U.S. patent application number 16/252354 was filed with the patent office on 2019-05-23 for nanoparticles comprising protein-polynucleotide complexes and for delivering protein based complexes.
The applicant listed for this patent is RoverMed BioSciences, LLC. Invention is credited to Vicci Korman, Gretchen M. Unger.
Application Number | 20190151470 16/252354 |
Document ID | / |
Family ID | 67301195 |
Filed Date | 2019-05-23 |
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United States Patent
Application |
20190151470 |
Kind Code |
A1 |
Unger; Gretchen M. ; et
al. |
May 23, 2019 |
NANOPARTICLES COMPRISING PROTEIN-POLYNUCLEOTIDE COMPLEXES AND FOR
DELIVERING PROTEIN BASED COMPLEXES
Abstract
This invention provides nanoparticles containing
protein-polynucleotide complexes and methods of manufacture and
methods of their use. These particles, when administered to a
subject in need, are capable of delivering these complexes to
target cells and target intracellular locations where they can
perform a therapeutic function. In some embodiments, this
therapeutic function includes gene editing, induction of gene
skipping, and regulation of gene expression. The instant
nanoparticles are generally formed by designing and synthesizing
the polynucleotide to according to its intended function, combining
it with a protein selected for its substrate specificity and
enzymatic function in a manner to form a polynucleotide-protein
complex, encapsulating the complexes by dispersion into a
water-insoluble surfactant system, optionally adding a targeting
ligand, and stabilizing the nanoparticles by crystallization of the
ligand to the surface of the nanoparticles.
Inventors: |
Unger; Gretchen M.; (Chaska,
ME) ; Korman; Vicci; (Minneapolis, MN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
RoverMed BioSciences, LLC |
St. Cloud |
MN |
US |
|
|
Family ID: |
67301195 |
Appl. No.: |
16/252354 |
Filed: |
January 18, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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62619885 |
Jan 21, 2018 |
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62264100 |
Dec 7, 2015 |
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62619882 |
Jan 21, 2018 |
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62619883 |
Jan 21, 2018 |
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62619881 |
Jan 21, 2018 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 15/90 20130101;
A61K 9/51 20130101; A61K 47/6907 20170801; C12N 2310/14 20130101;
C12N 15/111 20130101; A61P 11/00 20180101; A61K 48/0091 20130101;
A61K 9/5169 20130101; A61K 48/0008 20130101; A61K 48/00 20130101;
A61K 9/5146 20130101; A61K 48/0066 20130101; C12N 15/11 20130101;
C12N 2320/32 20130101; A61K 48/0058 20130101; C12N 9/22 20130101;
A61P 1/16 20180101; C12N 2310/20 20170501; A61K 47/62 20170801 |
International
Class: |
A61K 48/00 20060101
A61K048/00; A61K 9/51 20060101 A61K009/51; A61P 11/00 20060101
A61P011/00; A61P 1/16 20060101 A61P001/16; A61K 47/69 20060101
A61K047/69 |
Claims
1. A composition comprising nanoparticles comprising a
polynucleotide component, a protein component, a surfactant having
an HLB value of less than 6.0 units, optionally a hydrophilic
polymer, and optionally Li.sup.+ and Cs.sup.+, wherein: a) the
protein component and the polynucleotide component are capable of
functioning together in a biologic system as a biologic agent; b)
the protein component and the polynucleotide component form a
complex; c) the complex and the surfactant form a surfactant
micelle core; d) the optionally hydrophilic polymer, if present,
forms a shell around the micelle core; e) the nanoparticles have an
average diameter of less than about 50 nanometers, and f) the
biologic agent is optionally a therapeutic agent.
2. The composition of claim 1 wherein the biologic agent is a RISC
or a sgRNA-Cas complex.
3. The composition of claim 1 wherein the biologic agent is a PNA,
or an SGN.
4. The composition of claim 2 wherein the protein component is a
RISC associated protein and the polynucleotide component is a guide
strand and wherein the guide strand is a member selected from the
group consisting of ssRNA, siRNA, and miRNA.
5. The composition of claim 4 wherein the RISC-associated protein
is an argonaute protein.
6. The composition of claim 5 wherein the argonuate protein is
AGO-2.
7. The composition of claim 1 wherein the protein component is a
CRISPR associated protein and the polynucleotide component is
sgRNA.
8. The composition of claim 7 wherein the CRISPR associated protein
is Cas-9.
9. The composition of claim 7 wherein the CRISPR associated protein
is dCas-
10. The composition of claim 1, further comprising a donor template
DNA.
11. A method of treating a subject in need comprising administering
the composition of claim 1, wherein the subject has a genetic
mutation and wherein the polynucleotide is configured to correct
the genetic disease.
12. A method of treating a subject in need comprising administering
the composition of claim 10, wherein the subject has a genetic
mutation and wherein the polynucleotide and the donor template DNA
of the composition are configured to correct the genetic
disease.
13. A method of treating a subject in need comprising administering
a composition of claim 1, wherein the polynucleotide is configured
to inhibiting synthesis of a gene wherein the subject is affected
by an autosomal dominant heritable disease resulting from a
mutation in the gene.
14. The composition of claim 1, wherein the polynucleotide
component is configured to target the biologic agent to a gene that
is expressed in muscle, lung, or liver.
15. A composition comprising nanoparticles comprising a first
protein component, a second protein component different from the
first, a surfactant having an HLB value of less than 6.0 units,
optionally a hydrophilic polymer, and optionally Li+ and Cs+,
wherein: a) the first protein component and the second protein
component form a complex; b) the complex and the surfactant form a
surfactant micelle core; c) the optionally hydrophilic polymer, if
present, forms a shell around the micelle core; d) the
nanoparticles have an average diameter of less than about 50
nanometers, and e) the biologic agent is optionally a therapeutic
agent.
16. A method of treating a subject in need comprising administering
the composition of claim 12, wherein the polynucleotide and the
donor template DNA of the composition are configured to correct the
genetic disease.
17. A method of treating a subject in need comprising administering
a composition of claim 1, wherein the polynucleotide is configured
to inhibiting synthesis of a gene wherein the subject is affected
by an autosomal dominant heritable disease resulting from a
mutation in the gene.
18. The method of claim 12, wherein the genetic mutation is one
affecting one or more of lung function, liver function, and muscle
function.
19. A method of making a nanoparticle comprising: a) designing a
polynucleotide component and a protein component, wherein the
polynucleotide component targets a gene of interest and comprises
structural features necessary to interact functionally with the
protein component and wherein the polynucleotide component and the
protein component make a bioactive agent; b) combining the
polynucleotide component and the protein component in solution; c)
providing sufficient time for the protein component and the
polynucleotide component to form a complex; d) encapsulating the
complex by dispersion into a biocompatible, water-miscible solvent
to form inverted micelles or reverse micelles; e) optionally
coating the micelles with a ligand by mixing the ligand with the
micelles in an aqueous buffered-solution; and f) optionally,
stabilizing the ligand-coated micelles by mixing metal ions into
the aqueous buffered-solution, wherein the metal ions comprise
lithium pretreated with Cesium.
Description
I. CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims priority to and the benefit of U.S.
Provisional Application No. 62/619,881, filed on 21 Jan. 2018, U.S.
Provisional Application No. 62/619,882, filed on 21 Jan. 2018, U.S.
Provisional Application No. 62/619,883, filed on 21 Jan. 2018, U.S.
Provisional Application No. 62/619,885, filed on 21 Jan. 2018 and
U.S. Provisional Application No. 62/264,100, filed 30 Jan. 2018,
each of which are hereby incorporated by reference in their
entireties as if fully set forth herein.
II. FIELD OF THE INVENTION
[0002] The instant invention relates generally to the field of
therapy using nanoparticles to deliver biologic agents. More
specifically, the present invention relates to nanoparticles,
methods of their manufacture, and methods of use for delivering
biologic agents.
III. Background
[0003] Genome editing technologies have shown much potential in
their ability to change the genetic code of cells. These
technologies could enable novel insights in drug discovery and lead
to the development of next generation gene therapies.
[0004] One such genome editing technology is the so-called "CRISPR
technology, in which a targeting RNA (sgRNA) complexes with one or
more CRISPR associated proteins such as Cas9 and directs the
complex to the target where the nuclease activity of the Cas
protein cuts the DNA.
[0005] RNA interference (RNAi) is also a powerful tool to silence
or reduce the expression of a target gene and is mediated by small
single- or double-stranded RNA molecules. These molecules, such as
siRNAs, miRNAs and shRNAs, are important intermediaries in the RNAi
pathway that lead to degradation of specific mRNAs through the
RNA-induced silencing complex (RISC). During assembly of RISC, a
single strand of the RNA molecules binds to the protein Argonaute 2
(Ago2), a key component of RISC. This strand then guides RISC to
its complementary target mRNA, which is finally cleaved by the
RNase activity located in the Ago2 protein, triggering its
destruction.
[0006] Other gene editing complexes such as structure-guided
endonuclease gene editing technology and triplex-forming peptide
nucleic acid oligomers are being developed with promising
therapeutic utility.
[0007] However, the utility of these methods for human therapy is
fraught with challenges. There is a need for developing or
improving methods of delivering sufficient levels of these tools to
the proper target cells and to the proper intracellular
localization. While ongoing efforts seem to focus on delivering the
polynucleotide component(s), assembly in situ with the endogenous
protein components is often limiting. Thus there remains a need at
several levels for developing these tools for effecting gene
therapy.
[0008] Furthermore, despite significant research over a period of
more than 30 years, the development of proteins as therapeutic
agents has been hindered by problems in effective and efficient
delivery. Proteins induce inflammatory reactions in the blood and
are subject to significant enzymatic degradation both in vitro and
in vivo. Solutions to protect protein therapies are often thwarted
by the fragile nature of the protein and the challenges of
stabilizing while also preserving functionality.
