the linker is a polyethylene glycol (PEG) linker. Suitable protein linkers include polypeptides of between 4 amino acids and 60 amino acids in length (e.g., 4-50, 4-40, 4-30, 4-25, 4-20, 4-15, 4-10, 6-60, 6-50, 6-40, 6-30, 6-25, 6-20, 6-15, 6-10, 8-60, 8-50, 8-40, 8-30, 8-25, 8-20, or 8-15 amino acids in length).
In some embodiments, a subject linker is rigid (e.g., a linker that include one or more proline residues). One non-limiting example of a rigid linker is GAPGAPGAP (SEQ ID NO: 17). In some cases, a polypeptide linker includes a C residue at the N- or C-terminal end. Thus, in some case a rigid linker is selected from: GAPGAPGAPC (SEQ ID NO: 18) and CGAPGAPGAP (SEQ ID NO: 19).
Peptide linkers with a degree of flexibility can be used. Thus, in some cases, a subject linker is flexible. The linking peptides may have virtually any amino acid sequence, bearing in mind that flexible linkers will have a sequence that results in a generally flexible peptide. The use of small amino acids, such as glycine and alanine, are of use in creating a flexible peptide. The creation of such sequences is routine to those of skill in the art. A variety of different linkers are commercially available and are considered suitable for use. Example linker polypeptides include glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)n, GSGGSn (SEQ ID NO: 20), GGSGGSn (SEQ ID NO: 21), and GGGSn (SEQ ID NO: 22), where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers. Example linkers can comprise amino acid sequences including, but not limited to, GGSG (SEQ ID NO: 23), GGSGG (SEQ ID NO: 24), GSGSG (SEQ ID NO: 25), GSGGG (SEQ ID NO: 26), GGGSG (SEQ ID NO: 27), GSSSG (SEQ ID NO: 28), and the like. The ordinarily skilled artisan will recognize that design of a peptide conjugated to any elements described above can include linkers that are all or partially flexible, such that the linker can include a flexible linker as well as one or more portions that confer less flexible structure. Additional examples of flexible linkers include, but are not limited to: GGGGGSGGGGG (SEQ ID NO: 29) and GGGGGSGGGGS (SEQ ID NO: 30). As noted above, in some cases, a polypeptide linker includes a C residue at the N- or C-terminal end. Thus, in some cases a flexible linker includes an amino acid sequence selected from: GGGGGSGGGGGC (SEQ ID NO: 31), CGGGGGSGGGGG (SEQ ID NO: 32), GGGGGSGGGGSC (SEQ ID NO: 33), and CGGGGGSGGGGS (SEQ ID NO: 34).
In some cases, a subject polypeptide linker is endosomolytic. Endosomolytic polypeptide linkers include but are not limited to: KALA (SEQ ID NO: 35) and GALA (SEQ ID NO: 36). As noted above, in some cases, a polypeptide linker includes a C residue at the N- or C-terminal end. Thus, in some cases a subject linker includes an amino acid sequence selected from: CKALA (SEQ ID NO: 37), KALAC (SEQ ID NO: 38), CGALA (SEQ ID NO: 39), and GALAC (SEQ ID NO: 40).
(e.g., for conjugation via sulfhydryl chemistry, e.g., using a cysteine residue) (e.g., for conjugating a targeting ligand or glycopeptide to a linker, conjugating a targeting ligand or glycopeptide to an anchoring domain (e.g., cationic anchoring domain), conjugating a linker to an anchoring domain (e.g., cationic anchoring domain), and the like)
Disulfide Bond
Cysteine residues can form disulfide bonds under mild oxidizing conditions or at higher than neutral pH in aqueous conditions.
Thioether/Thioester Bond
Sulfhydryl groups of cysteine react with maleimide and acyl halide groups, forming stable thioether and thioester bonds respectively.
Azide—Alkyne Cycloaddition
This conjugation is facilitated by chemical modification of the cysteine residue to contain an alkyne bond, or by the use of an L-propargyl amino acid derivative (e.g., L-propargyl cysteine—pictured below) in synthetic peptide preparation (e.g., solid phase synthesis). Coupling is then achieved by means of Cu promoted click chemistry.
Examples of targeting ligands include, but are not limited to, those that include the following amino acid sequences:
RRRRRRRRR
MEGICRNRVTNNVKDVTKLVANLPKDYMITLKYVPGMDVLPSHCWISEMVVQLSD
RRRRRRRRRPEEGSGCSVRRRPYGCVLRAALVPLVAGLVICLVVCIQRFAQAQQQLPLESLGWDV
R
MGSS
HHHHHH
SSGLVPRGSHMDAVAVYHGKISRETGEKLLLATGLDGSYLLRDSESVPGVYCLCVL
RRRRRRRRR
SSGLVPRGSHMDAVAVYHGKISRETGEKLLLATGLDGSYLLRDSESVPGVYCLCVLY
The targeting ligands in the present disclosure can be designed diagnostically-responsively following identification of the receptor profile of targeted cells. These targeting ligands may be peptides, peptoids, antibodies, aptamers, or other receptor-specific targeting molecules. In many embodiments, these targeting ligands are derived from native proteins or protein fragments where X-ray crystal structure data of a given protein (or protein homologue), or docking simulations of a given ligand to a measured or predicted protein structure, are used. In other embodiments, the targeting ligands are derived from antibodies, ScFvs, and the like. In other embodiments, the targeting ligands are derived from a SELEX or phage-display RNA/DNA aptamer or peptide libraries, respectively. In other embodiments, the targeting ligands are derived from other methods of combinatorial library prep of a random or natively-derived sequence/structure of polymer sequences [including peptides, peptoids, nucleotides, poly(B-amino esters), modified PEG sequences, LNAs, MNAs, PNAs and the like]. The “targeting ligands” are intended to represent a holistic set of targeting molecules designed for conferring cellular specificity for a combination of cellular receptor profiles, and can be combinatorially evaluated with a variety of nanoparticle or conjugation chemistries to create a cell/tissue/organ-specific delivery system for a given payload or set of payloads (e.g. CRISPR, TALEN, mRNA, small molecules).
Multiple targeting ligands patterned in specific densities along with optional stealth and/or linear/brushed glycoprotein motifs (as described elsewhere) may also be used to increase biodistributions and cell specificity, by limiting serum adsorption (protein corona formation, see, e.g., h followed by ttps://followed by ww followed by w.natu followed by re. co followed by m/articles/s41467-017-00600-w) to the ligand surface which otherwise limits cell-specific uptake. Regulation of particle clearance by macrophages may also be achieved through “eat me” and “don't eat me” cues on the particle surface, whereby CD47 and SIRPα normally interact and limit macrophage clearance of healthy cells. Fragments or mimetics (e.g. antibodies) of SIRPα may be presented upon the particle surface in order to limit macrophage clearance. Similar fragments or mimetics may be used as “receptor antagonistic” ligands that limit receptor-mediated endocytosis on targeted cells, while secondary sets of ligands (homo or heterovalent) may engage another cell's endocytotic machinery and cell specificity. Nanoparticles used in this way may also serve as intermediaries to cell-cell signaling, forming cell junctions (e.g. endothelial cell-immune junctions and the like) with biased uptake and gene-, gene edit-, and/or drug-mediated modification in the endocytosis-biased ligand-receptor pairing (e.g. the target cell population for genetic/other cellular reprogramming, such as with an immune cell engineered with an affinity marker). In other words, coupled with techniques for limiting non-specific serum adsorption, these embodiments can facilitate cell-specific targeting ligands (or combination of ligands) to confer 1) cell-specificity, 2) limited non-specific clearance of nanomaterials, and 3) active inhibition of macrophage/other cell uptake and protein corona formation in vivo, with an optional capacity for 4) cell-cell junction formation and biased reprogramming of a single target cell population. Broadly, the methods and uses for anchoring these targeting ligands to a universal set of gene editing, gene therapy and small molecule modalities represent clear innovation beyond the state of the art, in addition to significant innovations in “smart” composite nanomaterials and their architectures thereof, as well as the manufacturing, simulation, design and screening components thereof.
In some cases, a targeting ligand is conjugated (e.g., in some cases with a cleavable linker) directly to a payload—to deliver the payload. In some cases a targeting ligand is fused to a charged domain (detailed elsewhere herein), e.g., where the charged domain interacts with a payload. In some cases, a targeting ligand is associated with (e.g., through electrostatic interactions, via direct conjugation, via lipids, and the like) a delivery vehicle such as a solid particle core nanoparticle or a nanoparticle having a core that comprises polymers (e.g., a nanoparticle having cationic/anionic polymers, a cationic polypeptide, and the like)—for example, for the targeted delivery of a payload. In some cases a targeting ligand can serve it's own purpose without delivering a payload—as an example, an IL2 fragment (or IL-2-PEG) can be used.
A variety of targeting ligands (e.g., as part of a subject delivery molecule, e.g., as part of a nanoparticle) can be used (e.g., at any desired surface density when used as part of a nanoparticle) and numerous different targeting ligands are envisioned. In some embodiments the targeting ligand is a fragment (e.g., a binding domain) of a wild type protein. For example, in some cases a peptide targeting ligand of a subject delivery molecule can have a length of from 4-50 amino acids (e.g., from 4-40, 4-35, 4-30, 4-25, 4-20, 4-15, 5-50, 5-40, 5-35, 5-30, 5-25, 5-20, 5-15, 7-50, 7-40, 7-35, 7-30, 7-25, 7-20, 7-15, 8-50, 8-40, 8-35, 8-30, 8-25, 8-20, or 8-15 amino acids). The targeting ligand can be a fragment of a wild type protein, but in some cases has a mutation (e.g., insertion, deletion, substitution) relative to the wild type amino acid sequence (i.e., a mutation relative to a corresponding wild type protein sequence). For example, a targeting ligand can include a mutation that increases or decreases binding affinity with a target cell surface protein. Once 5-200 amino acids (e.g., from 5-150, 5-100, 5-80, 15-200, 15-150, 15-100, 15-80, 30-200, 30-150, 30-100, 30-80, 50-200, 50-150, 50-100, or 50-80 amino acids) within a binding pocket of a given receptor are identified, libraries of peptide targeting ligands of from 4-50 amino acids (e.g., from 4-40, 4-35, 4-30, 4-25, 4-20, 4-15, 5-50, 5-40, 5-35, 5-30, 5-25, 5-20, 5-15, 7-50, 7-40, 7-35, 7-30, 7-25, 7-20, 7-15, 8-50, 8-40, 8-35, 8-30, 8-25, 8-20, or 8-15 amino acids) can be generated (e.g. 1, 2, 3, 4, 5, 10, 15, 30, 50 or 100 targeting ligands per receptor) with variable anchor and linker motifs and nanoparticle-binding chemistries. These libraries of peptide targeting ligands may be screened according to a variety of nanoparticle formulations as disclosed herein (e.g. variable D:L isomer ratios, molecular weights, charges and compositions of cationic/anionic polymers; lipid embodiments and alternative nanoparticle chemistries may also be used), either decorating a pre-formed particle or directly forming the particle through directed self-assembling interactions (e.g. electrostatic, DNA origami templates, etc.). The best performing particles, as determined by their physicochemical and biological properties (e.g. size, charge, payload stability, cellular internalization, cellular specificity, cellular gene expression/editing), can be selected and in some cases further iterated around for increased cell/tissue/organ-specific behavior.
In some cases the targeting ligand is an antigen-binding region of an antibody (F(ab)). In some cases the targeting ligand is an ScFv. “Fv” is the minimum antibody fragment which contains a complete antigen-recognition and binding site. In a two-chain Fv species, this region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. In a single-chain Fv species (scFv), one heavy- and one light-chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can associate in a “dimeric” structure analogous to that in a two-chain Fv species. For a review of scFv see Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).
In some cases a targeting ligand includes a viral glycoprotein, which in some cases binds to ubiquitous surface markers such as heparin sulfate proteoglycans, and may induce micropinocytosis (and/or macropinocytosis) in some cell populations through membrane ruffling associated processes. Poly(L-arginine) is another example targeting ligand that can also be used for binding to surface markers such as heparin sulfate proteoglycans.
In some cases a targeting ligand is coated upon a particle surface (e.g., nanoparticle surface) either electrostatically or utilizing covalent modifications to the particle surface or one or more polymers on the particle surface. In some cases, a targeting ligand can include a mutation that adds a cysteine residue, which can facilitate conjugation to a linker and/or an anchoring domain (e.g., cationic anchoring domain). For example, cysteine can be used for crosslinking (conjugation) via sulfhydryl chemistry (e.g., a disulfide bond) and/or amine-reactive chemistry.
In some cases, a targeting ligand includes an internal cysteine residue. In some cases, a targeting ligand includes a cysteine residue at the N- and/or C-terminus. In some cases, in order to include a cysteine residue, a targeting ligand is mutated (e.g., insertion or substitution), e.g., relative to a corresponding wild type sequence. As such, any of the targeting ligands described herein can be modified by inserting and/or substituting in a cysteine residue (e.g., internal, N-terminal, C-terminal insertion of or substitution with a cysteine residue).
By “corresponding” wild type sequence is meant a wild type sequence from which the subject sequence was or could have been derived (e.g., a wild type protein sequence having high sequence identity to the sequence of interest). In some cases, a “corresponding” wild type sequence is one that has 85% or more sequence identity (e.g., 90% or more, 92% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) over the amino acid stretch of interest. For example, for a targeting ligand that has one or more mutations (e.g., substitution, insertion) but is otherwise highly similar to a wild type sequence, the amino acid sequence to which it is most similar may be considered to be a corresponding wild type amino acid sequence.
A corresponding wild type protein/sequence does not have to be 100% identical (e.g., can be 85% or more identical, 90% or more identical, 95% or more identical, 98% or more identical, 99% or more identical, etc.) (outside of the position(s) that is modified), but the targeting ligand and corresponding wild type protein (e.g., fragment of a wild protein) can bind to the intended cell surface protein, and retain enough sequence identity (outside of the region that is modified) that they can be considered homologous. The amino acid sequence of a “corresponding” wild type protein sequence can be identified/evaluated using any convenient method (e.g., using any convenient sequence comparison/alignment software such as BLAST, MUSCLE, T-COFFEE, etc.).