[0009] Encapsulation of proteins is one of numerous protective
strategies to improve delivery. However, protein encapsulation
yields are often low, necessitating efforts to chemically stabilize
the protein but which can also significantly denature the protein.
Further, protein encapsulation approaches often must be evaluated
on an empirical, case-by-case basis, as the protective effects of
solutes are variable.
[0010] There is also therefore, a need for improved delivery of
protein therapeutics with high encapsulation yields and low
toxicity levels, in a modular system.
IV. SUMMARY OF THE INVENTION
[0011] Provided here are nanoparticles, methods of their
manufacture, and methods of use for delivering biologic agent. In
one embodiment, the instant invention is a composition of
nanoparticles comprising a polynucleotide component, a protein
component, a surfactant having an HLB value of less than 6.0 units,
optionally a hydrophilic polymer, optionally a ligand, and
optionally Li+ and Cs+, wherein: [0012] i) the protein component
and the polynucleotide component function as a complex in concert
as a biologic agent; [0013] ii) the protein component and the
polynucleotide component form a complex; [0014] iii) the complex
and the surfactant form a surfactant micelle core; [0015] iv) the
optional hydrophilic polymer forms a shell around the micelle core;
[0016] v) the nanoparticles have an average diameter of less than
about 50 nanometers; and [0017] vi) the biologic agent is
optionally a therapeutic agent.
[0018] In another embodiment, the instant invention comprises a
method of making a nanoparticle of the present invention.
[0019] In another embodiment, the instant invention comprises a
method of using the instant invention for example to treat a
subject in need comprising administering a nanoparticle of the
present invention to the subject where the protein component and
polynucleotide component are designed to alter gene expression of a
disease-relevant gene.
V. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] The foregoing and other objects, advantages and features of
the present invention, and the manner in which the same are
accomplished, will become more readily apparent upon consideration
of the following detailed description of the invention taken in
conjunction with the accompanying figures. and Examples, which
illustrate embodiments, wherein:
[0021] FIG. 1 is a TEM micrograph for Formula Ha;
[0022] FIG. 2 is Silver-stained gel of supernatants from reaction
mixtures;
[0023] FIG. 3 shows immunomicroscopy from RISC nanoparticles in 3D
hepatocyte cultures;
[0024] FIG. 4 shows immunohistochemistry of mouse liver;
[0025] FIG. 5 shows immunohistochemistry in 3D hepatocyte
cultures;
[0026] FIG. 6 shows FT-IR Scan for MilliQ Water;
[0027] FIG. 7 shows FT-IR Scan for Hepes 10% Lactitol hydrated;
[0028] FIG. 8 shows FT-IR Scan for Hepes 10% Lactitol partially
dehydrated;
[0029] FIG. 9 shows FT-IR Scan (Li.sup.+ Cs) Cl;
[0030] FIG. 10 shows FT-IR Scan for ASOR hydrated;
[0031] FIG. 11 shows FT-IR Scan for DMSO;
[0032] FIG. 11.5 shows TM-Diol Surfactant in 50% DMSO;
[0033] FIG. 12 shows FT-IR Scan for Ammonium Acetate Dried;
[0034] FIG. 13 shows FT-IR Scan for ASOR Sugar Nanocapsule
hydrated;
[0035] FIG. 13-2 shows FT-IR Scan for ASOR Sugar Dried Only 2nd
Run;
[0036] FIG. 14 shows FT-IR Scan for ASOR Sugar Nanocapsule
dehydrated;
[0037] FIG. 15 shows FT-IR Scan for ASOR Sugar Micelle
dehydrated;
[0038] FIG. 16 shows FT-IR Scan for ASOR RISC RNAi F7 Nanocapsule
dehydrated;
[0039] FIG. 17 shows FT-IR Scan for ASOR RISC RNAi F7 Micelle;
[0040] FIG. 18 shows FT-IR Scan for ASOR RISC 2RF7 Micelle;
[0041] FIG. 19 shows FT-IR Scan for ASOR RNAi F7 Nanocapsule
dehydrated;
[0042] FIG. 20 FT-IR Scan for ASOR RNAi F7 Micelle dehydrated;
[0043] FIG. 21 shows FT-IR Scan for ASOR Cas9 F7 Nanocapsule
hydrated;
[0044] FIG. 22 shows FT-IR Scan for ASOR Cas9 F7 Nanocapsule
dehydrated;
[0045] FIG. 23 shows FT-IR Scan for ASOR Cas9 Micelle
dehydrated;
[0046] FIG. 24 shows FT-IR Scan for ASOR dCas9 F7 Nanocapsule
dehydrated;
[0047] FIG. 25 shows FT-IR Scan for ASOR dCas9 Micelle
dehydrated;
[0048] FIG. 26 shows FT-IR Scan of TBG Erythitol Nanocapsule
hydrated;
[0049] FIG. 27 shows FT-IR Scan Tbg Erythritol Nanocapsule
dehydrated;
[0050] FIG. 28 shows "short-release" (in vivo) CUSP-RNP particle
DLS and TEM;
[0051] FIG. 29 shows CUSP RNP pilot study in mice;
[0052] FIG. 30 shows ASOR-coated CUSP-RNP for targeted liver
therapy;
[0053] FIG. 31 shows CUSP-mediated RNP delivery into target cells
via non-endosomal lipid raft path;
[0054] FIG. 32 shows cytokine levels of mice treated with CUSP-RNP
at highest doses: fast+slow release particles;
[0055] FIG. 33 shows transcript changes observed via qPCR for Group
10, but differed based on probe set design;
[0056] FIG. 34 shows CUSP RNP knocks down FVII protein in RNP pilot
study in vivo;
[0057] FIG. 35A shows isotopic bio distribution study in mice,
utilizing CUSP-encapsulated Dy-Dextran;
[0058] FIG. 35B shows co-localization of spCas9 protein cargo
(blue) and immuno-labeled CUSP shell;
[0059] FIG. 36 shows CUSP delivers RNP to nucleus;
[0060] FIG. 37 shows CUSP-RNP induces FVII transcript decrease as
measured by in situ hybridization;
[0061] FIG. 38 shows CUSP dCas9 RNP shows FVII protein inhibition
similar to CUSP Cas9 RNP in vitro; and
[0062] FIG. 39 shows neither dCas9 or Cas9 species shows mutational
activity by amplicon deep sequencing;
VI. DETAILED DESCRIPTION OF THE INVENTION
[0063] By way of illustrating and providing a more complete
appreciation of the present invention and many of the attendant
advantages thereof, the following detailed description and examples
are given concerning the nanoparticles, nanoparticle compounds,
compositions, methods of manufacture and methods to deliver
biological or other agents of the present invention
[0064] As used here, the following definitions and abbreviations
apply:
[0065] ASOR means asialoorosomucoid.
[0066] AGO means Argonaute and refers to, by way of non-limiting
examples, the mammalian Argonaute protein family currently known in
the art to consist of eight members, four of which are ubiquitously
expressed (Ago subfamily), with the remaining four (Piwi subfamily)
being expressed in germ cells. Similarly, Ago2 is useful in the
instant invention in gene silencing independent of such cleavage
activity, such as in translational repression. Argonaute can be an
Aquifex aeolicus, a Microsystis aeruginosa, a Clostridium
bartlettii, an Exiguobacterium, an Anoxybacillus flavithermus, a
Halogeometricum borinquense, a Halorubrum lacusprofundi, an
Aromatoleum aromaticum, a Thermus thermophilus, a Synechococcus, a
Synechococcus elongatus, or a Thermosynechococcus elogatus
Argonaute. Argonaute can be mammalian Argonaute, such as mouse
AGO2. Argonaute can refer to the wild-type or a modified form of
the Argonaute protein that can comprise an amino acid change such
as a deletion, insertion, substitution, variant, mutation, fusion,
chimera, or any combination thereof. The proteins referred to
herein may also be identified by their NCBI accession numbers; Ago
1, NP-036331; Ago2, NP-036286, Ago3, NP-079128, and Ago4,
NP-060099.
[0067] Biologic agents of the instant invention are, by way of
non-limiting example, gene editing agents, agents that affect or
modulate transcription or translation, guided endonuclease
machinery, or agents that cause other genetic or biochemical
changes in a biologic system.
[0068] Cas protein, or Cas, refers to CRISPR-associated proteins
and by non-limiting examples, Cas9 proteins, Cas9-like proteins
encoded by Cas9 orthologs, Cas9-like synthetic proteins, Cpf1
proteins, proteins encoded by Cpf1 orthologs, Cpf1-like synthetic
proteins, C2c1 proteins, C2c2 proteins, C2c3 proteins, and variants
and modifications thereof. In a preferred embodiment, a Cas protein
is a Class 2 CRISPR-associated protein, for example a Class 2 Type
II CRISPR-associated protein or a Class 2 Type V CRISPR-associated
protein. Each CRISPR-Cas protein interacts with one or more cognate
polynucleotide (typically RNA) to form a nucleoprotein complex
(typically a ribonucleoprotein complex).
[0069] Cas9 protein (or Cas9) as used herein refers to a Cas9
wild-type protein derived from Type II CRISPR-Cas9 systems,
modifications of Cas9 proteins, variants of Cas9 proteins, Cas9
orthologs, and combinations thereof. The term "dCas9" as used
herein refers to variants of Cas9 protein that are
nuclease-deactivated Cas9 proteins, also termed "catalytically
inactive Cas9 protein," or "enzymatically inactive Cas9."
[0070] Chimeric refers a molecule that is composed of both RNA and
DNA moieties that are naturally occurring or nucleotide analogs,
linked by phosphodiester, phosphorothioate, and/or any other
naturally occurring or synthetic linkage that permits the
nucleotides or analogs to retain their intended function.
[0071] Cognate, as used here, typically refers to a Cas protein and
one or more Cas polynucleotides that are able of forming a
nucleoprotein complex capable of site-directed binding to a target
nucleic acid complementary to the target nucleic acid binding
sequence present in one of the Cas polynucleotides.
[0072] Cpf1 protein refers to a Cpf1 wild-type protein derived from
Type V CRISPR-Cpf1 systems, modifications of Cpf1 proteins,
variants of Cpf1 proteins, Cpf1 orthologs, and combinations
thereof. The term "dCpf1" as used herein refers to variants of Cpf1
protein that are nuclease-deactivated Cpf1 proteins, also termed
"catalytically inactive Cpf1 protein", or "enzymatically inactive
Cpf1".
[0073] CRISPR means clustered regularly interspaced short
palindromic repeats.
[0074] dCas9 means endonuclease dead Cas9, also known as dead Cas9
and is a mutant form of Cas9 whose endonuclease activity is removed
through point mutations in its endonuclease domains.
[0075] DLS means Dynamic Light Scattering and, in connection with
data shown herein, means a Nicomp ZLS 380 instrument.
[0076] Donor template DNA, in reference to CRISPR technology or
RISC technology or, means DNA that is configured to provide a
template for DNA synthesis in regions that have been excised by
CRISPR technology or RISC technology.
[0077] DTT means dithiothreitol.
[0078] Flag means FLAG-tag comprising the amino acid residues
DYKDDDDK.
[0079] fpm means feet per minute.
[0080] Gene editing, as used herein, refers to the insertion,
deletion or replacement of nucleic acids in genomic DNA so as to
add, disrupt or modify the function of the product that is encoded
by a gene.
[0081] "Guide strand" refers to the single-stranded polynucleotide
that comprises at least 12 nucleotides that binds to an Argonaute
polypeptide or a related RISC complex and is capable of directing
NP complex to a target polynucleotide. The guide molecule can be a
DNA or an RNA or a chimeric molecule. The skilled artisan, with the
teaching herein, is readily able to design the guide strand,
typically optimizes for uniform hybridization energies across
sequences at sites of low target mRNA secondary structure while
siRNA design is more focused on optimizing a hybridization profile
across the sequence within the context of sequence "rules". Design
algorithms such as Soligo for antisense and SiRNA for siRNA are
publicly available.
[0082] In concert (as a biologic agent) means performing a biologic
function together as part of a complex or physical association or
interaction such as that which occurs in substrate-enzyme binding
or other physicochemical interactions.
[0083] Instant means, by way of non-limiting examples, when used as
is "instant nanoparticles" or "instant ligands" means the
nanoparticles or ligands of the present invention.
[0084] NP means nucleoprotein complex which is a
polynucleotide-protein complex. By non-limiting examples of such
complexes are Cas-sgRNA, RISC, PNA, and SGN complexes. Typically,
the polynucleotide portion is an RNA species, although DNA-protein
complexes and chimeric nucleic acid polymers are also contemplated
here.
[0085] PNA means Triplex-forming peptide nucleic acid oligomers.
PNAs refer to complexes in which, by way of non-limiting example,
the phosphate backbone of polynucleotides is replaced in its
entirety by repeating N-(2-aminoethyl)-glycine units and
phosphodiester bonds are replaced by peptide bonds. The various
heterocyclic bases are linked to the backbone by methylene carbonyl
bonds. PNAs maintain spacing of heterocyclic bases that are similar
to polynucleotides, but are achiral and neutrally charged
molecules. Peptide nucleic acids are comprised of peptide nucleic
acid monomers. PNA complexes have a charge-neutral peptide-like
backbone and nucleobases enabling hybridization with DNA and RNA
with high affinity. PNA/DNA/PNA triplexes recruit the cell's
endogenous DNA repair systems to initiate site-specific
modification of the genome when single-stranded "donor template
DNAs" are co-delivered as templates containing the desired sequence
modification. This technology, described by Rogers, et al., Proc.
Natl. Acad. Sci. USA, 99:16695-16700 (2002) is also contemplated to
be useful according to the present invention as taught by U.S.
20170283830. More recently, a review describing useful aspects of
this technology has been published by Siddiquee et al. in Adv Tech
Biol Med 2015, 3:2.
[0086] Polynucleotide, as used here, means a biopolymer composed of
any number of nucleic acid monomer of deoxyribonucleic acid or
ribonucleic acid and contemplates nature and modified or synthetic
nucleic acid monomers. "Polynucleotide components", as used herein,
means polynucleotides with the additional teaching set forth
elsewhere herein.
[0087] rAgo2 means recombinant protein argonaute-2.
[0088] RISC as used herein means RNA-induced silencing complex and,
by way of non-limiting example, a guide strand-AGO protein
complex.
[0089] RISC nanoparticles means the instant nanoparticles wherein
the biologic agent is a RISC.
[0090] RNAiF7 means a single stranded RNA polynucleotide
complementary to Factor VII (i.e. a guide RNA).
[0091] RNP means ribonucleic protein.
[0092] sgRNA-Cas nanoparticles means the instant nanoparticles
wherein the biologic agent is an sgRNA-Cas nucleoprotein
complex.
[0093] siF7 means a double stranded siRNA complementary to Factor
VII coding sequence.
[0094] SGN means the structure-guided endonuclease gene editing
technology, for example, where the protein component is a FEN-1
fusion (endonuclease) and where the polynucleotide is a guide DNA.
The guide DNA can be about 20 to about 50 nucleotides. The gene
editing function of SGN is described, for example, in Xu S, Cao S,
Zou B, Yue Y, Gu C, Chen X, et al. An alternative novel tool for
DNA editing without target sequence limitation: the
structure-guided nuclease (SGN). Genome Biol. 2016 and Varshney G
K, Burgess S M. DNA-guided genome editing using structure-guided
endonucleases. Genome Biology. 2016; 17:187.
doi:10.1186/s13059-016-1055-4.
[0095] sgRNA or gRNA means a single-guide RNA with about 20
nucleotides and directs Cas9 or dCas9 or other Cas proteins to
their targets and together make up the whole or part of the CRISPR
system.
Biologic Agents and Protein and Polynucleotide Components
[0096] Biologic agents can be any Biologic agent that comprises a
protein component comprising one or more proteins and a
polynucleotide component, comprising one or more polynucleotides,
where the biologic activity of the biologic agent requires at least
the co-action of the protein component and the polynucleotide
component.
[0097] Examples of such biologic agents are RNA-induced silencing
complexes ("RISC") which is a complex which incorporates one strand
of a single-stranded RNA (ssRNA) fragment (such as microRNA
[miRNA], or double-stranded small interfering RNA [siRNA]) and one
or more specific RISC-associated proteins.
[0098] Other examples of such biologic agents are complexes between
sgRNA and one or more CRISPR associated protein ("Cas").
[0099] Other examples are Triplex-forming peptide nucleic acid
oligomers.
[0100] Other examples are guide nucleic acid and FEN-1 using the
so-called structure-guided endonuclease technology.
[0101] By way of non-limiting example, protein and polynucleotide
component pairs (capable of forming a biologic agent) of the
instant invention comprise one or more of:
[0102] an argonaute protein and a guide strand where the guide
strand is single stranded RNA (ssRNA);
[0103] an argonaute protein and a guide strand where the guide
strand is a small interfering RNA (siRNA);
[0104] an argonaute protein and a guide strand where the guide
strand is microRNA (miRNA);
[0105] a Cas9 protein and a sgRNA;
[0106] a Cas9 protein, an sgRNA, and a donor template DNA;
[0107] a dCas9 protein and an sgRNA;
[0108] a Cpf1 protein and a sgRNA;
[0109] a PNA; and/or
[0110] FEN-1 and a guide nucleic acid.
[0111] It should be further understood that the instant biologic
agents are useful as therapeutic agents.
Biologic Agent Complex
[0112] Instant biologic agents are co-encapsulated according to the
instant invention in a configuration that may or may not represent
the configuration or complex structure that occurs in their
physiologically-active state. For example, it is well understood
that in RISC complexes and Cas-sgRNA complexes, there is a specific
interaction between the protein components and the polynucleotide
components that dictate their coordinate action. Nevertheless, this
complex not only co-delivers the protein components and the
polynucleotide components, these components are delivered in
immediate, physical proximity to one another and provide a means
for efficient transition to an optimal configuration. Moreover,
when formed as taught herein, nanoparticles are made with superior
efficacy and loading. For drafting convenience, the complex formed
between the protein components and the polynucleotide components in
the instant nanoparticles is also referred to as a "biologic
agent".
[0113] The biologic agent of the instant invention may be formed by
complexing the protein components and the polynucleotide components
by methods described below and further in the Examples. However,
with the teaching herein, the skilled artisan will readily
appreciate useful modifications of these methods.
[0114] With regard to the complex formed between the polynucleotide
component and the protein component as encapsulated in the
nanoparticles, in some embodiments, this complex is of the manner
an aggregate, meaning a heterogeneous interaction involving
different interactions ranging from specific, high affinity
interaction to non-specific interactions. In other embodiments, the
complex is made of one or more specific interactions such as an
enzyme binding to a substrate or a polymerase binding to a
polynucleotide acid.
Preparation of Nanoparticles
[0115] The instant nanoparticles are produced as taught by the
methodology, examples, and principles herein and also considering
certain general methods taught by Unger in U.S. Pat. No. 6,632,671
and US20160058706 and elsewhere. Provided below are non-limiting
embodiments of the nanoparticles and their preparation.
[0116] I. Reaction Binding of the Protein Component and the
Polynucleotide Component.
[0117] The protein component is suspended in buffer (e.g. at about
1 to 70% w/w) and the polynucleotide component is added (e.g. by
weight at 25-120% of protein component on a molar basis) in
sufficient volume to maintain protein weight percent at or below
25% w/v for reaction. The polynucleotide and protein components are
allowed to interact and complex by gentle mixing (e.g. about 3-8
minutes).