Examples of targeting ligands that can be used as part of a surface coat (e.g., as part of a delivery molecule of a surface coat) include, but are not limited to, those listed in Table 1. Examples of targeting ligands that can be used as part of a subject delivery molecule include, but are not limited to, those listed in Table 3 (many of the sequences listed in Table 3 include the targeting ligand (e.g., SNRWLDVK for row 2) conjugated to a cationic polypeptide domain, e.g., 9R, 6R, etc., via a linker (e.g., GGGGSGGGGS). Examples of amino acid sequences that can be included in a targeting ligand include, but are not limited to: NPKLTRMLTFKFY (SEQ ID NO: xx) (IL2), TSVGKYPNTGYYGD (SEQ ID NO: xx) (CD3), SNRWLDVK (Siglec), EKFILKVRPAFKAV (SEQ ID NO: xx) (SCF); EKFILKVRPAFKAV (SEQ ID NO: xx) (SCF), EKFILKVRPAFKAV (SEQ ID NO: xx) (SCF), SNYSIIDKLVNIVDDLVECVKENS (SEQ ID NO: xx) (cKit), and Ac-SNYSAibADKAibANAibADDAibAEAibAKENS (SEQ ID NO: xx) (cKit). Thus in some cases a targeting ligand includes an amino acid sequence that has 85% or more (e.g., 90% or more, 95% or more, 98% or more, 99% or more, or 100%) sequence identity with NPKLTRMLTFKFY (SEQ ID NO: xx) (IL2), TSVGKYPNTGYYGD (SEQ ID NO: xx) (CD3), SNRWLDVK (Siglec), EKFILKVRPAFKAV (SEQ ID NO: xx) (SCF); EKFILKVRPAFKAV (SEQ ID NO: xx) (SCF), EKFILKVRPAFKAV (SEQ ID NO: xx) (SCF), or SNYSIIDKLVNIVDDLVECVKENS (SEQ ID NO: xx) (cKit).
One non-limiting example of a multifunctional peptide sequence (variable anchor, linker and ligand domains with cell-specific matrix metalloprotease degradation behavior) is as follows:
KKKRKKKKRKGGGGSCGGGGSSFKFLFDIIKKIAES-[optional
This peptide serves many purposes:
KKKRKKKKRK—Anchor domain. Electrostatic-phase domain for genetic/protein payload condensation with importin-binding sequence for nuclear targeting. The N-terminus can also be utilized as a covalent modification to a small molecule drug, protein, or binding surface (as detailed elsewhere). Alternative sequences may be net-cationic, net-anionic, histone tail peptides, alternative NLS or subcellular trafficking/release sequences, and additional embodiments for reversible-charged and reversibly-binding electrostatic domains. This domain may also be replaced with a variety of covalent coupling techniques to alternative entities as described elsewhere.
GGGGSCGGGGSS—Flexible linker/spacer domain between electrostatic-phase domain and subsequent functional domain. This particular sequence includes a cysteine residue for linking to maleimide moieties. It may also be used to form cross-chain crosslinks between individual anchor-linker-ligand pairings. In this case, in contrast to H2A-3C and other cysteine-substituted histone tail peptides/cationic motifs utilized in our “core condensation” studies with cationic and anionic polypeptides, AlexaFluor594 occupies 100% of Cys residues on the linker domains. In alternative embodiments, the release of cross-chain crosslinks from a nanoparticle is believed to namely be mediated through glutathione activity and the stability of these complexes is shown elsewhere where mRNA condensation data (SYBR inclusion/exclusion curves) are used to show extended serum stability of nanoparticle complexes utilizing interspersed cysteine substitutions (e.g. cysteine-substituted histone tail peptides, cysteine-substituted anchor domains, cysteine-substituted linker domains, cysteine-stabilized ligand domains, and the like).
FKFL—Cathepsin B substrate for endosomal cleavage (bioresponsive domain may be customized for each cell/tissue/organ/cancer matrix metalloprotease [MMP] and/or other proteolytic enzymes (as detailed elsewhere).
FDIIKKIAES—Bioresponsive functional domain (ref: Discovery and Characterization of a Peptide That Enhances Endosomal Escape of Delivered Proteins in Vitro and in Vivo Margie Li, Yong Tao, Yilai Shu, Jonathan R. LaRochelle, Angela Steinauer, David Thompson, Alanna Schepartz, Zheng-Yi Chen, and David R. LiuJournal of the American Chemical Society 2015 137 (44), 14084-14093 DOI: 10.1021/jacs.5b05694). In this case a helical domain serves an endosomal escape function, however this particular peptide may have additional utility as well (
A targeting ligand (e.g., of a delivery molecule) can include the amino acid sequence RGD and/or an amino acid sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth in any one of SEQ ID NOs: 1-12. In some cases, a targeting ligand includes the amino acid sequence RGD and/or the amino acid sequence set forth in any one of SEQ ID NOs: 1-12. In some embodiments, a targeting ligand can include a cysteine (internal, C-terminal, or N-terminal), and can also include the amino acid sequence RGD and/or an amino acid sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth in any one of SEQ ID NOs: 1-12.
A targeting ligand (e.g., of a delivery molecule) can include the amino acid sequence RGD and/or an amino acid sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth in any one of SEQ ID NOs: 1-12 and 181-187. In some cases, a targeting ligand includes the amino acid sequence RGD and/or the amino acid sequence set forth in any one of SEQ ID NOs: 1-12 and 181-187. In some embodiments, a targeting ligand can include a cysteine (internal, C-terminal, or N-terminal), and can also include the amino acid sequence RGD and/or an amino acid sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth in any one of SEQ ID NOs: 1-12 and 181-187.
A targeting ligand (e.g., of a delivery molecule) can include the amino acid sequence RGD and/or an amino acid sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth in any one of SEQ ID NOs: 1-12, 181-187, and 271-277. In some cases, a targeting ligand includes the amino acid sequence RGD and/or the amino acid sequence set forth in any one of SEQ ID NOs: 1-12, 181-187, and 271-277. In some embodiments, a targeting ligand can include a cysteine (internal, C-terminal, or N-terminal), and can also include the amino acid sequence RGD and/or an amino acid sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth in any one of SEQ ID NOs: 1-12, 181-187, and 271-277.
In some cases, a targeting ligand (e.g., of a delivery molecule) can include an amino acid sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth in any one of SEQ ID NOs: 181-187, and 271-277. In some cases, a targeting ligand includes the amino acid sequence set forth in any one of SEQ ID NOs: 181-187, and 271-277. In some embodiments, a targeting ligand can include a cysteine (internal, C-terminal, or N-terminal), and can also include an amino acid sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth in any one of SEQ ID NOs: 181-187, and 271-277.
In some cases, a targeting ligand (e.g., of a delivery molecule) can include an amino acid sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth in any one of SEQ ID NOs: 181-187. In some cases, a targeting ligand includes the amino acid sequence set forth in any one of SEQ ID NOs: 181-187. In some embodiments, a targeting ligand can include a cysteine (internal, C-terminal, or N-terminal), and can also include an amino acid sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth in any one of SEQ ID NOs: 181-187.
In some cases, a targeting ligand (e.g., of a delivery molecule) can include an amino acid sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth in any one of SEQ ID NOs: 271-277. In some cases, a targeting ligand includes the amino acid sequence set forth in any one of SEQ ID NOs: 271-277. In some embodiments, a targeting ligand can include a cysteine (internal, C-terminal, or N-terminal), and can also include an amino acid sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth in any one of SEQ ID NOs: 271-277.
The terms “targets” and “targeted binding” are used herein to refer to specific binding. The terms “specific binding,” “specifically binds,” and the like, refer to non-covalent or covalent preferential binding to a molecule relative to other molecules or moieties in a solution or reaction mixture (e.g., an antibody specifically binds to a particular polypeptide or epitope relative to other available polypeptides, a ligand specifically binds to a particular receptor relative to other available receptors). In some embodiments, the affinity of one molecule for another molecule to which it specifically binds is characterized by a Kd (dissociation constant) of 10−5 M or less (e.g., 10−6 M or less, 10−7 M or less, 10−8 M or less, 10−9 M or less, 10−10 M or less, 10−11 M or less, 10−12 M or less, 10−13 M or less, 10−14 M or less, 10−15 M or less, or 10−16 M or less). “Affinity” refers to the strength of binding, increased binding affinity correlates with a lower Ka.
In some cases, the targeting ligand provides for targeted binding to a cell surface protein selected from a family B G-protein coupled receptor (GPCR), a receptor tyrosine kinase (RTK), a cell surface glycoprotein, and a cell-cell adhesion molecule. Consideration of a ligand's spatial arrangement upon receptor docking can be used to accomplish a desired functional selectivity and endosomal sorting biases, e.g., so that the structure function relationship between the ligand and the target is not disrupted due to the conjugation of the targeting ligand to the payload or anchoring domain (e.g., cationic anchoring domain). For example, conjugation to a nucleic acid, protein, ribonucleoprotein, or anchoring domain (e.g., cationic anchoring domain) could potentially interfere with the binding cleft(s).
Thus, in some cases, where a crystal structure of a desired target (cell surface protein) bound to its ligand is available (or where such a structure is available for a related protein), one can use 3D structure modeling and sequence threading to visualize sites of interaction between the ligand and the target. This can facilitate, e.g., selection of internal sites for placement of substitutions and/or insertions (e.g., of a cysteine residue).
As an example, in some cases, the targeting ligand provides for binding to a family B G protein coupled receptor (GPCR) (also known as the ‘secretin-family’). In some cases, the targeting ligand provides for binding to both an allosteric-affinity domain and an orthosteric domain of the family B GPCR to provide for the targeted binding and the engagement of long endosomal recycling pathways, respectively (e.g., see
G-protein-coupled receptors (GPCRs) share a common molecular architecture (with seven putative transmembrane segments) and a common signaling mechanism, in that they interact with G proteins (heterotrimeric GTPases) to regulate the synthesis of intracellular second messengers such as cyclic AMP, inositol phosphates, diacylglycerol and calcium ions. Family B (the secretin-receptor family or ‘family 2’) of the GPCRs is a small but structurally and functionally diverse group of proteins that includes receptors for polypeptide hormones and molecules thought to mediate intercellular interactions at the plasma membrane (see e.g., Harmar et al., Genome Biol. 2001; 2(12): REVIEWS3013). There have been important advances in structural biology as relates to members of the secretin-receptor family, including the publication of several crystal structures of their N-termini, with or without bound ligands, which work has expanded the understanding of ligand binding and provides a useful platform for structure-based ligand design (see e.g., Poyner et al., Br J Pharmacol. 2012 May; 166(1):1-3).
For example, one may desire to use a subject delivery molecule to target the pancreatic cell surface protein GLP1R (e.g., to target B-islets) using the Exendin-4 ligand, or a derivative thereof (e.g., a cysteine substituted Exendin-4 targeting ligand such as that presented as SEQ ID NO: 2). Because GLP1R is abundant within the brain and pancreas, a targeting ligand that provides for targeting binding to GLP1R can be used to target the brain and pancreas. Thus, targeting GLP1R facilitates methods (e.g., treatment methods) focused on treating diseases (e.g., via delivery of one or more gene editing tools) such as Huntington's disease (CAG repeat expansion mutations), Parkinson's disease (LRRK2 mutations), ALS (SOD1 mutations), and other CNS diseases. Targeting GLP1R also facilitates methods (e.g., treatment methods) focused on delivering a payload to pancreatic β-islets for the treatment of diseases such as diabetes mellitus type I, diabetes mellitus type II, and pancreatic cancer (e.g., via delivery of one or more gene editing tools).
When targeting GLP1R using a modified version of exendin-4, an amino acid for cysteine substitution and/or insertion (e.g., for conjugation to a nucleic acid payload) can be identified by aligning the Exendin-4 amino acid sequence, which is HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS
(SEQ ID NO. 1), to crystal structures of glucagon-GCGR (4ERS) and GLP1-GLP1R-ECD complex (PDB: 3IOL, using PDB 3 dimensional renderings, which may be rotated in 3D space in order to anticipate the direction that a cross-linked complex must face in order not to disrupt the two binding clefts. When a desirable cross-linking site (e.g., site for substitution/insertion of a cysteine residue) of a targeting ligand (that targets a family B GPCR) is sufficiently orthogonal to the two binding clefts of the corresponding receptor, high-affinity binding may occur as well as concomitant long endosomal recycling pathway sequestration (e.g., for improved payload release). The cysteine substitution at amino acid positions 10, 11, and/or 12 of SEQ ID NO: 1 confers bimodal binding and specific initiation of a Gs-biased signaling cascade, engagement of beta arrestin, and receptor dissociation from the actin cytoskeleton. In some cases, this targeting ligand triggers internalization of the nanoparticle via receptor-mediated endocytosis, a mechanism that is not engaged via mere binding to the GPCR's N-terminal domain without concomitant orthosteric site engagement (as is the case with mere binding of the affinity strand, Exendin-4 [31-39]).
In some cases, a subject targeting ligand includes an amino acid sequence having 85% or more (e.g., 90% or more, 95% or more, 98% or more, 99% or more, or 100%) identity to the exendin-4 amino acid sequence (SEQ ID NO: 1). In some such cases, the targeting ligand includes a cysteine substitution or insertion at one or more of positions corresponding to L10, S11, and K12 of the amino acid sequence set forth in SEQ ID NO: 1. In some cases, the targeting ligand includes a cysteine substitution or insertion at a position corresponding to S11 of the amino acid sequence set forth in SEQ ID NO: 1. In some cases, a subject targeting ligand includes an amino acid sequence having the exendin-4 amino acid sequence (SEQ ID NO: 1). In some cases, the targeting ligand is conjugated (with or without a linker) to an anchoring domain (e.g., a cationic anchoring domain).
As another example, in some cases a targeting ligand according to the present disclosure provides for binding to a receptor tyrosine kinase (RTK) such as fibroblast growth factor (FGF) receptor (FGFR). Thus in some cases the targeting ligand is a fragment of an FGF (i.e., comprises an amino acid sequence of an FGF). In some cases, the targeting ligand binds to a segment of the RTK that is occupied during orthosteric binding (e.g., see the examples section below). In some cases, the targeting ligand binds to a heparin-affinity domain of the RTK. In some cases, the targeting ligand provides for targeted binding to an FGF receptor and comprises an amino acid sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence KNGGFFLRIHPDGRVDGVREKS (SEQ ID NO: 4). In some cases, the targeting ligand provides for targeted binding to an FGF receptor and comprises the amino acid sequence set forth as SEQ ID NO: 4.
In some cases, small domains (e.g., 5-40 amino acids in length) that occupy the orthosteric site of the RTK may be used to engage endocytotic pathways relating to nuclear sorting of the RTK (e.g., FGFR) without engagement of cell-proliferative and proto-oncogenic signaling cascades, which can be endemic to the natural growth factor ligands. For example, the truncated bFGF (tbFGF) peptide (a.a.30-115), contains a bFGF receptor binding site and a part of a heparin-binding site, and this peptide can effectively bind to FGFRs on a cell surface, without stimulating cell proliferation. The sequences of tbFGF are KRLYCKNGGFFLRIHPDGRVDGVREKSDPHIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLLASK CVTDECFFFERLESNNYNTY (SEQ ID NO: 13) (see, e.g., Cai et al., Int J Pharm. 2011 Apr. 15; 408(1-2): 173-82).
In some cases, the targeting ligand provides for targeted binding to an FGF receptor and comprises the amino acid sequence HFKDPK (SEQ ID NO: 5) (see, e.g., the examples section below). In some cases, the targeting ligand provides for targeted binding to an FGF receptor, and comprises the amino acid sequence LESNNYNT (SEQ ID NO: 6) (see, e.g., the examples section below).
In some cases, a targeting ligand according to the present disclosure provides for targeted binding to a cell surface glycoprotein. In some cases, the targeting ligand provides for targeted binding to a cell-cell adhesion molecule. For example, in some cases, the targeting ligand provides for targeted binding to CD34, which is a cell surface glycoprotein that functions as a cell-cell adhesion factor, and which is protein found on hematopoietic stem cells (e.g., of the bone marrow). In some cases, the targeting ligand is a fragment of a selectin such as E-selectin, L-selectin, or P-selectin (e.g., a signal peptide found in the first 40 amino acids of a selectin). In some cases a subject targeting ligand includes sushi domains of a selectin (e.g., E-selectin, L-selectin, P-selectin).
In some cases, the targeting ligand comprises an amino acid sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence MIASQFLSALTLVLLIKESGA (SEQ ID NO: 7). In some cases, the targeting ligand comprises the amino acid sequence set forth as SEQ ID NO: 7. In some cases, the targeting ligand comprises an amino acid sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence MVFPWRCEGTYWGSRNILKLWVWTLLCCDFLIHHGTHC (SEQ ID NO: 8). In some cases, the targeting ligand comprises the amino acid sequence set forth as SEQ ID NO: 8. In some cases, targeting ligand comprises an amino acid sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence MIFPWKCQSTQRDLWNIFKLWGWTMLCCDFLAHHGTDC (SEQ ID NO: 9). In some cases, targeting ligand comprises the amino acid sequence set forth as SEQ ID NO: 9. In some cases, targeting ligand comprises an amino acid sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence MIFPWKCQSTQRDLWNIFKLWGWTMLCC (SEQ ID NO: 10). In some cases, targeting ligand comprises the amino acid sequence set forth as SEQ ID NO: 10.
Fragments of selectins that can be used as a subject targeting ligand (e.g., a signal peptide found in the first 40 amino acids of a selectin) can in some cases attain strong binding to specifically-modified sialomucins, e.g., various Sialyl Lewis' modifications/O-sialylation of extracellular CD34 can lead to differential affinity for P-selectin, L-selectin and E-selectin to bone marrow, lymph, spleen and tonsillar compartments. Conversely, in some cases a targeting ligand can be an extracellular portion of CD34. In some such cases, modifications of sialylation of the ligand can be utilized to differentially target the targeting ligand to various selectins.
In some cases, a targeting ligand according to the present disclosure provides for targeted binding to E-selectin. E-selectin can mediate the adhesion of tumor cells to endothelial cells and ligands for E-selectin can play a role in cancer metastasis. As an example, P-selectin glycoprotein-1 (PSGL-1) (e.g., derived from human neutrophils) can function as a high-efficiency ligand for E-selectin (e.g., expressed by the endothelium), and a subject targeting ligand can therefore in some cases include the PSGL-1 amino acid sequence (or a fragment thereof the binds to E-selectin). As another example, E-selectin ligand-1 (ESL-1) can bind E-selectin and a subject targeting ligand can therefore in some cases include the ESL-1 amino acid sequence (or a fragment thereof the binds to E-selectin). In some cases, a targeting ligand with the PSGL-1 and/or ESL-1 amino acid sequence (or a fragment thereof the binds to E-selectin) bears one or more sialyl Lewis modifications in order to bind E-selectin. As another example, in some cases CD44, death receptor-3 (DR3), LAMP1, LAMP2, and Mac2-BP can bind E-selectin and a subject targeting ligand can therefore in some cases include the amino acid sequence (or a fragment thereof the binds to E-selectin) of any one of: CD44, death receptor-3 (DR3), LAMP1, LAMP2, and Mac2-BP.
In some cases, a targeting ligand according to the present disclosure provides for targeted binding to P-selectin. In some cases PSGL-1 can provide for such targeted binding. In some cases a subject targeting ligand can therefore in some cases include the PSGL-1 amino acid sequence (or a fragment thereof the binds to P-selectin). In some cases, a targeting ligand with the PSGL-1 amino acid sequence (or a fragment thereof the binds to P-selectin) bears one or more sialyl Lewis modifications in order to bind P-selectin.
In some cases, a targeting ligand according to the present disclosure provides for targeted binding to a target selected from: CD3, CD8, CD4, CD28, CD90, CD45f, CD34, CD80, CD86, CD19, CD20, CD22, CD47, CD3-epsilon, CD3-gamma, CD3-delta; TCR Alpha, TCR Beta, TCR gamma, and/or TCR delta constant regions; 4-1BB, OX40, OX40L, CD62L, ARP5, CCR5, CCR7, CCR10, CXCR3, CXCR4, CD94/NKG2, NKG2A, NKG2B, NKG2C, NKG2E, NKG2H, NKG2D, NKG2F, NKp44, NKp46, NKp30, DNAM, XCR1, XCL1, XCL2, ILT, LIR, Ly49, IL2R, IL7R, IL10R, IL12R, IL15R, IL18R, TNFα, IFNγ, TGF-β, and α5β1
In some cases, a targeting ligand according to the present disclosure provides for targeted binding to a transferrin receptor. In some such cases, the targeting ligand comprises an amino acid sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence THRPPMWSPVWP (SEQ ID NO: 11). In some cases, targeting ligand comprises the amino acid sequence set forth as SEQ ID NO: 11.
In some cases, a targeting ligand according to the present disclosure provides for targeted binding to an integrin (e.g., α5β1 integrin). In some such cases, the targeting ligand comprises an amino acid sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence RRETAWA (SEQ ID NO: 12). In some cases, targeting ligand comprises the amino acid sequence set forth as SEQ ID NO: 12. In some cases, the targeting ligand comprises an amino acid sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence RGDGW (SEQ ID NO: 181). In some cases, targeting ligand comprises the amino acid sequence set forth as SEQ ID NO: 181. In some cases, the targeting ligand comprises the amino acid sequence RGD.
In some cases, a targeting ligand according to the present disclosure provides for targeted binding to an integrin. In some such cases, the targeting ligand comprises an amino acid sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence GCGYGRGDSPG (SEQ ID NO: 182). In some cases, the targeting ligand comprises the amino acid sequence set forth as SEQ ID NO: 182. In some cases such a targeting ligand is acetylated on the N-terminus and/or amidated (NH2) on the C-terminus.
In some cases, a targeting ligand according to the present disclosure provides for targeted binding to an integrin (e.g., a5133 integrin). In some such cases, the targeting ligand comprises an amino acid sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence DGARYCRGDCFDG (SEQ ID NO: 187). In some cases, the targeting ligand comprises the amino acid sequence set forth as SEQ ID NO: 187.
In some embodiments, a targeting ligand used to target the brain includes an amino acid sequence from rabies virus glycoprotein (RVG) (e.g., YTIWMPENPRPGTPCDIFTNSRGKRASNGGGG (SEQ ID NO: 183)). In some such cases, the targeting ligand comprises an amino acid sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as SEQ ID NO: 183. As for any of targeting ligand (as described elsewhere herein), RVG can be conjugated and/or fused to an anchoring domain (e.g., 9R peptide sequence). For example, a subject delivery molecule used as part of a surface coat of a subject nanoparticle can include the sequence YTIWMPENPRPGTPCDIFTNSRGKRASNGGGGRRRRRRRRR (SEQ ID NO: 180).
In some cases, a targeting ligand according to the present disclosure provides for targeted binding to c-Kit receptor. In some such cases, the targeting ligand comprises an amino acid sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as SEQ ID NO: 184. In some cases, the targeting ligand comprises the amino acid sequence set forth as SEQ ID NO: 184.
In some cases, a targeting ligand according to the present disclosure provides for targeted binding to CD27. In some such cases, the targeting ligand comprises an amino acid sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as SEQ ID NO: 185. In some cases, the targeting ligand comprises the amino acid sequence set forth as SEQ ID NO: 185.
In some cases, a targeting ligand according to the present disclosure provides for targeted binding to CD150. In some such cases, the targeting ligand comprises an amino acid sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as SEQ ID NO: 186. In some cases, the targeting ligand comprises the amino acid sequence set forth as SEQ ID NO: 186.
In some embodiments, a targeting ligand provides for targeted binding to KLS CD27+/IL-7Ra-/CD150+/CD34-hematopoietic stem and progenitor cells (HSPCs). For example, a gene editing tool(s) (described elsewhere herein) can be introduced in order to disrupt expression of a BCL11a transcription factor and consequently generate fetal hemoglobin. As another example, the beta-globin (HBB) gene may be targeted directly to correct the altered E7V substitution with a corresponding homology-directed repair donor DNA molecule. As one illustrative example, a CRISPR/Cas RNA-guided polypeptide (e.g., Cas9, CasX, CasY, Cpf1) can be delivered with an appropriate guide RNA such that it will bind to loci in the HBB gene and create double-stranded or single-stranded breaks in the genome, initiating genomic repair. In some cases, a Donor DNA molecule (single stranded or double stranded) is introduced (as part of a payload) and is release for 14-30 days while a guide RNA/CRISPR/Cas protein complex (a ribonucleoprotein complex) can be released over the course of from 1-7 days.
In some embodiments, a targeting ligand provides for targeted binding to CD4+ or CD8+ T-cells, hematopoietic stem and progenitor cells (HSPCs), or peripheral blood mononuclear cells (PBMCs), in order to modify the T-cell receptor. For example, a gene editing tool(s) (described elsewhere herein) can be introduced in order to modify the T-cell receptor. The T-cell receptor may be targeted directly and substituted with a corresponding homology-directed repair donor DNA molecule for a novel T-cell receptor. As one example, a CRISPR/Cas RNA-guided polypeptide (e.g., Cas9, CasX, CasY, Cpf1) can be delivered with an appropriate guide RNA such that it will bind to loci in the TCR gene and create double-stranded or single-stranded breaks in the genome, initiating genomic repair. In some cases, a Donor DNA molecule (single stranded or double stranded) is introduced (as part of a payload). It would be evident to skilled artisans that other CRISPR guide RNA and donor sequences, targeting beta-globin, CCR5, the T-cell receptor, or any other gene of interest, and/or other expression vectors may be employed in accordance with the present disclosure.
In some embodiments, a targeting ligand is a nucleic acid aptamer. In some embodiments, a targeting ligand is a peptoid.
Also provided are delivery molecules with two different peptide sequences that together constitute a targeting ligand. For example, in some cases a targeting ligand is bivalent (e.g., heterobivalent). In some cases, cell-penetrating peptides and/or heparin sulfate proteoglycan binding ligands are used as heterobivalent endocytotic triggers along with any of the targeting ligands of this disclosure. A heterobivalent targeting ligand can include an affinity sequence from one of targeting ligand and an orthosteric binding sequence (e.g., one known to engage a desired endocytic trafficking pathway) from a different targeting ligand.
In some cases, targeting ligands are identified by screening (also described in more detail elsewhere herein). The term “top-performing” targeting ligands can be used to mean the targeting ligands that perform best in the assays when comparted to other ligands of the screen. The criteria used to determine which ligands are “top-performing” can be any convenient criteria. Examples of such parameters can include physical and/or biological measures of performance. Examples can include transfection efficiency, cell specificity, etc. In some cases, the “top-performing” ligands are the top 50 (e.g., top 40, top 30, top 20, top 15, top 10, or top 5) performing ligands. In some cases, the “top-performing” ligands are the top 30 (e.g., top 20, top 15, top 10, or top 5) performing ligands. In some cases, the “top-performing” ligands are the top 15, e.g., top 10 or top 5) performing ligands. In some cases, the “top-performing” ligands are the top performing 20% of ligands (e.g., top 10% or top 5%) (e.g., if 1000 ligands were screened, the top-performing 20% would be the top 200 performing 200). In some cases, the “top-performing” ligands are the top performing 10% of ligands (e.g., top 5% or top 2% or top 1%) (e.g., if 1000 ligands were screened, the top-performing 10% would be the top performing 100 ligands). In some cases, the “top-performing” ligands are the top performing 5% of ligands (e.g., top 2% or top 1%) (e.g., if 1000 ligands were screened, the top-performing 5% would be the top performing 50 ligands). In some cases, the “top-performing” ligands are the top performing 2% of ligands (e.g., top 1%) (e.g., if 1000 ligands were screened, the top-performing 2% would be the top performing 20 ligands).