[0118] The protein component and the polynucleotide component of
the biologic agent are selected by the skilled artisan according to
biochemical, biophysical, genetic, and physiologic considerations.
Typical polynucleotide lengths are 16 to 105 nt.
[0119] The protein component and polynucleotide component are
generally incubated at about 10 to 120% polynucleotide to protein
on a molar basis; e.g. at about 50%.
[0120] After complexation, the biologic agent is immediately added
to the surfactant to create a reverse micelle.
[0121] II. Encapsulation.
[0122] An aqueous solution of protein component--polynucleotide
complex is encapsulated by dispersing the biologic agent into a
biocompatible, water-miscible solvent using a biocompatible,
water-insoluble surfactant system suitable for preparation of an
inverted or reverse micelle.
[0123] Suitable surfactant systems are well-known in the
formulation arts as surface-active materials that are essentially
hydrophobic and characterized by a hydrophile-lipophile balance
(HLB) of less than about 6, a critical micelle concentration (CMC)
of less than about 200 .mu.M, or a critical packing diameter
greater than 1. In one embodiment, the HLB value is between about 3
and 8, between about 3 and 6, and between about 4 and 6. In one
embodiment, the hydrophobic is not biologically toxic as may be
determined for example in cell culture testing. Suitable
surface-active materials are non-ionic and thus not amphiphilic. In
some embodiments, the HLB is less than about 5. Hydrophobic
surfactants and hydrophobic, water-miscible solvents suitable for
preparing reverse micelles are described in Pashley & Karaman
(2004, In Applied Colloid and Surface Chemistry, John Wiley, pp.
60-85), Rosen (2004, in Surfactants and Interfacial Phenomena, John
Wiley), The Handbook of Industrial Surfactants (1993, Ash, ed.,
Gower Pub), and Perry's Chemical Engineer's Handbook (1997, Perry
& Green, 7th Ed., McGraw Hill Professional), incorporated
herein by reference.
[0124] In some embodiments, the surfactant component may be
2,4,7,9-tetramethyl-5-decyn-4,7-diol(TM-diol), blends of
2,4,7,9-tetramethyl-5-decyn-4,7-diol(TM-diol), molecules having one
or more acetylenic dial groups, cetyl alcohol, or any combination
of any of these. In some embodiments, water-miscible solvents
comprising food or USP grade oils, such as DMSO, DMF, castor oil,
or any combination thereof, may be used. In one embodiment, a
hydrophobic surfactant can be 2,4,7,9-tetramethyl-5-decyn-4,7-diol
(TM-diol) or preparations thereof, such as Surfynol SE (Air
Products), used in a concentration of up to about 15% by weight on
protein, and a water-miscible solvent can be DMSO. The
concentration of surfactant selected should be sufficient to
prepare an optically clear nanoemulsion, but not so much as to
induce aggregation, since aggregation may lead to overly large
nanoparticles.
[0125] IV. Optional Ligand Coating.
[0126] The micelles can be coated with ligand by mixing one or more
ligands with an aqueous buffer (e.g. about pH=7.4, 7-7.5) dilution
of the composition (nanoparticles) of the previous step. In some
embodiments, ligands can be mixed with nanoparticles in a ratio (by
weight) of about 1:500 to about 1:0.1 of ligand to biologic agent,
depending upon factors including the targeting moiety and the rate
at which the nanoparticle is desired to dissolve or disassemble. In
one embodiment, the weight ratio is about 1:80 (that is, about
1/80th) of ligand to biologic agent. In one embodiment, the weight
ratio is about 1:40 of targeting moiety to biologic agent.
[0127] V. Stabilization.
[0128] Optionally, ligand-coated nanoparticles are further
stabilized. To further stabilize the ligand-adsorbed nanoparticle,
the aqueous suspension of nanoparticles coated with one or more
ligands can be mixed into an aqueous solution of metal ions (i.e.,
a "stabilization solution or receiving bath") capable of
precipitating, crystallizing, or iontophoretic exchange with the
coated nanoparticles. Representative, non-limiting examples of
solutes that can be used to form coated nanoparticles include ionic
species derived from elements listed in the periodic table. Ions
may be included in the aqueous stabilization composition in a range
from about, for example, 0.1 part per trillion to about 1 M. An
adequate amount of ion should be included, such that the coated
nanoparticles are sufficiently contacted with ions, but not so much
that aggregation occurs, which may lead to overly large
nanoparticles.
[0129] In one embodiment, a stabilization (or crystallization or
receiving) solution can comprise about 10 mM Ca.sup.2+ and about
125 mM Li.sup.+. If ultrapure reagents are used in the
stabilization solution, very small amounts (e.g., less than about 1
mM) of ions such as Ba, Fe, Mg, Sr, and Bi may be added to optimize
stabilization of the coated nanoparticles. In one embodiment, when
the nanoparticles are prepared with sterile water, 126 mM of
Li.sup.+ is pre-treated with 2.5 ppb of Cs.sup.+ for increased
stability. In one embodiment, a stabilization solution includes
10.5 mM Ca.sup.2+, 125 mM Li.sup.+ (pre-mixed with 2.5 ppb
Cs.sup.+), 0.042 mM Ba.sup.2+, 4 nM Bi.sup.2+ with 7 nM Mg.sup.2+,
0.88 nM Sr.sup.2+, (all ultrapure, all prepared as stock solutions
with sterile water, except Sr.sup.2+, and Mg.sup.2+ which are
prepared with laboratory grade water, all metals are used as
chloride salts, total bath volume approximately 36 ml). Flexibility
of the system is demonstrated by for example nanoparticles showing
high levels of cellular uptake that have been synthesized at
lithium levels about 10-fold lower than those described here (data
not shown). The artisan will understand that a variety of
counter-ions can be used with these metals in the stabilization
solution, such as chloride, sulfate, and nitrate.
[0130] In one embodiment, the stabilization solution comprises
lithium pretreated with Cesium (Cs).
[0131] This stabilization is associated with changes in polymorphic
form, as evidenced by substantive differences in melting point,
thermal spectra and FTIR spectra.
[0132] VI. Storage.
[0133] The ligand-coated nanoparticle may be used immediately or
dried and reconstituted in the future.
Nanoparticle Sizings
[0134] The instant nanoparticles have an average size of less than
about 50 nm. Optionally, the D.sub.90 (that is, the minimum size
which is greater than the diameter of 90% of the particles) is
about 50 nm, or optionally about 45 nm, or optional about 40 nm, or
optionally about 35 nm, or optionally about 30 nm. In one
embodiment, the instant nanoparticles are on average between about
2 and about 50 nanometers in diameter.
Ligands
[0135] Nanoparticles of the instant invention can optionally
include a polymer shell comprising a ligand or targeting moiety for
targeting the nanoparticles to a specific biological compartment,
tissue, cell-type, or subcellular compartment.
[0136] In one embodiment, the instant nanoparticles provide a means
for targeting the nanoparticles to a given tissue or cellular
target, without the steps of chelating, conjugating, or covalently
attaching the ligand or targeting moiety to the polymer coated
nanoparticle or to the surfactant micelle.
[0137] In one embodiment the instant nanoparticles comprise a
hydrophobic surface for adsorbing ligands including hydrophilic
ligands in a manner that does not require complex chemistry
development and is not limited by the ligand-size constraints
associated with for example nanoparticles comprising ligands
conjugated to or within said nanoparticles. Accordingly, one having
skill in the art will understand that, with judicious selection of
a targeting moiety based upon the intended target and methods and
compositions known in the art, the inventive nanoparticles are
capable of delivering bioactive agents to predetermined target
tissue and cells.
[0138] Ligands, by way of non-limiting example, can be natural or
synthetic nucleic acids, proteins, peptides, small molecules, etc.,
such as asialoglycoprotein, insulin, low density lipoprotein,
growth factors, galactose, lectin, folate, and monoclonal and
polyclonal antibodies directed against cell surface molecules
etc.
[0139] The skilled artisan, with the teaching herein, will readily
recognize such ligands in the literature, sometimes referred to as
targeting factors, cell surface membrane receptor associated
targeting factors, and other terms of art. For example, U.S.
20030138432 gives many examples of useful ligands in the art and
describes methods of using such ligands as a targeting factor. U.S.
Pat. No. 7,716,030 further teaches methods of designing targeting
ligands.
[0140] By way of another example, tenfibgen can be a ligand of the
instant invention and cause the nanoparticle to target tenascin
receptors.
[0141] Other non-limiting examples include asialoorosomucoid (ASOR)
and hyaluronan.
sgRNA-Cas Nanoparticle
[0142] In one embodiment of the instant invention, the protein
component is a Cas protein and the polynucleotide component is
sgRNA. With the instant invention, it is only now possible to
modulate gene expression through the CRISPR technology in a manner
with one or more of greater precision, efficacy, higher therapeutic
levels, cellular targeting, subcellular targeting, modular and
versatile structure, and shelf stability. sgRNA-Casa nanoparticles
can further comprise template DNA. It is clear, according to the
examples contained herein and according to insight by the mind of
the inventor, that the technology is operable over a wide range of
Cas proteins (and Cas-like proteins) and over a wide range of guide
nucleic acids.
RISC Nanoparticles
[0143] In one embodiment of the instant invention, the protein
component is any RISC protein such as an argonaute protein and the
polynucleotide component is as guide RNA. With the instant
invention, it is only now possible to modulate gene expression
through the RISC technology (or RNAi technology) in a manner with
one or more of greater precision, efficacy, higher therapeutic
levels, cellular targeting, subcellular targeting, modular and
versatile structure, and shelf stability. It is clear according to
the examples contained herein and according to insight by the mind
of the inventor, that the technology is operable over a wide range
of RISC proteins and RISC like proteins and for a wide range of
guide nucleic acids.