Anchoring Domain
In some embodiments, a delivery molecule includes a targeting ligand conjugated to an anchoring domain (e.g., cationic anchoring domain, an anionic anchoring domain). In some cases a subject delivery vehicle includes a payload that is condensed with and/or interacts electrostatically or covalently with the anchoring domain (e.g., a delivery molecule can be the delivery vehicle used to deliver the payload). In some cases the surface coat of a nanoparticle includes such a delivery molecule with an anchoring domain, and in some such cases the payload is in the core (interacts with the core) of such a nanoparticle. In some cases, the payload is a small molecule or biologic covalently attached to anchoring domain. See the above section describing charged polymer polypeptide domains for additional details related to anchoring domains.
In some cases, an outer layer (surface layer) can include motifs that lend stealth functionality, limiting protein corona formation, and complement activity. These motifs may be composed of carbohydrate functionalized peptides, polysialic acid, hyaluronic acid, poly(ethylene glycol) or any other hydrated biopolymers.
Alternative Packaging (e.g., Lipid Formulations)
In some embodiments, a subject core (e.g., including any combination of components and/or configurations described above) is part of a lipid-based delivery system, e.g., a cationic lipid delivery system (see, e.g., Chesnoy and Huang, Annu Rev Biophys Biomol Struct. 2000, 29:27-47; Hirko et al., Curr Med Chem. 2003 Jul. 10(14):1185-93; and Liu et al., Curr Med Chem. 2003 Jul. 10(14):1307-15). In some cases a subject core (e.g., including any combination of components and/or configurations described above) is not surrounded by a sheddable layer. As noted above a core can include an anionic polymer composition (e.g., poly(glutamic acid)), a cationic polymer composition (e.g., poly(arginine), a cationic polypeptide composition (e.g., a histone tail peptide), and a payload (e.g., nucleic acid and/or protein payload).
In some cases in which the core is part of a lipid-based delivery system, the core was designed with timed and/or positional (e.g., environment-specific) release in mind. For example, in some cases the core includes ESPs, ENPs, and/or EPPs, and in some such cases these components are present at ratios such that payload release is delayed until a desired condition (e.g., cellular location, cellular condition such as pH, presence of a particular enzyme, and the like) is encountered by the core (e.g., described above). In some such embodiments the core includes polymers of D-isomers of an anionic amino acid and polymers of L-isomers of an anionic amino acid, and in some cases the polymers of D- and L-isomers are present, relative to one another, within a particular range of ratios (e.g., described above). In some cases the core includes polymers of D-isomers of a cationic amino acid and polymers of L-isomers of a cationic amino acid, and in some cases the polymers of D- and L-isomers are present, relative to one another, within a particular range of ratios (e.g., described above). In some cases the core includes polymers of D-isomers of an anionic amino acid and polymers of L-isomers of a cationic amino acid, and in some cases the polymers of D- and L-isomers are present, relative to one another, within a particular range of ratios (e.g., described above). In some cases the core includes polymers of L-isomers of an anionic amino acid and polymers of D-isomers of a cationic amino acid, and in some cases the polymers of D- and L-isomers are present, relative to one another, within a particular range of ratios (e.g., described elsewhere herein). In some cases the core includes a protein that includes an NLS (e.g., described elsewhere herein). In some cases the core includes an HTP (e.g., described elsewhere herein).
Cationic lipids are nonviral vectors that can be used for gene delivery and have the ability to condense plasmid DNA. After synthesis of N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride for lipofection, improving molecular structures of cationic lipids has been an active area, including head group, linker, and hydrophobic domain modifications. Modifications have included the use of multivalent polyamines, which can improve DNA binding and delivery via enhanced surface charge density, and the use of sterol-based hydrophobic groups such as 3B-[N—(N′,N′-dimethylaminoethane)-carbamoyll cholesterol, which can limit toxicity. Helper lipids such as dioleoyl phosphatidylethanolamine (DOPE) can be used to improve transgene expression via enhanced liposomal hydrophobicity and hexagonal inverted-phase transition to facilitate endosomal escape. In some cases a lipid formulation includes one or more of: DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, DLin-MC3-DMA, 98N12-5, C12-200, a cholesterol a PEG-lipid, a lipidopolyamine, dexamethasone-spermine (DS), and disubstituted spermine (D2S) (e.g., resulting from the conjugation of dexamethasone to polyamine spermine). DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, 98N12-5, C12-200 and DLin-MC3-DMA can be synthesized by methods outlined in the art (see, e.g, Heyes et. al, J. Control Release, 2005, 107, 276-287; Semple et. al, Nature Biotechnology, 2010, 28, 172-176; Akinc et. al, Nature Biotechnology, 2008, 26, 561-569; Love et. al, PNAS, 2010, 107, 1864-1869; international patent application publication WO2010054401; all of which are hereby incorporated by reference in their entirety.
Examples of various lipid-based delivery systems include, but are not limited to those described in the following publications: international patent publication No. WO2016081029; U.S. patent application publication Nos. US20160263047 and US20160237455; and U.S. Pat. Nos. 9,533,047; 9,504,747; 9,504,651; 9,486,538; 9,393,200; 9,326,940; 9,315,828; and 9,308,267; all of which are hereby incorporated by reference in their entirety.
As such, in some cases a subject core is surrounded by a lipid (e.g., a cationic lipid such as a LIPOFECTAMINE transfection reagent). In some cases a subject core is present in a lipid formulation (e.g., a lipid nanoparticle formulation). A lipid formulation can include a liposome and/or a lipoplex. A lipid formulation can include a Spontaneous Vesicle Formation by Ethanol Dilution (SNALP) liposome (e.g., one that includes cationic lipids together with neutral helper lipids which can be coated with polyethylene glycol (PEG) and/or protamine).
A lipid formulation can be a lipidoid-based formulation. The synthesis of lipidoids has been extensively described and formulations containing these compounds can be included in a subject lipid formulation (see, e.g., Mahon et al., Bioconjug Chem. 2010 21:1448-1454; Schroeder et al., J Intern Med. 2010 267:9-21; Akinc et al., Nat Biotechnol. 2008 26:561-569; Love et al., Proc Natl Acad Sci USA. 2010 107:1864-1869; and Siegwart et al., Proc Natl Acad Sci USA. 2011 108:12996-3001; all of which are incorporated herein by reference in their entirety). In some cases a subject lipid formulation can include one or more of (in any desired combination): 1,2-Dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC); 1,2-Dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE); N-[1-(2,3-Dioleyloxy)prophyl]N,N,N-trimethylammonium chloride (DOTMA); 1,2-Dioleoyloxy-3-trimethylammonium-propane (DOTAP); Dioctadecylamidoglycylspermine (DOGS); N-(3-Aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1 (GAP-DLRIE); propanaminium bromide; cetyltrimethylammonium bromide (CTAB); 6-Lauroxyhexyl ornithinate (LHON); 1-(2,3-Dioleoyloxypropyl)-2,4,6-trimethylpyridinium (20c); 2,3-Dioleyloxy-N-[2(sperminecarboxamido-ethyl]-N,N-dimethyl-1 (DOSPA); propanaminium trifluoroacetate; 1,2-Dioleyl-3-trimethylammonium-propane (DOPA); N-(2-Hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1 (MDRIE); propanaminium bromide; dimyristooxypropyl dimethyl hydroxyethyl ammonium bromide (DMRI); 3.beta.-[N—(N′,N′-Dimethylaminoethane)-carbamoyl]cholesterol DC-Chol; bis-guanidium-tren-cholesterol (BGTC); 1,3-Diodeoxy-2-(6-carboxy-spermyl)-propylamide (DOSPER); Dimethyloctadecylammonium bromide (DDAB); Dioctadecylamidoglicylspermidin (DSL); rac-[(2,3-Dioctadecyloxypropyl)(2-hydroxyethyl)]-dimethylammonium (CLIP-1); chloride rac-[2(2,3-Dihexadecyloxypropyl (CLIP-6); oxymethyloxy)ethyl]trimethylammonium bromide; ethyldimyristoylphosphatidylcholine (EDMPC); 1,2-Distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA); 1,2-Dimyristoyl-trimethylammonium propane (DMTAP); O,O′-Dimyristyl-N-lysyl aspartate (DMKE); 1,2-Distearoyl-sn-glycero-3-ethylphosphocholine (DSEPC); N-Palmitoyl D-erythro-sphingosyl carbamoyl-spermine (CCS); N-t-Butyl-N0-tetradecyl-3-tetradecylaminopropionamidine; diC14-amidine; octadecenolyoxy[ethyl-2-heptadecenyl-3 hydroxyethyl] imidazolinium (DOTIM); chloride N1-Cholesteryloxycarbonyl-3,7-diazanonane-1,9-diamine (CDAN); 243-[bis(3-aminopropyl)amino]propylaminol-N-[2-[di(tetradecl]amino]-2-oxoethyl]acetamide (RPR209120); ditetradecylcarbamoylme-ethyl-acetamide; 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA); 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane; DLin-KC2-DMA; dilinoleyl-methyl-4-dimethylaminobutyrate; DLin-MC3-DMA; DLin-K-DMA; 98N12-5; C12-200; a cholesterol; a PEG-lipid; a lipiopolyamine; dexamethasone-spermine (DS); and disubstituted spermine (D25).
As noted above, in some cases methods and compositions of the disclosure can be diagnostically responsive (i.e., designed based on information such as RNA and/or protein expression data from the individual being treated). As such, design of the delivery vehicle (e.g., selection of an appropriate nanoparticle targeting ligand) and/or payload (e.g., choice of a particular promoter for expressing a heterologous RNA and/or protein) can be tailored to the specific characteristics of a patient's disease. This may be accomplished in a diagnostically responsive manner, e.g., after biopsy and analysis of the retrieved tissue/cells.
In some cases, the information used from an individual when designing a diagnostically responsive formulation is information from high throughput methodologies such as high throughput/next generation RNA or DNA sequencing methods (e.g., nanopore sequencing, 454 pyrophosphate sequencing, single molecule Heliscope sequencing, nano-array sequencing, SOLiD sequencing, Illumina/Solexa sequencing, Ion Torrent sequencing, Single-molecule real-time (SMRT) sequencing, and the like—see, e.g., Reuter et al., Mol Cell. 2015 May 21; 58(4):586-97). In some cases, the information used from an individual when designing a diagnostically responsive formulation is information from high throughput proteomic technologies (e.g., Mass spectrometry (MS)-based high-throughput proteomics, antibody arrays, peptide arrays, ligand/receptor-based arrays, and the like—see, e.g., Zhang et al., Annu Rev Anal Chem (Palo Alto Calif.). 2014; 7:427-54; Paczesny et al., Proteomics Clin Appl. 2018 Oct. 11:e1800145). In some cases, the information used is the identity of (e.g., a list of) proteins and/or nucleic acids that are highly expressed, enriched, and/or specifically expressed in diseased tissue such as cancer cells. In some such cases, the information used includes or is even limited to cell surface proteins that are highly expressed, enriched, and/or specifically expressed in diseased tissue such as cancer cells.
While the information used from an individual can be from high throughput methodologies, such information is not necessary in all cases. For example, in some cases, a disease such as a particular type of cancer can classified into subgroupings based on previously determined diagnostic assays. In some cases, such assays can be used to identify a desired protein and/or nucleic acid (e.g., a surface protein) that is highly expressed, enriched, and/or specifically expressed in diseased tissue such as cancer cells.
The information used from an individual can in some cases include identification of one or more of: (1) highly expressed, enriched, and/or specifically expressed surface protein(s) (e.g., receptors); (2) a promoter(s) that is highly expressed, enriched, and/or specifically expressed; and (3) highly expressed, enriched, and/or specifically expressed proteolytic enzyme(s) (e.g. MMPs, cathepsins).
A subject delivery vehicle such as a nanoparticle and/or payload can then be designed based on the individual's information (e.g., diagnosis/classification, based on an identified enriched surface protein in a target cell/tissue/organ). As examples:
A novel approach for modeling and predicting ideal target sequences in a desired cell, tissue, organ or cancer target is outlined whereby a database containing RNAseq and/or proteomics data is compared against expression patterns in all available datasets for healthy tissues. This allows for generating various means of establishing the selectivity of a given receptor/surface protein targeting approach. In this example, data was gathered from the GTEx portal and Human Protein Atlas.
The identified proteins above may represent ligand and/or receptor and/or structural homologues of concomitant ligand/receptor/secretome profiles of target cell populations. In other words, a target cell/tissue/organ will contain a certain set of overexpressed genes. In the above examples, several cancer-enriched markers are shown for a variety of cancer markers based on transcriptomics and/or proteomics data from the Human Protein Atlas, as compared to healthy tissues/organs through selection algorithms detailed throughout this application. In the above examples, crystal structures represent a ligand OR a receptor OR a secreted protein for a given receptor profile or secreted microenvironment of a cell/tissue/organ. Ligands may represent locally secreted (e.g. lung-cancer-enriched) proteins and protein fragments thereof, in order to take part in an autocrine and/or paracrine signaling environment that is cell, tissue, organ, and/or cancer enriched, or to mimic physicochemical properties that are ideal for that environment (e.g. Surfactant protein B being a mucoadsorptive molecule, as shown in
In an illustrative example of keratin 31 (
Domains of 30-80 amino acids may also be ligated together (e.g. through native chemical ligation) in order to assemble larger proteins that typically can only be synthesized recombinantly. This offers the advantage of controlling protein folding in stages and sequentially assembling proteins with appropriate tertiary and quaternary structures. Such techniques of peptide synthesis may also be utilized for assembling protein components of gene editing materials such as TALENs, whereby 31-33 amino acid RVD (repeat variable diresidue) sequences may be synthesized and subsequently “daisy chained” together through native chemical ligation (
In the following examples (
In this illustrative example, sequences from one protein align highly with the signaling domain of another protein. Even in the absence of structural data on the entire protein, the relevant portion for designing a peptide targeting ligand can be predicted and modeled with high precision and accuracy across various protein classes. The need for large tertiary structures to align is eliminated when binding motifs between peptide ligands and their cognate receptors represent small portions of the overall protein. In some cases, techniques such as those described in: AlQuraishi M, Cell Syst. 2019 Apr. 24; 8(4):292-301. Epub 2019 Apr. 17; can be used (e.g., in some cases when the designed candidate protein 20 or more amino acids in length). Such techniques can be used to compare the structure of larger sequences when structural data is limited or not available prior to extracting and optimizing smaller binding sequences
In the following protein sequence alignment script (EMBOSS Needle), human and mouse SCF isoform 1 are found to have 89.7% sequence similarity (
Any combination of the above personalized techniques can be used. For example, diagnostic information can be used to select a targeting ligand (and/or desired cell type to target), a promoter, and cargo. On the other hand, a more generalized cargo can be delivered in a personalized (diagnostically responsive) way by delivering the cargo using a delivery vehicle (e.g., a nanoparticle) that has a targeting ligand this is personalized. Likewise, a specific personalized cargo (e.g., a gene-editing cargo that edits a T cell receptor) can be delivered using a delivery vehicle that does not include a personalized targeting ligand—e.g., a delivery vehicle such as a nanoparticle can be delivered by local inject such as intratumoral injection. A combination of promoters and protease-specific sequences may also be utilized to increase cell, tissue, organ and/or cancer-specific release and activity of a given payload.
In some cases, a subject method is not molecularly tailored to a particular individual based on diagnostic information (e.g., genotype/phenotypic evaluation). For example, localization can in some cases be achieved via direct local injection (e.g., into a tumor). In some cases, delivery is not personalized (is not diagnostically responsive). For example, in some cases a subject delivery vehicle (e.g., a nanoparticle) is delivered without using a targeting ligand, promoter or protease domain that was designed based on the patient's profile. For example, in some cases a delivery vehicle is delivered via passive delivery (e.g., systemic delivery or local delivery such as injection) so that it accumulates in a target tissue such as a tumor.
The tumor (or organ/tissue) microenvironment's pathophysiology and immunological milieu also present a set of hurdles for successful immunotherapy and/or nanoparticle targeting. The tumor microenvironment (TME) is a complex and dynamic circuit of malignant and non-malignant cell interactions. Due to the TME's hypoxic and inflammatory setting, antigen presenting cells in the TME can fail to activate the immune system. Malignant cells are also known to recruit T regulatory cells and myeloid derived suppressor cells as well as promote production of IL-10, vascular endothelial growth factor, indoleamine 2,3-dioxygenase, TGF-0, and other immunosuppressive chemokines. Delivery vehicles such as nanoparticles of this disclosure can be used to suppress the production of these and other factors through delivery of siRNA or miRNA that target the immunosuppressive signals such as chemokines. On the other hand, delivery vehicles (such as nanoparticles) of this disclosure can be used to deliver, as a payload, a nucleic acid that encodes a secreted protein, e.g., pro-inflammatory signs such as a cytokine.
In some embodiments, delivery of the payload results in expression and secretion of a protein of interest (a protein such as a cytokine that modulates the local tumor microenvironment after secretion). In other embodiments, “secretomimetic” ligands may confer favorable characteristics to nanoparticles designed to function in a specific secretome environment (e.g.
Payloads that lead to cancer cell cytotoxicity (including any variants thereof that retain their cytotoxic function)
In some embodiments, a delivery vehicle (e.g., a nanoparticle such as a targeted nanoparticle) is used to influence protein expression and/or cell surface composition of a target cell such as a cancerous tissue thereby bolstering the adaptive immune response and overcoming physiological hurdles faced in the treatment of solid tumors. Thus, in some embodiments delivery of a payload results in expression and presentation of a protein of interest (e.g., an affinity marker) on the surface of the cell.
In some cases the affinity marker is a protein presented on the cell surface that is highly immunogenic and is a “non-self” domain. This approach can bypass the central tolerance in the thymus. Delivery using non-viral delivery vehicles such as nanoparticles mitigates barriers faced by viral delivery because nanoparticles do not express immunogenic epitopes on their surface and are stealth from the immune system until interaction with the targeted cancer cells.
As such, in some cases a payload is an affinity marker (or a nucleic acid encoding same). The term “affinity marker” is used herein to refer to a polypeptide presented on the cell surface (e.g., via forced heterologous expression in a target cell such as a cancer cell) that may elicit an endogenous adaptive immune response (against the affinity marker) and/or may act as a target for T-Cell therapy. In some cases an affinity marker is a naturally existing membrane protein, and in some cases an affinity marker is a chimeric polypeptide in which a membrane anchored region (e.g., a transmembrane domain) is fused to an extracellular portion that elicits an endogenous immune response or is targeted with T-cells that are engineered to recognize the affinity marker.
Thus, in some cases cancerous tissue can be “programmed” to present a distinct surface marker as a domain that is subsequently targeted by immune cells, triggering an adaptive immune response across many tumor subclonal populations. This approach presents an improvement to TCR or CAR engineering, and other single-marker targeted immuno-oncology approaches, in that the affinity marker (in some cases delivered via nanoparticle) induces a tumor-wide expression of adaptive immune learning cues. For particularly complex cancers with a diversity of clonal subpopulations, this leads to a more robust learning response and improved treatment. Additionally, the in vivo utility of this approach limits the need for complex and cumbersome autologous and allogeneic cell transplantation procedures.
In some cases cancerous tissue is programmed to present a distinct antigen as a functional domain that is subsequently targeted by an engineered (e.g., cytotoxic) T cell. The T Cell can possess a TCR or CAR that is specific to the antigen, and may be engineered ex vivo or in vivo.
An affinity marker payload can be delivered using any delivery vehicle. In some cases the delivery vehicle is a subject nanoparticle (e.g., a nanoparticle that includes a targeting ligand and/or a core comprising an anionic polymer composition, a cationic polymer composition, and a cationic polypeptide composition). In some cases the affinity marker is delivered using a delivery vehicle with a targeting ligand and in some cases using a delivery vehicle without a targeting ligand (e.g., the delivery vehicle can be delivered using local administration such as intratumoral injection).
An affinity marker payload can be delivered using personalized delivery (descried in more detail elsewhere herein)—meaning, e.g., that it can be delivered using a delivery vehicle designed using information from the individual/patient. For example, in some cases an affinity marker payload is delivered using a delivery vehicle with a targeting ligand and/or a promoter that was selected based on an individual's/patient's diagnostic evaluation. In some cases a subject affinity marker is a diagnostically responsive surface protein—meaning that the surface protein was determined to be enriched on the surface of cancer cells of an individual/patient or even specifically expressed by such cells.
In some cases, the affinity marker can stimulate innate immune activity (i.e., the affinity marker can be recognized by endogenous immune cells as signal of non-self, and this can trigger an endogenous immune system response against cells expressing that beacon). In some cases T-cells engineered to target the affinity marker can be co-administered (either in series or in parallel) with the delivery vehicle. The affinity marker may be any protein or protein fragment with a known protein-protein interaction, including endogenous human proteins, viral proteins, and synthetic de novo proteins. In some cases, an affinity marker engages a direct signaling cascade (for example, but not limited to—with a CAR-T/TCR).
In some cases, an affinity marker is a synthetic chimeric protein that includes a membrane anchor fused (e.g., via a linker—various linkers are described elsewhere herein and can be used in an affinity marker) to a functional domain that is displayed extracellularly by the cell that expresses it. Tables 17 and 18 provide examples of membrane anchors and extracellular polypeptides that can be used as part of an affinity marker. These “anchors” may represent conserved transmembrane domains of extracellularly-presenting affinity marker sequences, or sequence alignments for machine learning approaches for determining optimal ligand-receptor docking for a given cell/tissue/organ with one of these classes of proteins or homologues enriched. Rather than de novo modeling of ligand-receptor interactions, this approach allows for rapid design and synthesis of a targeting ligand or library of targeting ligands (e.g. selectively mutated amino acid residues and/or peptoid and/or synthetic amino acid and/or alternative polymer/glycoprotein modifications upon a native peptide or glycoprotein sequence). De novo modeling and synthesis approaches may also be used, either as part of selected mutagenesis libraries or alternative means of combinatorial/library prep. (e.g. SELEX, phage display, and similar techniques). These techniques are further enhanced by a modular nanoparticle, nanomaterials and gene editing/gene delivery platform approach for efficiently delivering these synthetic markers (e.g. affinity markers, transmembrane anchor domains detailed elsewhere) to specified cells/tissues/organs/cancers.
Examples of extracellular domains that can be used as part of an affinity marker domains are detailed through the sets of ligands and receptors outlined within this disclosure. In other words, a non-limiting example includes any ligand or receptor pairing outlined herein (or otherwise determined through proteomics and/or transcriptomics of a given cell population—or otherwise identifiable cell-specific markers) can be utilized to create an affinity marker. Many such pairings are detailed herein.
In some examples, an already-overexpressed protein may be further hyper-expressed within a target cell/tissue/organ/cancer type. For example, a transmembrane domain that is uniquely and/or differentially expressed within a target tumor (e.g. a transmembrane domain with high cell/tissue/organ specificity indices) may be used as a sequence that further includes an extracellular affinity domain (as detailed elsewhere) or a signaling domain (as with introduction of a GPCR, DREADD, or chimeric receptor). These extracellular domains may serve as affinity domains for chimerically-modified immune cells (or other cells, such as stem cells), and may be coupled to enhanced or suppressed immune/stem cell/other circulatory cell homing (e.g. chemotaxis) or signaling (e g enhanced killing response of a CD8+ T cell subpopulation, NK cell subpopulation; enhanced affinity of an antigen-presenting cell subpopulation).
These affinity domains may include any variants thereof that maintain their immune-stimulating function, as well as a multitude of immunogenic markers such as viral protein fragments and patient-defined preexisting immunity/allergy/immune-response-generating peptide/glycopeptide/lipopeptide/glycolipid sequences. A cancer neoantigen may also serve as an extracellular domain. Engagement of dendritic cells and other antigen-presenting cells (APCs, including gamma delta (γδ) T cells, as part of this platform is further detailed within this disclosure as a method and use for personalized immunotherapies. These personalized immunotherapies are designed to be in vivo, ex vivo, or through a combination of ex vivo and in vivo approaches, whereby a subject nanoparticle or delivery vehicle is administered with affinity for a patient's cancer or a specific subtype of cells that require secondary beaconing by an alternative cell subpopulation (e.g. senescent cells being targeted to generate affinity for an extracellularly presenting domain of an engineered stem cell engraftment. Other methods for regenerative therapies can be envisaged. An optional, secondary subject nanoparticle or delivery vehicle may be utilized to introduce a “standardized docking domain” into a specific immune subpopulation or combination of immune subpopulations, or alternatively to a specific “interactive cell population” whereby the interactive cell population is intended to have a signaling and/or chemotactic effect with its local environment and the secondarily targeted set of cells.
Advances in rapid DNA synthesis technology further facilitate these innovations, whereby cancer-diagnostic determined (e.g. diagnostically-responsive transcriptomic and cell surface proteomic) transmembrane sequences may be introduced into a patient (following DNA synthesis or mRNA amplification/synthesis of the appropriate sequence) as part of a nanoparticle-administered immunotherapy, whereby the transmembrane domain (“cell/tissue/organ/cancer personalized transmembrane domain”) serves as a further anchor for an affinity domain (a ligand or receptor or fragment thereof as outlined elsewhere in this disclosure) and is encoded by the delivered DNA. Numerous library-generation DNA approaches may be utilized to combinatorially screen top-performing nanoparticle candidates delivering a variety of transgenes to a cell, tissue, organ or cancer type, and evaluate directed mutagenic libraries. For example, a large TCR mutagenic library may be utilized and transfected into T cells to establish optimal cancer-killing effects of a given recognition and signaling domain. Gene editing approaches and gene insertion approaches may be utilized as well, whereby donor DNA templates are customized for each patient and can be combinatorially or singly evaluated for their 1) gene insertion efficiency and/or 2) phenotypic effect. Rapid DNA synthesis may be coupled to existing peptide, polymer and/or ligand/anchor/linker libraries and is further supported by rapid peptide synthesis and predictive ligand-receptor modeling with optional high-throughput fluid-handling robotic workflows in the case of nanoparticle synthesis or library preparation with a variety of drug/RNA/DNA/protein-ligand conjugation techniques. Top-performing nanomedicine candidates can readily be applied to microfluidic and millifluidic scale-up techniques as well as parallel arrays of microfluidic devices for milligram-to-kilogram scale synthesis. Newly synthesized (e.g. high-throughput synthesized) peptide sequences may be coupled to anchor-linker or anchor libraries (detailed elsewhere) through numerous means further facilitated by flow-based synthesis and fluid-handling techniques. These peptide or ligand-polymer sequences may be combinatorially assembled with a variety of genetic, protein or small molecule payloads, as well as directly chemically conjugated to numerous surfaces and reactive domains, to enable multimodal and “super-personalized” diagnostically-responsive therapies. The ligands used herein and their associated anchors and linkers may also be introduced to recombinant protein sequences (e.g. recombinant Cas9-ligand, recombinant TALEN-ligand, recombinant recombinase-ligand) or modified nucleic acids/PNAs/MNAs/LNAs (e.g. modRNA-ligand, PNA-ligand, DNA-PNA-ligand, RNA-DNA-ligand, and the like) either homovalently or heterovalently through the methods and uses described herein (the “diagnostically-responsive” workflows. Combinatorial genes with DNA/RNA/PNA/LNA barcodes may also be used to create large pooled libraries of nanoparticles that can be subsequently sequenced in target cells, allowing for each formulation to have its own tag for subsequent identification in cell, organ-on-chip or animal models.
As noted above, in some cases, introduction of a payload encoding/carrying an affinity marker into a target cell results in the expression of the affinity marker on the surface of a targeted cell such as a cancer cell. In some such cases, this is coupled with a T-cell therapy in which T cells are engineered to recognize the affinity marker. The T cells can be introduced into the individual as part of a T cell therapy (after being engineer in vitro/ex vivo to express the desired receptor), or the T cells can be engineered endogenously (edited in vivo) in the individual. To accomplish the engineering, the T cell receptor (TCR) locus (e.g., alpha, beta, delta, and/or gamma subunit) of T cells can be edited so that the T cells express an engineered receptor that can specifically bind to the desired affinity marker. T cells can also be engineered to express a chimeric antigen receptor (CAR). Either way, the engineered T cells specifically recognize and target those cells that were targeted to express the affinity marker.