Other Embodiments
[0144] It is specifically contemplated by the inventor that the
instant technology is useful when the protein component is any
protein component involving endonuclease activity that interacts
biologically and physically with a polynucleotide in a complex and
where the interaction is part of a biologic unit or cellular
machinery. With the disclosure here, the skilled artisan is now
able to encapsulate for example proteins and polynucleotides with
diverse physicochemical properties.
[0145] In one embodiment, the protein component is an enzymatically
active protein and the polynucleotide component is a substrate for
the enzymatically active protein component.
[0146] In another embodiment, the protein component is an
enzymatically active protein and the polynucleotide component is a
substrate for the enzymatically active protein component and the
two components are complexed in the nanoparticle by an
enzyme-substrate interaction.
[0147] In another embodiment, the polynucleotide and protein
components comprise PNA complexes and optionally further comprise
donor DNA.
[0148] In one embodiment, the protein and polynucleotide components
are not conjugated to each other.
[0149] In one embodiment, the instant nanoparticles comprise a
surfactant wherein such surfactant is not amphiphilic.
[0150] In one embodiment, the instant nanoparticles do not require
cholesterol for assembly; in other embodiments the instant
nanoparticles do not contain cholesterol.
[0151] In some embodiments, the instant nanoparticles do not
comprise polyethylene glycol (PEG) or derivatives thereof. In some
embodiments, the instant nanoparticles do not comprise
polyethylenimine (PEI) or derivatives thereof.
Uses of Instant Nanoparticles
[0152] Nanoparticles of the present invention are useful for gene
editing in vitro, ex vivo, and in vivo. Instant nanoparticles are
useful for gene editing in subjects, e.g. any plant or animal
recipient of the administered nanoparticles. Other non-limiting
examples are mammals such as humans, non-human primates, vertebrate
animals, rodents, and the like. Non-limiting examples of ex vivo
systems useful in combination with instant nanoparticles are those
where experimentation is done in or on tissue from an organism in
an external environment.
[0153] In some embodiments, the instant nanoparticles are useful
for providing therapies for diverse genetic diseases.
[0154] Certain embodiments are useful for probing gene function in
vivo (e.g. in animal models).
[0155] Certain embodiments are useful for identifying molecular
targets in animal models of disease. The skilled artisan will
readily envision other utilities.
[0156] Significantly, 80-90% of protein mutations responsible for
human disease arise from the substitution, deletion, or insertion
of single nucleotides. Sg-Cas nanoparticles, when further
containing an appropriate donor template DNA, are useful for
correcting such genetic defects.
[0157] Sg-Cas nanoparticles or RISC nanoparticles are useful to
induce gene silencing at the transcriptional level. Sg-Cas
nanoparticles are useful to induce gene silencing at the
translational level. Accordingly, such nanoparticles of the instant
invention are useful for preventing synthesis of defective gene
products; e.g. to treat autosomal dominant diseases.
[0158] Sg-Cas nanoparticles according to the instant invention are
useful to induce exon skipping to treat, for example, Duchene
muscular dystrophy.
[0159] In certain embodiments, instant nanoparticles may be
topically administered to intact, unbroken skin.
[0160] In other embodiments, nanoparticles may be topically
administered without the use of physical methods and/or external
enzymatic penetration enhancers and/or external chemical
penetration enhancers, thus potentially reducing or avoiding cell
damage, treatment pain, and other adverse reactions at the site of
treatment.
[0161] In one embodiment, the invention provides methods of
treating a subject having a disease of the liver. Such methods
generally include the steps of administering a composition of
liver-specific nanoparticles to a subject having a disease of the
liver. It is a feature of one embodiment of the invention that the
instant nanoparticles comprising a ligand shell can be targeted to
and bind to liver cells and the binding of the nanoparticles to the
liver cells results in the delivery of the pharmaceutical agent to
the liver cells. Representative diseases of the liver include,
without limitation, alpha-1-antitrypsin deficiency, Wilson's
disease, familial hypercholesterolemia, ornithine transcarbamylase
deficiency, phenylketonuria, peroxisome diseases, and familial
amyloidosis.
[0162] In one embodiment, the invention provides for methods of
mediating site-directed repair of a genomic mutation in liver cells
of a subject.
[0163] In one embodiment, the compositions of the present invention
may be administered by a number of routes, depending upon whether
local or systemic treatment is desired and upon the area to be
treated. In one embodiment, administration may be topical
(including ophthalmic, vaginal, rectal, intranasal, transdermal),
oral or parenteral. In one embodiment, topical treatment comprises
treating a subject diagnosed with Pachyonychia Congenita disease.
Parenteral administration includes intravenous administration,
subcutaneous, intraperitoneal or intramuscular injection,
intratumoral, or intrathecal or intraventricular administration.
Without wishing to be bound by theory, the flexibility of particle
(composition) administration options is enabled, in part, by the
small size and low surface charge of the inventive and highly
stable nanoparticle, allowing the particle and its drug cargo to
traverse biologic barriers and size-limited structures such as the
bloodstream wall, lymphatic channels, and the skin to reach
cellular and molecular targets.
[0164] In one embodiment, the target is an in vitro biological
system such as in vitro tissues or cells. In one embodiment,
inventive non-ionic micelles and ligand-coated micelles are used
for high-throughput testing of primary cells cultured
3-dimensionally.
[0165] In one embodiment, nanoparticles bearing protein
combinations enable an ex vivo treatment of dendritic cells for
subsequent readministration to a patient.
[0166] In one embodiment, nanoparticles bearing protein
combinations enable an ex vivo treatment of dendritic cells for
subsequent readministration to a patient for purposes of
vaccination as part of a treatment protocol for solid and
hemapoietic malignancies.
[0167] In one embodiment, instant nanoparticles are administered in
to the airway epithelium. In one embodiment, the nanoparticles
carry a bioactive agent useful for treating any one of the
following genetic disorders affecting the lungs:
Acropectorovertebral dysplasia F form, Acute interstitial
pneumonia, Allergic bronchopulmonary aspergillosis, Alpha-1
antitrypsin deficiency, Alveolar capillary dysplasia, Arterial
tortuosity syndrome, Asbestosis, Autoimmune pulmonary alveolar
proteinosis, Beryllium disease, Blau syndrome, Brain-lung-thyroid
syndrome, Bronchiolitis obliterans, Bronchiolitis obliterans
organizing pneumonia, Bronchogenic cyst, Bronchopulmonary
dysplasia, Cantu syndrome, Catamenial pneumothorax, Children's
interstitial lung disease, Chronic granulomatous disease, Chronic
thromboembolic pulmonary hypertension, Classical-like Ehlers-Danlos
syndrome, Coal worker's pneumoconiosis, Congenital diaphragmatic
hernia, Congenital lobar emphysema, Congenital pulmonary alveolar
proteinosis, Congenital pulmonary lymphangiectasia, Congenital
tracheomalacia, Cornelia de Lange syndrome, Costocoracoid ligament
congenitally short, Cranioectodermal dysplasia, CREST syndrome,
Cryptogenic organizing pneumonia, Cutis laxa, autosomal dominant,
Cutis laxa, autosomal recessive type 1, Cystic fibrosis, Cystic
medial necrosis of aorta, Diffuse cutaneous systemic sclerosis,
Diffuse idiopathic pulmonary neuroendocrine cell hyperplasia,
Diffuse panbronchiolitis, Donnai-Barrow syndrome, Eisenmenger
syndrome, Ellis-Van Creveld syndrome, Emanuel syndrome,
Enthesitis-related juvenile idiopathic arthritis, Eosinophilic
granulomatosis with polyangiitis, Familial hypocalciuric
hypercalcemia type 1, Familial hypocalciuric hypercalcemia type 2,
Familial hypocalciuric hypercalcemia type 3, Familial mixed
cryoglobulinemia, Familial thoracic aortic aneurysm and dissection,
Familial thyroglossal duct cyst, Feingold syndrome, Fetal akinesia
deformation sequence, Fibrosing mediastinitis, Froster-Huch
syndrome, Game Friedman Paradice syndrome, Gaucher disease type 1,
Gaucher disease type 2, Gaucher disease type 3, Geroderma
osteodysplastica, Goodpasture syndrome, Granulomatosis with
polyangiitis, Hashimoto-Pritzker syndrome, Hemangiomatosis,
familial pulmonary capillary, Henoch-Schonlein purpura, Hereditary
fibrosing poikiloderma with tendon contractures, myopathy, and
pulmonary fibrosis, Hypercoagulability syndrome due to
glycosylphosphatidylinositol deficiency, Idiopathic acute
eosinophilic pneumonia, Idiopathic pulmonary fibrosis, Idiopathic
pulmonary hemosiderosis, Intrahepatic cholestasis of pregnancy,
Jeune syndrome, Juvenile dermatomyositis, Juvenile polymyositis,
Kabuki syndrome, Kaolin pneumoconiosis, Kartagener syndrome,
Laryngoonychocutaneous syndrome--See Epidermolysis bullosa, Lethal
congenital contracture syndrome 1, Limited cutaneous systemic
sclerosis, Limited systemic sclerosis, Loeys-Dietz syndrome,
Loeys-Dietz syndrome type 1, Loeys-Dietz syndrome type 2,
Loeys-Dietz syndrome type 3, Loeys-Dietz syndrome type 4, Lung
agenesis, Lymphangioleiomyomatosis, Manouvrier syndrome, Meconium
aspiration syndrome, Microphthalmia syndromic 9, Microscopic
polyangiitis, Mixed connective tissue disease, Mounier-Kuhn
syndrome, Multifocal fibrosclerosis, Multisystemic smooth muscle
dysfunction syndrome, Niemann-Pick disease type B, Nocardiosis,
Nontuberculous mycobacterial lung disease, Novak syndrome,
Occipital horn syndrome, Orofaciodigital syndrome 4, PAGOD
syndrome, Pallister-Killian mosaic syndrome, Pentalogy of Cantrell,
Peroxisomal biogenesis disorders, Primary ciliary dyskinesia,
Psoriatic juvenile idiopathic arthritis, Pulmonary alveolar
microlithiasis, Pulmonary sequestration, Pulmonary venoocclusive
disease, Recurrent respiratory papillomatosis, Respiratory distress
syndrome, infant, Sarcoidosis--Not a rare disease, SCARF syndrome,
Short rib-polydactyly syndrome type 3, Short rib-polydactyly
syndrome type 1, Short rib-polydactyly syndrome type 2, Short
rib-polydactyly syndrome type 4, Silicosis, Simpson-Golabi-Behmel
syndrome, Sprengel deformity, Sudden infant death with dysgenesis
of the testes syndrome, Systemic onset juvenile idiopathic
arthritis, Thoracic dysplasia hydrocephalus syndrome,
Thoracolaryngopelvic dysplasia, Thoracomelic dysplasia, Tracheal
agenesis, Tracheobronchomalacia, Tracheobronchopathia
osteoplastica, Vascular Ehlers-Danlos syndrome, Wilson-Mikity
syndrome, Wolf-Hirschhorn syndrome, Wrinkly skin syndrome, Yellow
nail syndrome, Young syndrome.