As one example, a NY-ESO antigen sequence may be inserted into cancer cells, and a corresponding NY-ESO-targeted TCR may be used with gamma delta (γδ) T cells in order to create an enhanced antigen-presenting effect following T cell distribution within the target cancer. Other antigen-presenting cells or αβ T cells may also be utilized.
In some cases, an affinity marker can be used to aid cell engraftment (e.g., stem cell engraftment when administering stem cells to a patient). Thus, in some cases, an affinity includes a functional domain that grants a cell affinity to a tissue, organ, or tissue environment of interest (e.g., when the affinity marker is expressed on the cell's surface). This is of particular interested for use in regenerative medicine applications where this may promote proper engraftment of cells in the desired environment and in the desired phenotype. For example, expanded stem cells can lose their phenotypic surface presentation and can be unable to migrate and/or engraft properly. They can also become trapped in the liver, lung, and/or spleen. Because of this, sometimes as little as 1% can reach the target tissue/disease area. In addition, direct injection of cells at the target organ can include a risk of hemorrhage and other complications associated with the administration method. Cell survival is also a shortcoming. To the contrary, affinity markers can promote adhesion to proper tissue compartment so that proper engraftment is achieved, as well as promote migration from the site of administration to the target organ thereby mitigating problems associated with expansion of both autologous and allogeneic stem cells. Thus, in some cases, affinity markers are expressed on stem cells that can be used in adoptive cell transfer. The stem cells can be any stem cell (e.g., endoderm, ectoderm, mesoderm stem cells; hematopoietic stem cells; mesenchymal stem cells; neural stem cells; endocrine precursors; and the like). When using stem cells for such applications, the stem cells can in some cases differentiate into any desired cell/tissue type (e.g., cartilage, bone, cardiomyocytes, neurons, adipocytes, osteoblasts, hepatocytes, myoblasts, neuron-like cells, and the like). The target organs/tissues can include, e.g., kidney, AKI administered for tubular endothelial cell repair, inflamed bowel, lung, bone, bone marrow, ischemic tissue, myocardial infarct damaged tissue, wounds, and the like. Such applications can be used for, e.g., diabetes, beta cell pathologies, myocardial infarction, brain trauma, and multiple sclerosis. Examples can include, e.g., migratory receptors of the CXC, CC, XC, CX3C families (e.g., CCR1, CCR2, CCR7, CXCR4/SDF-1, CX3CR1, CXCR6, c-met, CD44), which respond to proteins such as CXCL9, CXCL16, CCL20, CCL25, HGF, MCP-3, CXCL12, and HIF. In some case, e.g., when using hematopoietic stem cells, example proteins can include CCR1, CCR4, CCR7, CXCR5, and CCR10. In some cases stem cells can be used for their immunomodulatory abilities due to their ability to secrete a wide variety of growth factors and cytokines, with a subset that may have a profound effect on modulating immune response.
In some cases a subject method includes using a delivery vehicle to deliver a payload to a target cell, e.g., via administration to an individual, via transfection, via a nanoparticle, via a delivery molecule, etc. In some cases two or more different payloads are introduced into the cell as part of the same delivery vehicle (e.g., nanoparticle, delivery molecule, etc.). The payload can be delivered to any desired target cell, e.g., any desired eukaryotic cell such as a cancer cell.
In some cases the target cell is in vitro (e.g., the cell is in culture), e.g., the cell can be a cell of an established tissue culture cell line. In some cases the target cell is ex vivo (e.g., the cell is a primary cell (or a recent descendant) isolated from an individual, e.g. a patient). In some cases, the target cell is in vivo and is therefore inside of (part of) an organism.
A delivery vehicle may be introduced to a subject (i.e., administered to an individual) via any of the following routes: systemic, local, parenteral, subcutaneous (s.c.), intravenous (i.v.), intracranial (i.c.), intraspinal, intraocular, intradermal (i.d.), intramuscular (i.m.), intralymphatic (id.), or into spinal fluid. The components may be introduced by injection (e.g., systemic injection, direct local injection, local injection into or near a tumor and/or a site of tumor resection, etc.), catheter, or the like. Examples of methods for local delivery (e.g., delivery to a tumor and/or cancer site) include, e.g., by bolus injection, e.g. by a syringe, e.g. into a joint, tumor, or organ, or near a joint, tumor, or organ; e.g., by continuous infusion, e.g. by cannulation, e.g. with convection (see e.g. US Application No. 20070254842, incorporated here by reference).
The number of administrations of treatment to a subject may vary. Introducing a delivery vehicle into an individual may be a one-time event; but in certain situations, such treatment may elicit improvement for a limited period of time and require an on-going series of repeated treatments. In other situations, multiple administrations of a delivery vehicle may be required before an effect is observed. As will be readily understood by one of ordinary skill in the art, the exact protocols depend upon the disease or condition, the stage of the disease and parameters of the individual being treated.
A “therapeutically effective dose” or “therapeutic dose” is an amount sufficient to effect desired clinical results (i.e., achieve therapeutic efficacy). A therapeutically effective dose can be administered in one or more administrations. For purposes of this disclosure, a therapeutically effective dose of a payload is an amount that is sufficient, when administered to the individual, to palliate, ameliorate, stabilize, reverse, prevent, slow or delay the progression of a disease state/ailment.
In some cases, the target cell is a mammalian cell (e.g., a rodent cell, a mouse cell, a rat cell, an ungulate cell, a cow cell, a sheep cell, a pig cell, a horse cell, a camel cell, a rabbit cell, a canine (dog) cell, a feline (cat) cell, a primate cell, a non-human primate cell, a human cell). Any cell type can be targeted, and in some cases specific targeting of particular cells depends on the presence of targeting ligands (e.g., as part of a surface coat of a nanoparticle, as part of a delivery molecule, etc), where the targeting ligands provide for targeting binding to a particular cell type. For example, cells that can be targeted include but are not limited to bone marrow cells, hematopoietic stem cells (HSCs), long-term HSCs, short-term HSCs, hematopoietic stem and progenitor cells (HSPCs), peripheral blood mononuclear cells (PBMCs), myeloid progenitor cells, lymphoid progenitor cells, T-cells, B-cells (e.g., via targeting CD19, CD20, CD22), NKT cells, NK cells, dendritic cells, monocytes, granulocytes, erythrocytes, megakaryocytes, mast cells, basophils, eosinophils, neutrophils, macrophages (e.g., via targeting CD47 via SIRPα-mimetic peptides), erythroid progenitor cells (e.g., HUDEP cells), megakaryocyte-erythroid progenitor cells (MEPs), common myeloid progenitor cells (CMPs), multipotent progenitor cells (MPPs), hematopoietic stem cells (HSCs), short term HSCs (ST-HSCs), IT-HSCs, long term HSCs (LT-HSCs), endothelial cells, neurons, astrocytes, pancreatic cells, pancreatic β-islet cells, muscle cells, skeletal muscle cells, cardiac muscle cells, hepatic cells, fat cells, intestinal cells, cells of the colon, and cells of the stomach.
Examples of various applications (e.g., for targeting neurons, cells of the pancreas, hematopoietic stem cells and multipotent progenitors, etc.) are discussed above, e.g., in the context of targeting ligands. For example, hematopoietic stem cells and multipotent progenitors can be targeted for gene editing (e.g., insertion) in vivo. Even editing 1% of bone marrow cells in vivo (approximately 15 billion cells) would target more cells than an ex vivo therapy (approximately 10 billion cells) and in many cases (such as with sickle cell disease) the pathology will innately positively select for a cell chimerism (e.g. the targeted and edited cell populations expanding preferentially due to survival-enhancing pleiotropic effects of HBB edits). In vivo applications are amenable to repeat dosing with a non-viral platform consisting of native human protein fragments and other targeting ligand/constituent polymer designs that are unlikely to be immunogenic, and can particularly benefit from techniques for selective expansion either through direct programming e.g. a stem cell differentiation factor, or pleiotropic effects as outlined above). As another example, pancreatic cells (e.g., (3 islet cells) can be targeted, e.g., to treat pancreatic cancer, to treat diabetes, etc. In an exemplary embodiment, pancreatic B islets in Type I diabetes, if engineered to be less prone to autoimmunity, would also innately experience positive selection vs. non-targeted cells following treatment similarly to HSCs edited to be free of the sickle cell trait. As another example, somatic cells in the brain such as neurons can be targeted (e.g., to treat indications such as Huntington's disease, Parkinson's (e.g., LRRK2 mutations), and ALS (e.g., SOD1 mutations) and may experience enhanced survival or stem cell renewal following treatment). Additionally, targeted cells may have multiple genetic, protein, or small molecule instructions delivered to them, whereby edited or modified cells will experience asymmetrical cell division (e.g. enhanced cell division) in response to growth-stimulatory or cell differentiation cues (e.g. IL2 mRNA or mRNA/DNA/molecules encoding a cytokine/chemokine activity in immune cells; SCF, NGF, or other growth factor/Yamanaka factor mRNA or mRNA/DNA/molecules encoding a cell differentiation cue in stem cell poopulations, etc.). In some cases neural targeting can be achieved through direct intracranial injections. In other cases treatment of a cancer may be presented following resection of a tumor, to cause local environmental programming. Other local injection approaches may be utilized with or without ligand targeting in order to provide local effects and optional multimodal programming (e.g. gene edit+mRNA, gene edit+small molecules, mRNA+DNA, and the like).
As another example, endothelial cells and cells of the hematopoietic system (e.g., megakaryocytes and/or any progenitor cell upstream of a megakaryocyte such as a megakaryocyte-erythroid progenitor cell (MEP), a common myeloid progenitor cell (CMP), a multipotent progenitor cell (MPP), a hematopoietic stem cells (HSC), a short term HSC (ST-HSC), an IT-HSC, a long term HSC (LT-HSC)—see, e.g.,
Methods and compositions of this disclosure can be used to treat any number of diseases, including any disease that is linked to a known causative mutation, e.g., a mutation in the genome. For example, methods and compositions of this disclosure can be used to treat sickle cell disease, B thalassemia, HIV, myelodysplastic syndromes, JAK2-mediated polycythemia vera, JAK2-mediated primary myelofibrosis, JAK2-mediated leukemia, and various hematological disorders. As additional non-limiting examples, the methods and compositions of this disclosure can also be used for B-cell antibody generation, immunotherapies (e.g., delivery of a checkpoint blocking reagent), and stem cell differentiation applications.
In some embodiments, a targeting ligand provides for targeted binding to KLS CD27+/IL-7Ra-/CD150+/CD34-hematopoietic stem and progenitor cells (HSPCs). For example, the beta-globin (HBB) gene may be targeted directly to correct the altered E7V substitution with an appropriate donor DNA molecule. As one illustrative example, a CRISPR/Cas RNA-guided polypeptide (e.g., Cas9, CasX, CasY, Cpf1) can be delivered with an appropriate guide RNA(s) such that it will bind to loci in the HBB gene and cut the genome, initiating insertion of an introduced donor DNA. In some cases, a Donor DNA molecule (single stranded or double stranded) is introduced (as part of a payload) and is release for 14-30 days while a guide RNA/CRISPR/Cas protein complex (a ribonucleoprotein complex) can be released over the course of from 1-7 days.
In some embodiments, a targeting ligand provides for targeted binding to CD4+ or CD8+ T-cells, hematopoietic stem and progenitor cells (HSPCs), or peripheral blood mononuclear cells (PBMCs), in order to modify the T-cell receptor. For example, a gene editing tool(s) (described elsewhere herein) can be introduced in order to modify the T-cell receptor. The T-cell receptor may be targeted directly and substituted with a corresponding homology-directed repair donor DNA molecule for a novel T-cell receptor. As one example, a CRISPR/Cas RNA-guided polypeptide (e.g., Cas9, CasX, CasY, Cpf1) can be delivered with an appropriate guide RNA(s) such that it will bind to loci in the HBB gene and cut the genome, initiating insertion of an introduced donor DNA. It would be evident to skilled artisans that other CRISPR guide RNA and donor sequences, targeting beta-globin, CCR5, the T-cell receptor, or any other gene of interest, and/or other expression vectors may be employed in accordance with the present disclosure.
In some cases, a subject method is used to target a locus that encodes a T cell receptor (TCR), which in some cases has nearly 100 domains and as many as 1,000,000 base pairs with the constant region separated from the V(D)J regions by 100,000 base pairs or more.
In some cases insertion of the donor DNA occurs within a nucleotide sequence that encodes a T cell receptor (TCR) protein. In some such cases the donor DNA encodes amino acids of a CDR1, CDR2, or CDR3 region of the TCR protein. See, e.g., Dash et al., Nature. 2017 Jul. 6; 547(7661):89-93. Epub 2017 Jun. 21; and Glanville et al., Nature. 2017 Jul. 6; 547(7661):94-98. Epub 2017 Jun. 21.
In some cases a subject method is used to insert genes while placing them under the control of (in operable linkage with) specific enhancers as a fail-safe to genome engineering. If the insertion fails, the enhancer is disrupted leading to the subsequent gene and any possible indels being unlikely to express. If the gene insertion succeeds, a new gene can be inserted with a stop codon at its end, which is particularly useful for multi-part genes such as the TCR locus. In some cases, the subject methods can be used to insert a chimeric antigen receptor (CAR) or other construct into a T-cell, or to cause a B-cell to create a specific antibody or alternative to an antibody (such as a nanobody, shark antibody, etc.).
In some cases the donor DNA includes a nucleotide sequence that encodes a chimeric antigen receptor (CAR). In some such cases, insertion of the donor DNA results in operable linkage of the nucleotide sequence encoding the CAR to an endogenous T-cell promoter (i.e., expression of the CAR will be under the control of an endogenous promoter). In some cases the donor DNA includes a nucleotide sequence that is operably linked to a promoter and encodes a chimeric antigen receptor (CAR)—and thus the inserted CAR will be under the control of the promoter that was present on the donor DNA.
In some cases the donor DNA includes a nucleotide sequence encoding a cell-specific targeting ligand that is membrane bound and presented extracellularly. In some cases, insertion of said donor DNA results in operable linkage of the nucleotide sequence encoding the cell-specific targeting ligand to an endogenous promoter. In some cases the donor DNA includes a promoter operably linked to the sequence that encodes a cell-specific targeting ligand that is membrane bound and presented extracellularly—and therefore, after insertion of the donor DNA, expression of the membrane bound targeting ligand will be under the control of the promoter that was present on the donor DNA.
In some embodiments, insertion of a donor DNA occurs within a nucleotide sequence that encodes a T cell receptor (TCR) Alpha or Delta subunit. In some cases, insertion of a donor DNA occurs within a nucleotide sequence that encodes a TCR Beta or Gamma subunit. In some cases a subject method and/or composition includes two donor DNAs. In some such cases insertion of one donor DNA occurs within a nucleotide sequence that encodes a T cell receptor (TCR) Alpha or Delta subunit and insertion of the other donor DNA occurs within a nucleotide sequence that encodes a T cell receptor (TCR) Beta or Gamma subunit.
In some embodiments, insertion of a donor DNA occurs within a nucleotide sequence that encodes a T cell receptor (TCR) Alpha or Delta subunit constant region. In some cases insertion of a donor DNA occurs within a nucleotide sequence that encodes a T cell receptor (TCR) Beta or Gamma subunit constant region. In some cases a subject method and/or composition includes two donor DNAs. In some such cases insertion of one donor DNA occurs within a nucleotide sequence that encodes a T cell receptor (TCR) Alpha or Delta subunit constant region and insertion of the other donor DNA occurs within a nucleotide sequence that encodes a T cell receptor (TCR) Beta or Gamma subunit constant region.
In some embodiments, insertion of a donor DNA occurs within a nucleotide sequence that functions as a T cell receptor (TCR) Alpha or Delta subunit promoter. In some cases insertion of a donor DNA occurs within a nucleotide sequence that functions as a T cell receptor (TCR) Beta or Gamma subunit promoter. In some cases a subject method and/or composition includes two donor DNAs. In some such cases insertion of one donor DNA occurs within a nucleotide sequence that functions as a T cell receptor (TCR) Alpha or Delta subunit promoter and insertion of the other donor DNA occurs within a nucleotide sequence that functions as a T cell receptor (TCR) Beta or Gamma subunit promoter.
In some embodiments, insertion of a sequence of the donor DNA occurs within a nucleotide sequence that encodes a T cell receptor (TCR) Alpha or Gamma subunit. In some cases, insertion of a sequence of the donor DNA occurs within a nucleotide sequence that encodes a TCR Beta or Delta subunit. In some cases a subject method and/or composition includes two donor DNAs. In some such cases insertion of one sequence of the donor DNA occurs within a nucleotide sequence that encodes a T cell receptor (TCR) Alpha or Gamma subunit and insertion of the sequence of the other donor DNA occurs within a nucleotide sequence that encodes a T cell receptor (TCR) Beta or Delta subunit.
In some embodiments, insertion of a sequence of the donor DNA occurs within a nucleotide sequence that encodes a T cell receptor (TCR) Alpha or Gamma subunit constant region. In some cases insertion of a sequence of the donor DNA occurs within a nucleotide sequence that encodes a T cell receptor (TCR) Beta or Delta subunit constant region. In some cases a subject method and/or composition includes two donor DNAs. In some such cases insertion of one sequence of the donor DNA occurs within a nucleotide sequence that encodes a T cell receptor (TCR) Alpha or Gamma subunit constant region and insertion of the sequence of the other donor DNA occurs within a nucleotide sequence that encodes a T cell receptor (TCR) Beta or Delta subunit constant region.
In some embodiments, insertion of a sequence of the donor DNA occurs within a nucleotide sequence that functions as a T cell receptor (TCR) Alpha or Gamma subunit promoter. In some cases insertion of a sequence of the donor DNA occurs within a nucleotide sequence that functions as a T cell receptor (TCR) Beta or Delta subunit promoter. In some cases a subject method and/or composition includes two donor DNAs. In some such cases insertion of one sequence of the donor DNA occurs within a nucleotide sequence that functions as a T cell receptor (TCR) Alpha or Gamma subunit promoter and insertion of the sequence of the other donor DNA occurs within a nucleotide sequence that functions as a T cell receptor (TCR) Beta or Delta subunit promoter.
In some embodiment, insertion of a donor DNA results in operable linkage of the inserted donor DNA with a T cell receptor (TCR) Alpha, Beta, Gamma or Delta endogenous promoter. In some cases, the donor DNA comprises a protein-coding nucleotide sequence that is operably linked to a TCR Alpha, Beta, Gamma or Delta promoter such that after insertion, the protein-coding sequence will remain operably linked to (under the control of) the promoter present in the donor DNA. In some cases insertion of said donor DNA results in operable linkage of the inserted donor DNA (e.g., a protein-coding nucleotide sequence such as a CAR, TCR-alpha, TCR-beta, TCR-gamma, or TCR-Delta sequence) with a CD3 or CD28 promoter. In some cases the donor DNA includes a protein-coding nucleotide sequence that is operably linked to a promoter (e.g., a T-cell specific promoter). In some cases insertion of the donor DNA results in operable linkage of the inserted donor DNA with an endogenous promoter (e.g., a stem cell specific or somatic cell specific endogenous promoter). In some cases the donor DNA includes a nucleotide sequence that encodes a reporter protein (e.g., fluorescent protein such as GFP, RFP, YFP, CFP, a near-IR and/or far red reporter protein, etc., e.g., for evaluating gene editing efficiency). In some cases the donor DNA includes a protein-coding nucleotide sequence (e.g., one that encodes all or a portion of a TCR protein) that does not have introns.
In some cases a subject method (and/or subject compositions) can be used for insertion of sequence for applications such as insertion of fluorescent reporters (e.g., a fluorescent protein such green fluorescent protein (GFP)/red fluorescent protein (RFP)/near-IR/far-red, and the like), e.g., into the C- and/or N-termini of any encoded protein of interest such as transmembrane proteins.
In some embodiments, insertion of the nucleotide sequence of the donor DNA into the cell's genome results in operable linkage of the inserted sequence with an endogenous promoter (e.g., (i) a T-cell specific promoter; (ii) a CD3 promoter; (iii) a CD28 promoter; (iv) a stem cell specific promoter; (v) a a somatic cell specific promoter; (vi) a T cell receptor (TCR) Alpha, Beta, Gamma or Delta promoter; (v) a B-cell specific promoter; (vi) a CD19 promoter; (vii) a CD20 promoter; (viii) a CD22 promoter; (ix) a B29 promoter; and (x) a T-cell or B-cell V(D)J-specific promoter). In some cases the nucleotide sequence, of the insert donor composition, that is inserted includes a protein-coding sequence that is operably linked to a promoter (e.g., (i) a T-cell specific promoter; (ii) a CD3 promoter; (iii) a CD28 promoter; (iv) a stem cell specific promoter; (v) a somatic cell specific promoter; (vi) a T cell receptor (TCR) Alpha, Beta, Gamma or Delta promoter; (v) a B-cell specific promoter; (vi) a CD19 promoter; (vii) a CD20 promoter; (viii) a CD22 promoter; (ix) a B29 promoter; and (x) a T-cell or B-cell V(D)J-specific promoter).
In some embodiments the nucleotide sequence that is inserted into the cell's genome encodes a protein. Any convenient protein can be encoded—examples include but are not limited to: a T cell receptor (TCR) protein; a CDR1, CDR2, or CDR3 region of a T cell receptor (TCR) protein; a chimeric antigen receptor (CAR); a cell-specific targeting ligand that is membrane bound and presented extracellularly; a reporter protein (e.g., a fluorescent protein such as GFP, RFP, CFP, YFP, and fluorescent proteins that fluoresce in far red, in near infrared, etc.). In some embodiments the nucleotide sequence that is inserted into the cell's genome encodes a multivalent (e.g., heteromultivalent) surface receptor (e.g., in some cases where a T-cell is the target cell). Any convenient multivalent receptor could be used and non-limiting examples include: bispecific or trispecific CARS and/or TCRs, or other affinity tags on immune cells. Such an insertion would cause the targeted cell to express the receptors. In some cases multivalence is achieved by inserting separate receptors whereby the inserted receptors function as an OR gate (one or the other triggers activation), or as an AND gate (receptor signaling is co-stimulatory and homovalent binding won't activate/stimulate cell, e.g., a targeted T-cell). A protein encoded by the inserted DNA (e.g., a CAR, a TCR, a multivalent surface receptor) can be selected such that it binds to (e.g., functions to target the cell, e.g., T-cell to) one or more targets selected from: CD3, CD8, CD4, CD28, CD90, CD45f, CD34, CD80, CD86, CD19, CD20, CD22, CD47, CD3-epsilon, CD3-gamma, CD3-delta; TCR Alpha, TCR Beta, TCR gamma, and/or TCR delta constant regions; 4-1BB, OX40, OX40L, CD62L, ARP5, CCR5, CCR7, CCR10, CXCR3, CXCR4, CD94/NKG2, NKG2A, NKG2B, NKG2C, NKG2E, NKG2H, NKG2D, NKG2F, NKp44, NKp46, NKp30, DNAM, XCR1, XCL1, XCL2, ILT, LIR, Ly49, IL2R, IL7R, IL 10R, IL12R, IL15R, IL18R, TNFα, IFNγ, TGF-β, and α5β1.
As noted elsewhere herein, one advantage of delivering multiple payloads as part of the same package (delivery vehicle) is that the efficiency of each payload is not diluted. In some embodiments a two different payloads are payloads of the same delivery vehicle. In some embodiments, a donor DNA and/or one or more gene editing tools (e.g., as described elsewhere herein) is delivered in combination with (e.g., as part of the same package/delivery vehicle, where the delivery vehicle does not need to be a nanoparticle of the disclosure) a protein (and/or a DNA or mRNA encoding same) and/or a non-coding RNA that increases genomic editing efficiency. In some embodiments, one or more gene editing tools is delivered in combination with (e.g., as part of the same package/delivery vehicle, where the delivery vehicle does not need to be a nanoparticle of the disclosure) a protein (and/or a DNA or mRNA encoding same) and/or a non-coding RNA that controls cell division and/or differentiation. For example, in some cases one or more gene editing tools is delivered in combination with (e.g., as part of the same package/delivery vehicle, where the delivery vehicle does not need to be a nanoparticle of the disclosure) a protein (and/or a DNA or mRNA encoding same) and/or a non-coding RNA that controls cell division. In some cases one or more gene editing tools is delivered in combination with (e.g., as part of the same package/delivery vehicle, where the delivery vehicle does not need to be a nanoparticle of the disclosure) a protein (and/or a DNA or mRNA encoding same) and/or a non-coding RNA that controls differentiation. In some cases, one or more gene editing tools is delivered in combination with (e.g., as part of the same package/delivery vehicle, where the delivery vehicle does not need to be a nanoparticle of the disclosure) a protein (and/or a DNA or mRNA encoding same) and/or a non-coding RNA that biases the cell DNA repair machinery.
As noted above, in some cases the delivery vehicle does not need to be a nanoparticle of the disclosure. For example, in some cases the delivery vehicle is viral and in some cases the delivery vehicle is non-viral. Examples of non-viral delivery systems include materials that can be used to co-condense multiple nucleic acid payloads, or combinations of protein and nucleic acid payloads. Examples include, but are not limited to: (1) lipid based particles such as zwitterionic or cationic lipids, and exosome or exosome-derived vesicles; (2) inorganic/hybrid composite particles such as those that include ionic complexes co-condensed with nucleic acids and/or protein payloads, and complexes that can be condensed from cationic ionic states of Ca, Mg, Si, Fe and physiological anions such as O2-, OH, PO43-, SO42-; (3) carbohydrate delivery vehicles such as cyclodextrin and/or alginate; (4) polymeric and/or co-polymeric complexes such as poly(amino-acid) based electrostatic complexes, poly(Amido-Amine), and cationic poly(B-Amino Ester); and (5) virus like particles (e.g., protein and nucleic acid based). Examples of viral delivery systems include but are not limited to: AAV, adenoviral, retroviral, and lentiviral.
Also within the scope of the disclosure are kits. For example, in some cases a subject kit can include one or more of (in any combination) any of the components discussed above, e.g.,: (i) a donor DNA; (ii) one or more gene editing tools; (iii) a targeting ligand, (iv) a linker, (v) a targeting ligand conjugated to a linker, (vi) a targeting ligand conjugated to an anchoring domain (e.g., with or without a linker), (vii) an agent for use as a sheddable layer (e.g., silica), (viii) a payload, e.g., a an siRNA or a transcription template for an siRNA or shRNA; a gene editing tool, a donor DNA, and the like, (ix) a polymer that can be used as a cationic polymer, (x) a polymer that can be used as an anionic polymer, (xi) a polypeptide that can be used as a cationic polypeptide, e.g., one or more HTPs, and (xii) a subject viral or non-viral delivery vehicle. In some cases, a subject kit can include instructions for use. Kits typically include a label indicating the intended use of the contents of the kit. The term label includes any writing, or recorded material, e.g., computer-readable media, supplied on or with the kit, or which otherwise accompanies the kit.
Nanoparticle formulations have 13+parameters optimized for a specific payload and biological condition through iterative screening. These parameters include, but are not limited to (
The number of all possible formulations even when limiting each parameter to only a few options becomes intractable for exhaustive screening. Several techniques can be employed to constrain the search heuristic, which integrates aspects of genetic algorithms, stochastic gradient descent, and simulated annealing. Screening consists of two phases: an initial ‘broad’ screen with generic formulations, followed by a set of ‘deep’ iterative screens.
The first phase of screening samples a diverse set of possible particle architectures to sparsely cover the entire search space with initial values. The initial formulations are a combination of preformulated benchmark particles and generated formulations with uniform step changes in a given parameter. Characterization of these initial formulations in terms of physicochemical properties (such as diameter and charge) and biological activity (such as uptake percentage, uptake rate, gene expression, and toxicity) provides a data signature of the particles, the components of which are individually weighted and summed with a performance scoring function.