Unexpected Results
[0168] Through insight of the inventors, many of the challenges of
gene therapy are now overcome by the delivering bio-machinery
complexes (or portions of bio-machinery complexes) to cells and
tissues of interest instead of merely delivering one or more
bio-active components. This delivery of such machinery components
has now been made possible by the inventors' technological
breakthroughs which enable complexing of proteins with
polynucleotides in a compact, stable, and non-toxic nanoparticle
that optionally may be targeted to specific tissues and cells.
Thus, the long-recognized need for effective gene-editing has now
been met by the instant invention.
[0169] As demonstrated herein, or as observed in the mind of the
inventor, the instant nanoparticles have one or more of the
following surprising superior results: [0170] a. When administered
to a subject in need, they demonstrate superior efficacy when
compared to nanoparticles lacking the protein component; [0171] b.
Demonstrate reduce immunogenicity; [0172] c. Result in delivery to
relevant subcellular locations; [0173] d. Provide a means to supply
functional machinery components to cell types that otherwise are
deficient in them; [0174] e. Provide higher therapeutic levels at
the target tissues; and/or f. Show increased stability in vivo and
upon storage
VII. EXAMPLES
Example 1--Production of Nanoparticles and Comparators
[0175] Illustrative nanoparticles are generated as follows. These
examples describe how some colloidal formulations of diverse cargos
and biocompatible polymers may be generated, for subsequent in vivo
and in vitro administration, Nanoparticles are prepared by the
"dispersion atomization" method described in U.S. Pat. No.
6,632,671, which is incorporated herein by reference in its
entirety, using modifications as described herein. The instant
protein components are recombinantly prepared at PNA Bio
(dCas9-NLS), Novaprotein (Cas9; E67) and Active Motif
(Flag-Argonaute 2, 31886). Polynucleotide preparations are
synthesized as described in Table 1.
TABLE-US-00001 TABLE 1 Polynucleotide component preparations Seq
No. Name Description Sequence (5'-3') Manufacturer 1 RNAi F7
Single-strand chimeric 5'-gta aga ctt gag a 2o'ME[UGA Trilink
UCC]-(propyl)-3' 1 2RF7 Modified single-strand chimeric
5'-g2o'ME[U]a aga ctt gag a Trilink 2o'ME[UGA UCC]-(propyl)-3' 2
siF7 Double-stranded siRNA 5'.quadrature.GGAUCAUCUCAAGUCUUAC
Dharmacon TT-3' (p) 3 crF7 Single guide (sg) RNA equaling 19
AAGCACAUGGUGUCCUACAC Trilink nt targeting sequenc (BOLD) +
GUUUUAGAGCUAGAAAUAGCAAGUUAAAA Biotechnologies tracer RNA
scaffolding sequence UAAGGCUAGUCCGUUAUCAACUUGAAAAA modified by the
MS method of GUGGCACCGAGUCGGUGCUUUU Porteus (2015 NBT). *p denotes
passenger strand, sense strand or mRNA target region. Lower case
denotes phosphodiester DNA, upper case RNA.
[0176] Nanoparticles were made and designated as set forth in Table
2
TABLE-US-00002 TABLE 2 Nanoparticle Designations Protein
Polynucleotide ligand compnent component other Formual A ASOR rAgo2
Formulas B ASOR rAgo2 RNAiF7-50 Formula C ASOR rAgo2 RNAiF7-100
Formula Da ASOR rAgo2 2RF7- Formula E ASOR RNAiF7 Formula F ASOR
siF7 Formula G ASOR sugar Formula Ha ASOR Cas9 crF7-short
crystalization period Formula Hb ASOR Cas9 crF7-extended
crystalization Formula I ASOR dCas9 crF7
[0177] Briefly, to prepare each formula below, the following
procedures were used:
[0178] Formula A, (ASOR rAgo2, MW 106 kDa). About 31.25 .mu.g of
Flag-Ago2 (from insect cells) is resuspended into about 100 .mu.l
of reaction buffer (prepared using sterile water as about 20 mM
Hepes, about ph 7.4, about 5% ethylene glycol, about 1 mM DTT)
following washing with reaction buffer through about a 30 kDa MWCO
filter (Vivaspin 6, Sartorius). This protein component is then
dispersed using a water-insoluble surfactant system (2, 4, 7,
9-tetramethyl-5-decyn-4, 7-diol (TM-diol; SE-30 (Air Products),
about 1 .mu.g in about 50% DMSO. Following emulsification with a
water-miscible solvent (DMSO), by adding about 150 .mu.l of DMSO,
vortexing, and subsequently placing in bath sonicator for about 15
minutes, the micelles are then inverted and diluted by the addition
of about 700 .mu.l of PBS, also prepared in sterile water.
[0179] The resultant hydrophobic micelles are coated
(non-covalently) by the addition of about 0.4 .mu.g of
Asialoorosomucoid (Athens Research), placed in a bath sonicator for
about 30 minutes, is transferred to a 5 ml polypropylene tube, and
is diluted up to about 3 ml with PBS, prepared with sterile water,
then is atomized with a manual actuator using an approximately 250
.mu.m diameter orifice with feed pressure of less than about 10 psi
into a salt receiving solution of sterile water containing
primarily Li.sup.+ (about 31.4 mg Li.sup.+ (premixed with about 2.5
ppb Cs.sup.+ on Li.sup.+), about 15.0 mg Ca.sup.2+, about 210 .mu.g
Ba.sup.2+, about 29 .mu.g Bi.sup.2+ with about 6.12 ng Mg.sup.2+,
about 2.76 ng Sr.sup.2+, (all ultrapure, all are prepared as stock
solutions with sterile water except Sr.sup.2+ and Mg.sup.2+ are
prepared with laboratory grade water, all metals are used as
chloride salts, total bath volume approximately 32 ml). The total
reaction volume is approximately 36 ml. The level of the following
metals tested for in the sterile water used to prepare the
stabilization solution is determined to be less than about 0.2
parts per million in sum total: aluminum, arsenic, barium, cadmium,
chromium, copper, iron, lead, manganese, nickel, rubidium, sulfur,
vanadium, and zinc.
[0180] The premixing step comprises adding Cs.sup.+ at about 0.1
.mu.g/1 ml to about 4M Li.sup.+, at about 2.5 ppm Cs.sup.+ to
Li.sup.+ by weight, in sterile water in a 50 ml tube, and rotating
for about 2 minutes. Following cold-room incubation (at about
4.degree. C.) on a roller mill at about 0.5 rpm in 40 ml
round-bottomed tubes for about 4 hours, which further stabilizes
the coated micelles in the salt solution, the sub-50 nm
nanoparticles are recovered by centrifugation at about
20,000.times.g at about 4.degree. C. for about 1 hrs and are
resuspended in about 10 mM Hepes+10% lactitol, about pH=7.4, (at a
concentration of about 0.125 .mu.g/.mu.l), transferred to a 2 ml
conical, and spun down at maximum speed for about 5 minutes at
about 4.degree. C., and is washed by resuspending pellet in about
Hepes/10% lactitol, and is sterilized through a 0.2 .mu.m filter,
and frozen at about -20.degree. C.
[0181] In all formulations described in the instant example, a
small amount (about 1% of coating weight) of Syrian Hamster IgG is
added in tracer amounts into the ligand coat to enable
immunodetection of nanoparticle uptake by anti-Syrian Hamster
antibodies. Average particle size is less than about 50 nm, as
measured Dynamic Light Scattering (DLS) on a Nicomp ZLS 380
instrument. Particle size is measured as about 2.+-.0.4 nm, a count
rate of about 84 KHz with population volume of about 99.8% along
with a surface charge of about 5.6+/-0.5 mV per manufacturer's
instruction.
[0182] Formula B, (ASOR rAgo2 RNAiF7-50), Approximately 50% of the
Ago2 is co-encapsulated with the polynucleotide component as in
Formula A and as follows. About 31.25 .mu.g of Flag-Ago2 (from
insect cells) is resuspended into about 100 .mu.l of reaction
buffer (prepared using sterile water as about 20 mM Hepes, at about
ph 7.4, about 5% ethylene glycol, about 1 mM DTT) following washing
with reaction buffer through an about 30 kD MWCOa filter (Vivaspin
6, Sartorius). The protein component (Ago2) in an amount of about
295 pmol is then reacted with an amount of polynucleotide (guide
RNA, i.e. RNAi F7, Sequence 1 from Table 1) by slow trituration
about every two minutes over about a six minute period. This is
generally sufficient to drive the complexation such that about or
greater than 50% of the protein is complexed. RNAi F7 is chimeric
guide strand (single-stranded) from siFVII, (Akinc, et. al, 2009
Mol Ther 17(5)872-879) and listed as Sequence 1. Backbone chemistry
is described in its entirety in U.S. Pat. No. 9,132,148,
incorporated herein in its entirety.
[0183] Polynucleotide-protein complex is then dispersed using a
water-insoluble surfactant system (2, 4, 7,
9-tetramethyl-5-decyn-4, 7-diol (TM-diol; Surfynol SE (Air
Products), about 1 .mu.g in about 50% DMSO. Following
emulsification with a water-miscible solvent (DMSO), by adding
about 150 .mu.l of DMSO, vortexing, and subsequently .mu.lacing in
bath sonicator for about 15 minutes, the micelles are then inverted
and diluted by the addition of about 700 .mu.l of PBS.
[0184] The resultant hydrophobic micelles are coated
(non-covalently) by the addition of about 0.4 .mu.g of
Asialoorosomucoid (Athens Research), are .mu.laced in a bath
sonicator for about 30 minutes, are transferred to a 5 ml
polypropylene tube, and are diluted up to about 3 ml with PBS, then
they are atomized with a manual actuator using an approximately 250
.mu.m diameter orifice with feed pressure of less than about 10 psi
into a salt receiving solution of sterile water containing
primarily Li.sup.+ (about 31.4 mg Li.sup.+ (premixed with about 2.5
ppb Cs.sup.+ on Li.sup.+), about 15.0 mg Ca.sup.2+, about 210 .mu.g
Ba.sup.2+, about 29 .mu.g Bi.sup.2+ with about 6.12 ng Mg.sup.2+,
about 2.76 ng Sr.sup.2+, (all ultrapure, all are prepared as stock
solutions with sterile water except Sr.sup.2+ and Mg.sup.2+
prepared with laboratory grade water, all metals are used as
chloride salts, total bath volume approximately 32 ml). The total
reaction volume is approximately 36 ml.
[0185] Following cold-room incubation at about (4.degree. C.) on a
roller mill at about 0.5 rpm in 40 ml round-bottomed tubes for
about 4 hours, which further stabilizes the coated micelles in the
salt solution, the approximately sub-50 nm nanoparticles are
recovered by centrifugation at about 20,000.times.g at 4.degree. C.
for about 1 hrs and are resuspended in about 10 mM Hepes+10%
lactitol, at about pH=7.4, (at a concentration of about 0.125
.mu.g/.mu.l), is transferred to a 2 ml conical, and spun down at
maximum speed for about 5 minutes at about 4.degree. C., are washed
by resuspending pellet in about Hepes/10% lactitol, sterilized
through an about 0.2 .mu.m filter, and frozen at about -20.degree.
C.
[0186] Average particle size for Formula B is less than about 50 nm
as measured by DLS. Particle size is measured as about 11.+-.2 nm,
a count rate of about 24 KHz with population volume of about 99.6%
along with a surface charge of about 3.+-.0.6 meV per
manufacturer's instruction.
[0187] Formula C, (ASOR rAgo2 RNAiF7-100) is formulated similarly
to Formula B except that the Ago 2 protein is reacted with the
polynucleotide comprising chimeric single-stranded poly RNAi F7 in
an amount calculated to achieve about 110% of completion of the
reaction in that about 325 pmol of the polynucleotide was reacted
with about 295 pmol of Ago2 to initiate a core to support formation
of a micelle. For characterization, average particle size for
Formula C is less than about 50 nm, as measured by DLS. Particle
size is measured as about 19.+-.2 nm, a count rate of about 26 kHz
with population volume of about 98.6% along with a surface charge
of about 2.9+/-0.1 mV per manufacturer's instruction.
[0188] Formula D, (ASOR rAgo2 2RF7) is formulated similarly to
Formula C with the change that the polynucleotide (guide strand)
has a different backbone chemistry (2RF7, Sequence 1). 2RF7 is
distinguished from RNAi F7 by modifying the typical nucleotide in
position 2 from the 5' end to a 2'-O-Methyl Ribonucleotide. For
characterization, average particle size for Formula C is less than
about 50 nm, as measured by DLS. Particle size is measured as about
14.+-.2 with population volume of about 99.5% along with a surface
charge of about 0.3+/-1 mV per manufacturer's instruction.
[0189] Formula E, (ASOR RNAiF7 6115 Da MW). About 250 .mu.g of
about 20 mer, chimeric, single-stranded guide strand RNAiF7 (Table
1) is formulated generally as in Formula A, by first complexing the
polynucleotide component with about 62.5 .mu.g of about 11,800 Da
MW polyornithine (Sigma), then dispersing using a water-insoluble
surfactant system (2, 4, 7, 9-tetramethyl-5-decyn-4, 7-diol
(TM-diol; SE-30 (Air Products), at about 7.5 .mu.g in about 50%
DMSO. Following emulsification with a water-miscible solvent
(DMSO), by adding about 150 .mu.l of DMSO, vortexing, and
subsequently .mu.lacing in bath sonicator for about 15 minutes, the
resultant micelles are then inverted and diluted by adding about
700 .mu.l of PBS, also prepared in sterile water. Following the
addition of about 3.125 .mu.g of ASOR to coat micelles as described
in Formula A, the resultant hydrophobic micelles are then processed
as generally described in Formula A with the following changes in
receiving bath component weights: about 2.42 ng Mg.sup.2+, about
10.92 ng Sr.sup.2+. For characterization, average particle size for
Formula E is less than about 50 nm, as measured by DLS. Particle
size is measured per manufacturer's instruction at about 10.+-.1 nm
with population volume of about 99.2% along with a surface charge
of about 0.2+/-0.4 mV.
[0190] Formula F, (ASOR siF7, 13,800 Da MW nominal) about 250 .mu.g
of about 21 mer, unmodified, double-stranded RNA polynucleotide
(siFVII, Akinc, et. al, 2009 Mol Ther 17(5)872-879), is first
complexed with about 54 .mu.g Beta-cyclodextrin (Sigma), then is
dispersed into about 100 .mu.l of sterile water using a
water-insoluble surfactant system (2, 4, 7,
9-tetramethyl-5-decyn-4, 7-diol (TM-diol; SE-30 (Air Products)),
about 7.5 .mu.g in about 50% DMSO.
[0191] Following the addition of about 3.125 .mu.g of ASOR to coat
micelles as described in Formula A, the resultant hydrophobic
micelles are then processed as generally described in Formula A
with the following changes in receiving bath component weights:
about 10.72 ng Mg.sup.2+, about 212.5 ng Sr.sup.2+. For
characterization, average particle size for Formula E is less than
about 50 nm, as measured by DLS. Particle size is measured per
manufacturer's instruction at about 11.+-.2 nm with population
volume of about 99.4% along with a surface charge of about
0.2.+-.0.7 meV.
[0192] Formula G, (ASOR sugar), about 500 .mu.g of erythritol (MW
122.12) is dispersed into about 100 .mu.l of sterile water using a
water-insoluble surfactant system (2, 4, 7,
9-tetramethyl-5-decyn-4, 7-diol (TM-diol; SE (Air Products)), about
8.75 .mu.g in about 50% DMSO.
[0193] Following the addition of about 6.25 .mu.g of ASOR to coat
micelles as described in Formula A, the resultant hydrophobic
micelles are then processed as generally described in Formula A
with the following changes in receiving bath component weights:
about 5.75 ng Mg.sup.2+, about 10.36 ng Sr.sup.2+, For
characterization, average particle size for Formula E is less than
about 50 nm, as measured by DLS. Particle size is measured per
manufacturer's instruction at about 13.3.+-.1.9 nm with population
volume of about 99% along with a surface charge of about
0.04.+-.0.75 meV.
[0194] Formula Ha, (ASOR Cas9 F7-short). The polynucleotide
component used here is the recombinant Cas9 endonuclease
(Novoprotein E365, pl 9.1, 163 kDa MW) and is reacted with, as the
polynucleotide, guide RNA (F7-short). This creates a species with
an overall negative charge. The equimolar amount of the about 105
nt guide RNA carries approximately 3.25 nmol (-) charge per .mu.g
from phosphate groups, while the protein component carries about a
+22 charge per molecule as reported from sequence and supported by
it high isoelectric point. Thus, for the about 31.25 .mu.g
reaction, an imbalance of negative charge is operable following
protein-substrate binding, e.g. about 4290 pmol of (+) charge from
about 195 pmol of Cas9 is not balanced by .about.22680 of phosphate
charge from about 216 pmol of sgRNA.
[0195] We compare the impact of charge neutralization as is
commonly practiced in art for nucleic acid cargos (See Formulas E,
F). Surprisingly, added charge neutralization during the micelle
formation step (about 26%, Spermine), (about 55.5%, Spermidine)
does not promote capsule formation. Instead, best results are
obtained with no externally-mediated charge neutralization in the
manner of a sugar cargo which is electrically neutral (See Formula
G). This unexpected course of events indicates that a substantially
different mechanism is in effect in the synthesis of nanoparticles
containing substrate-reacted enzymatic proteins.
[0196] In Formula Ha, recombinant Cas9 is coencapsulated with the
polynucleotide component at about 110% molar completion of the
reaction in a sequence similar to Formula A as follows. About 31.25
.mu.g of recombinant Cas9 (from E. Coli) is resuspended into about
100 .mu.l of reaction buffer (prepared using sterile water as about
20 mM Hepes, at about ph 7.4, about 5% ethylene glycol, about 1 mM
DTT). The protein component Cas9 (.about.195 pmol) is then reacted
with an amount of guide RNA (crF7, Table 1) sufficient to drive
about 110% completion (.about.216 pmol) of the reaction by slow
trituration every two minutes over a six minute period. The
polynucleotide component is crF7 guide RNA (single-stranded, about
MW 33,528) listed as Sequence 3 in Table 1.
[0197] The protein-polynucleotide complex is then dispersed using a
water-insoluble surfactant system (2, 4, 7,
9-tetramethyl-5-decyn-4, 7-diol (TM-diol; SE (Air Products), about
1 .mu.g in about 50% DMSO. Following emulsification with a
water-miscible solvent (DMSO), by adding about 150 .mu.l of DMSO,
vortexing, and subsequently .mu.lacing in bath sonicator for 15
minutes, the micelles are then inverted and diluted by the addition
of about 700 .mu.l of PBS.