For optimization purposes, a particle can be described as being a feature vector in formulation parameter space that an unknown function maps to a vector in scoring space. The objective of an iterative optimization strategy would then be to increment a formulation's parameters to increase and ultimately maximize a particle's score. Subsequent rounds of optimization utilize this paradigm. A machine learning-based approach can be used to both approximate the unknown objective function and generate changes to candidate formulations. In this phase of screening, candidate formulations can be robotically synthesized, characterized, and a subset of top performers can be selected. In the simplest embodiment, this subset can be a threshold percentage of the highest aggregate scores. In other cases, selection and deselection criteria can be used to filter the list of candidate formulations. Example criteria are selecting no particles with diameter above 600 um, or selecting particles with a lower aggregate score if their expression efficiency is in the top 10% of the round. Each formulation in this subset can then be iterated into several variations incrementing different parameters to generate the next full round of candidate formulations.
The algorithm uses the error difference between predicted performance and measured performance, in addition to the accumulation of data points from all previous rounds of screening, to refine the estimation of the objective function leading to improved predictions and optimizations over time. As rounds progress, the size of the parameter change from a parent formulation to its offspring formulations is progressively limited to allow for stable convergence and finer optimization. This method facilitates reasonably optimal formulations in an exponential search space while being sufficiently efficient to achieve rapid turnaround.
tSNE (t-Distributed Stochastic Neighbor Embedding), PCA (Principal component analysis) and other forms of modeling nanoparticle multiparametric data via unsupervised learning (e.g., input=formulation, output=bio and nano characterization) can be used, whereby top performing and/or “most interesting” formulation clusters (i.e., formulation clusters of interest) are automatically selected and iterated around (e.g., for one or more additional rounds of screening). In some such cases, a nanoparticle or gene barcode can be used as as one of the variables in the method (e.g., tSNE), where one can optionally investigate data such as mRNA-Seq data, and then aggregate how each specific cell sub population type behaves with the nanoparticle in terms of any desired parameter(s) (e.g., survival, uptake, expression, and the like).
Theranostics
Theranostic (e.g. MRI, PET or CT contrast agent) nanoparticles may be utilized to determine biodistributions of given targeting ligand approaches. The nanoparticles may also be fluorescently labeled with near IR, far red or other dyes in order to be used for in vivo fluorescent imaging, or determination of uptake following biopsy of blood/cells/tissue(s)/organ(s). Gadolinium and other MRI/PET/CT contrast agents may also be tethered to ligands to establish baseline human biodistributions of ligand-targeting approaches. A library of “diagnostically-responsive” nanoparticles may be administered to the patient following a diagnosis, and a secondary biopsy or in vivo imaging technique (as detailed above) may be used to determine which variants achieved the desired uptake/expression in a given cell population or distribution to a given tissue/organ population. Subsequently, therapeutic modalities may be administered utilizing theranostically-identified ligand variants.
Other Uses
Generating Drug-Peptide Conjugates
In all experiments, the following instrumentation was used:
Genomics: Sanger sequencing was outsourced to GENEWIZ following PCR amplification of target genetic loci, and uploaded to Synthego's ICE analysis tool in parallel to internal computational data evaluation
Flow Cytometer: Attune NxT with Flow Cytometer
Procedures were performed within a sterile, dust free environment (BSL-II hood). Gastight syringes were sterilized with 70% ethanol before rinsing 3 times with filtered nuclease free water, and were stored at 4° C. before use. Surfaces were treated with RNAse inhibitor prior to use.
A first solution (an anionic solution) was prepared by combining the appropriate amount of payload (in this case plasmid DNA (EGFP-N1 plasmid) with an aqueous mixture (an ‘anionic polymer composition’) of poly(D-glutamic Acid) and poly(L-glutamic acid). This solution was diluted to the proper volume with 10 mM Tris-HCl at pH 8.5. A second solution (a cationic solution), which was a combination of a ‘cationic polymer composition’ and a ‘cationic polypeptide composition’, was prepared by diluting a concentrated solution containing the appropriate amount of condensing agents to the proper volume with 60 mM HEPES at pH 5.5. In this case, the ‘cationic polymer composition’ was poly(L-arginine) and the ‘cationic polypeptide composition’ was 16 μg of H3K4(me3) (tail of histone H3, tri methylated on K4).
Precipitation of nanoparticle cores in batches less than 200 μl can be carried out by dropwise addition of the condensing solution to the payload solution in glass vials or low protein binding centrifuge tubes followed by incubation for 30 minutes at 4° C. For batches greater than 200 μl, the two solutions can be combined in a microfluidic format (e.g., using a standard mixing chip (e.g. Dolomite Micromixer) or a hydrodynamic flow focusing chip). Optimal input flowrates can be determined such that the resulting suspension of nanoparticle cores is monodispersed, exhibiting a mean particle size below 100 nm. In many embodiments, a robotic fluid handling approach is utilized to perform sequential addition of peptides to payloads as detailed elsewhere.
In one case, the two equal volume solutions from above (one of cationic condensing agents and one of anionic condensing agents) were prepared for mixing. For the solution of cationic condensing agents, polymer/peptide solutions were added to one protein low bind tube (eppendorf) and were then diluted with 60 mM HEPES (pH 5.5) to a total volume of 100 μl (as noted above). This solution was kept at room temperature while preparing the anionic solution. For the solution of anionic condensing agents, the anionic solutions were chilled on ice with minimal light exposure. 10 μg of nucleic acid in aqueous solution (roughly 1 μg/μl) and 7 μg of aqueous poly (D-Glutamic Acid) [0.1%] were diluted with 10 mM Tris-HCl (pH 8.5) to a total volume of 100 μl (as noted above).
Each of the two solutions was filtered using a 0.2 micron syringe filter and transferred to its own Hamilton 1 ml Gastight Syringe (Glass, (insert product number). Each syringe was placed on a Harvard Pump 11 Elite Dual Syringe Pump. The syringes were connected to appropriate inlets of a Dolomite Micro Mixer chip using tubing, and the syringe pump was run at 120 μl/min for a 100 μl total volume. The resulting solution included the core composition (which now included nucleic acid payload, anionic components, and cationic components).
To coat the core with a sheddable layer, the resulting suspension of nanoparticle cores was then combined with a dilute solution of sodium silicate in 10 mM Tris HCl (pH8.5, 10-500 mM) or calcium chloride in 10 mM PBS (pH 8.5, 10-500 mM), and allowed to incubate for 1-2 hours at room temperature. In this case, the core composition was added to a diluted sodium silicate solution to coat the core with an acid labile coating of polymeric silica (an example of a sheddable layer). To do so, 10 μl of stock Sodium Silicate (Sigma) was first dissolved in 1.99 ml of Tris buffer (10 mM Tris pH=8.5, 1:200 dilution) and was mixed thoroughly. The Silicate solution was filtered using a sterile 0.1 micron syringe filter, and was transferred to a sterile Hamilton Gastight syringe, which was mounted on a syringe pump. The core composition from above was also transferred to a sterile Hamilton Gastight syringe, which was also mounted on the syringe pump. The syringes were connected to the appropriate inlets of a Dolomite Micro Mixer chip using PTFE tubing, and the syringe pump was run at 120 μl/min. In other embodiments, poly(glutamic acid) (0.1% and 0.15% w/v) in either pH 5.5 HEPES or pH 7.4 Tris was utilized following the initial core formation in place of silica.
Stabilized (coated) cores can be purified using standard centrifugal filtration devices (100 kDa Amicon Ultra, Millipore) or dialysis in 30 mM HEPES (pH 7.4) using a high molecular weight cutoff membrane. In many cases, no purification is necessary following electrostatic assembly. In the case of silica-coated particles, the stabilized (coated) cores were purified using a centrifugal filtration device. The collected coated nanoparticles (nanoparticle solution) were washed with dilute PBS (1:800) or HEPES and filtered again (the solution can be resuspended in 500 μl sterile dispersion buffer or nuclease free water for storage). Effective silica coating was demonstrated. The stabilized cores had a size of 110.6 nm and zeta potential of −42.1 mV (95%).
Addition of a surface coat (also referred to as an outer shell), sometimes referred to as “surface functionalization,” was accomplished by electrostatically grafting ligand species (in this case Rabies Virus Glycoprotein fused to a 9-Arg peptide sequence as a cationic anchoring domain—‘RVG9R’) to the negatively charged surface of the stabilized (in this case silica coated) nanoparticles. Beginning with silica coated nanoparticles that were filtered and resuspended in dispersion buffer or water, the final volume of each nanoparticle dispersion was determined, as was the desired amount of polymer or peptide to add such that the final concentration of protonated amine group was at least 75 uM. The desired surface constituents were added and the solution was sonicated for 20-30 seconds prior to incubate for 1 hour. Centrifugal filtration was performed at 300 kDa (the final product can be purified using standard centrifugal filtration devices, e.g., 300-500 kDa from Amicon Ultra Millipore, or dialysis, e.g., in 30 mM HEPES (pH 7.4) using a high molecular weight cutoff membrane), and the final resuspension was in either cell culture media or dispersion buffer. In some cases, optimal outer shell addition yields a monodispersed suspension of particles with a mean particle size between 50 and 150 nm and a zeta potential between 0 and −10 mV. In this case, the nanoparticles with an outer shell had a size of 115.8 nm and a Zeta potential of −3.1 mV (100%).
Nanoparticles were synthesized at room temperature, 37C or a differential of 37C and room temperature between cationic and anionic components. Solutions were prepared in aqueous buffers utilizing natural electrostatic interactions during mixing of cationic and anionic components. At the start, anionic components were dissolved in Tris buffer (30 mM-60 mM; pH=7.4-9) or HEPES buffer (30 mM, pH=5.5) while cationic components were dissolved in HEPES buffer (30 mM-60 mM, pH=5-6.5).
Specifically, payloads (e.g., genetic material (RNA or DNA), genetic material-protein-nuclear localization signal polypeptide complex (ribonucleoprotein), or polypeptide) were reconstituted in a basic, neutral or acidic buffer. For analytical purposes, the in some experiments the payload was manufactured to be covalently tagged with or genetically encode a fluorophore. With pDNA payloads, a Cy5-tagged peptide nucleic acid (PNA) specific to AGAGAG tandem repeats was used to fluorescently tag fluorescent reporter vectors and fluorescent reporter-therapeutic gene vectors. A timed-release component that may also serve as a negatively charged condensing species (e.g. poly(glutamic acid)) was also reconstituted in a basic, neutral or acidic buffer. Targeting ligands with a wild-type derived or wild-type mutated targeting peptide conjugated to a linker-anchor sequence were reconstituted in acidic buffer. In the case where additional condensing species or nuclear localization signal peptides were included in the nanoparticle, these were also reconstituted in buffer as 0.03% w/v working solutions for cationic species, and 0.015% w/v for anionic species. Experiments were also conducted with 0.1% w/v working solutions for cationic species and 0.1% w/v for anionic species. All polypeptides, except those complexing with genetic material, were sonicated for ten minutes to improve solubilization.
Rationale: In the previous experiments (
RNP=Cas9+LL224 (TRAC) guide
2 NP Prep Plates: single-layer and multi-layer
Overnight (˜12 h) transfection
Transfection in serum free media
blow Day 1 (uptake)—all
HEK293 Genomics Day 3/4 (TRAC editing)—grew out to Day 7 for genomics
Order of addition:
Order 1—RNP>[H2A>PLE/PDE layer]>EED>LIGAND
Order 2—RNP>[H2A>PLE/PDE layer]>LIGAND>EED
Order 3—RNP>[H2A>PLE/PDE layer]>LIGAND/EED
Dose of EE peptide: (0, 0.15, 0.3) molar ratio
3 orders of addition
3 EE Concentration (0, 0.15 0.3 mole fraction), all using AF594 tagged EE peptide+Stock EE AF594 is at 0.1%
2 nucleases
3 orders of addition
3 EE Doses (0, 0.15, 0.3 mole fraction)
5 ligands—CD8-Peg-9R, CD8-9R, PLR10, PLK10-PEG22, CD4-9R
1 transfection time (overnight)
One Buffer (HEPES pH 5.5)—this buffer produced slightly better ICE scores in the 3B.1.1.1 HEK-GFP cells See
Enhancing the Cutting Efficiency of Cas9 Protein through Systematic Nanoparticle Formulation: Data Driven Example
For many of the embodiments shown herein, the effect that different buffers and pH levels have on Cas9 aggregation was evaluated prior to formation of subsequent nanoparticles (
D. RNP+DNA>CPP>PLE>PLR10 (control group)
F. DNA+PLE mix>CPP+RNP mix>PLR10
G. CPP+RNP mix>DNA+PLE mix>PLR10
In the following flow cytometry data, an Attune NxT flow cytometer was used to determine cellular uptake of EGFP-Cas9 RNPs formed with a variety of heteromultivalent ligand coats transfected in human primary T cells with flow cytometry performed at 24 h. These studies were performed prior to subsequent core and ligand density optimization studies where cellular transfection efficiencies of Cas9 RNP-bearing nanoparticles exceeds 90% in CD4+ T cells. In these initial experiments, in human primary T cells as well as AF594 AND GFP+ cells following formulator app generated robotic code (
Aspects, including embodiments, of the present subject matter described above may be beneficial alone or in combination, with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting aspects of the disclosure are provided below. As will be apparent to those of ordinary skill in the art upon reading this disclosure, each of the individually numbered aspects may be used or combined with any of the preceding or following individually numbered aspects. This is intended to provide support for all such combinations of aspects and is not limited to combinations of aspects explicitly provided below. It will be apparent to one of ordinary skill in the art that various changes and modifications can be made without departing from the spirit or scope of the invention.
1. A method of generating a targeting ligand that can be used to target cells, tissues, or organs of interest, the method comprising:
This application is a continuation of International Patent Application No. PCT/US20/31188, filed May 1, 2020, which claims the benefit of U.S. Provisional Patent Application No. 62/842,400, filed May 2, 2019, both of which are incorporated herein by reference in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| 62842400 | May 2019 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US20/31188 | May 2020 | US |
| Child | 17453336 | US |