[0198] Following the addition of about 0.4 .mu.g of ASOR to coat
micelles as described in Formula A, the resultant hydrophobic
micelles are then processed as generally described in Formula A
except that the receiving bath component weights are about 5.75 ng
Mg.sup.2+ and about 2.6 ng Sr.sup.2+. For characterization, average
particle size for Formula E is less than about 50 nm, as measured
by DLS. Particle size is measured per manufacturer's instruction at
about 23.4.+-.4.3 nm with population volume of about 91.2% along
with a surface charge of about 0.03.+-.0.78 mV. TEM for Formula Ha
is illustrated in FIG. 1 and shows a fractal crystalline morphology
with a visible protein ligand corona.
[0199] Formula Hb, A more stable crystalline variant of Formula Ha
is synthesized by incubating ASOR ligand-coated micelles in the
receiving bath for about 36 hours rather than about 4 hours, The
receiving bath component weights are about 5.23 ng Mg.sup.2+, about
2.36 ng Sr.sup.2+ and without bismuth. In this case, the receiving
bath volume is about 40.5 and the total reaction volume is about
44.4 ml. For characterization, average particle size for Formula E
is less than about 50 nm, as measured DLS. Particle size is
measured per manufacturer's instruction at about 19.+-.3.2 nm with
population volume of about 97.1% along with a surface charge of
about 0.07.+-.0.73 meV.
[0200] Formula Hc is formulated similarly to Formula Ha with the
change that about 31.25 ug of a non-canonical Typell-B Cas9,
without a bi-lobe nuclease structure, e.g., from F. Novicida is
substituted in as the enzymatic protein pre-reacted with it RNA
binding partners. Average particle size for Formula Hc is less than
about 50 nm, as measured Dynamic Light Scattering (DLS) on a Nicomp
ZLS 380 instrument. Zeta potential is measured, based on
manufacturer's instructions, and determined to approximately
neutral.
[0201] Formula Hd is formulated similarly to Formula Hc with the
change that non-canonical Type V Cpf1 is substituted for
non-canonical Type II-B Cas9.
[0202] Formula I, (ASOR dCas9 F7), Formula I is formulated
similarly to Formula Ha with the change that the protein component
is double mutant recombinant spCas9 with about 6.times.his tag and
NLS from SV40 N-terminus ((D10A/H840A; PNA Bio #CD03) at about 31.
about 25 .mu.g. Average particle size for Formula I is less than
V50 nm, as measured by DLS. Particle size is measured as about 2K
14+/-2 nm with population volume of about 98.9% along with a
surface charge of about 0.6+/-0.8 mV per manufacturer's
instruction.
[0203] Formula J (Tenfibgen Chymotrypsin-Trypsin). Formula J is
formulated similarly to Formula A with the change that about 31.25
.mu.g of trypsin-chymotrypsin mixture (in the activity ratio 6:1)
having enzymatic activity of about 2000 AU/mg was substituted for
the enzyme-substrate combination and tenfibgen (MW 26,500 Da, the
fibrinogen fragment of Tenascin-C, fully incorporated from U.S.
Ser. No. 14/844,828) is substituted for ASOR. Following the
addition of about 0.4 .mu.g of Tenfibgen to coat micelles as
described in Formula A, the resultant hydrophobic micelles are then
processed as generally described in Formula A with the following
changes in receiving bath concentrations: about 6.38 ng Mg.sup.2+,
about 28.75 ng Sr.sup.2+. Particles are incubated with rolling as
described in Formula A with the change of about 48 hours of
incubation time before centrifugation and purification. In separate
batch runs, non-ionic micelles without ligand coating and
ligand-coated micelles without crystallization are also produced.
Average particle size for Formula J is less than about 50 nm, as
measured Dynamic Light Scattering (DLS) on a Nicomp ZLS 380
instrument. Zeta potential is measured as approximately neutral per
manufacturer's instruction.
[0204] Formula K (Tenfibgen Erythritol), about 500 .mu.g of
erythritol (MW 122.12) is dispersed into about 100 .mu.l of sterile
water using a water-insoluble surfactant system (2, 4, 7,
9-tetramethyl-5-decyn-4, 7-diol (TM-diol; SE (Air Products), about
8.75 .mu.g in about 50% DMSO. Following the addition of about 6.25
ug of Tenfibgen to coat micelles as described in Formula J, the
resultant hydrophobic micelles are then processed as generally
described in Formula A with the following changes in receiving bath
concentrations: about 6.38 ng Mg2.sup.+, about 28.75 ng Sr2.sup.+.
Particles are incubated with rolling as described in Formula A with
the change of about 48 hours of incubation time before
centrifugation and purification. For characterization, average
particle size for Formula E is less than about 50 nm, as measured
Dynamic Light Scattering (DLS) on a Nicomp ZLS 380 instrument.
[0205] Thus, we have described in some embodiments compositions and
methods for formulation of nanoparticles to deliver
protein-polynucleotide complexes where the nanoparticles may be
nonionic, ligand-coated or crystalline ligand-coated. The species
demonstrate similar desirable properties across a range of diverse
cargos and chemistries.
Example 2--Demonstration that Polynucleotide--Protein Complex
Encapsulation into Nanoparticles Enhances Incorporation into
Crystalline Nanoparticles
[0206] To date, intracellular enzymes have not been used
effectively for in vivo therapy by systemic in vivo delivery. Such
use is constrained by enzyme chemical and mechanical fragility
through the process of formulation, exposure to denaturing
excipients, and exposure during administration to denaturing
proteinases in the bloodstream, tissues and intracellularly. It has
been discovered, quite unexpectedly, that the instant
polynucleotide components may be used to facilitate incorporation
of an enzyme into a nanoparticles to create a supramolecular
crystalline complex.
[0207] Moreover, efforts at systemic delivery of nucleoprotein
complexes as the biologic agents taught here as pre-formed entities
have not been successful until the development of the instant
invention.
[0208] In this example, the protein components was Ago2, a 104 kDa
endonuclease was coupled with the polynucleotide component of a
short, approximately 22 mer, RNA "guide strand" (in this example, a
chimeric guide strand) against murine Coagulation Factor VII to
provide specificity for specific cleavage of the target mRNA, to
inhibit production of the target Factor VII.
[0209] Encapsulation of the Ago2-guide strand complex (at about 50%
molar loading) is compared with 1) protein component alone (Formula
A), 2) anti-Factor VII guide strand--Ago complex with about 50%
molar complexation (Formula B), 3) anti-Factor VII guide strand
with Ago2 complex with about 110% molar complexation (Formula C)
and 4) anti-Factor VII guide strand--Ago2 complex with about 110%
molar complexation using an alternate backbone chemistry for the
polynucleotide chimeric guide strand.
[0210] Following formulation, nanoparticles are characterized by
DLS and zeta potential. Supernatants from the lithium
crystallization solution (mother liquor) were buffer-exchanged
using an about 5 kDa MWCO ultrafilter (Vivaspin 20, Sartorius).
Equal volumes are normalized to the control (Ago2 and excipients
without crystallization but filtered) from each of the
approximately 100 .mu.l retentates are electrophoresed on an about
4-12% gradient bis-tris gel (Invitrogen) using a Tris-glycine
buffer and detected for protein by silver staining (Pierce).
[0211] The results, summarized in Table 3, show a direct
correspondence between polynucleotide component and protein
component complexation and incorporation into the crystalline
supramolecular nanoparticle complex as less protein was measured in
the reaction supernatant with increased completion of the specific
binding reaction. For the protein component alone, subjected to
nanoencapsulation, a protein band running at the same approximate
100 kDa level as control Ago2 and Ago2 admixed in excipients
without further processing is visibly indicating no incorporation
into particles that would be in the pellet. Dynamic light
scattering (DLS) before any nanoencapsulation, Ago2 protein showed
a particle size distribution of about 6.+-.1 nm (about 95%); about
35.+-.6 nm (about 4.6% population volume).
[0212] Following the nanoencapsulation process, in the product
stream at equivalent volumes, only about a 2 nm species with a high
primary incidence (about 98.5% population volume) was measured by
DLS along with a high count rate (about 84 KHz) suggesting a
degradation for the naked about 6 nm Ago2 protein species. Lower
count rates and species larger than about 6 nm appeared as unimodal
peaks in reacted Ago2 capsules (Table 3). Between about 50% and
100% of full binding reaction, the residual Ago2 band in the
supernatant disappeared indicating a direct correspondence between
extent of reaction and feasibility for successful incorporation
into inventive nanopartices. We conclude interactions between the
protein component and polynucleotide component in the complex
provides a means for greatly enhancing incorporation into
crystalline nanoparticle supramolecular complexes.
TABLE-US-00003 TABLE 3 Incorporation results for polynucleotide
complexation into nanoparticle Protein Particle Band in Size by
Count rate supernatant DLS (nm); from densitometry Population
equivalent Formula, Cargo (1, FIG. 2) volume (%) volumes Ago 2
standard (0.6 ug) 70.6 Ago 2 + excipients, no 39.2 processing
Formula A , Ago 2 protein 19.6 2 .+-. 0.4 (99.8%) 84 kHZ Form. B,
RISC - 50% 6.0 11 .+-. 2 (99.6%) 24 kHZ reacted Form. C, RISC -
100% 2.41 19 .+-. 2 (98.6%) 26 kHZ reacted Form D, RISC 2RF7 - 0.31
14 .+-. 2 (99.5%) 18 kHZ 100%
[0213] Ago2 standard on gel is about 0.6 .mu.g, Supernatant
concentrates represented approximately 25% of the total reaction
supernatant. Densitometry is perfomed in NIH Image J using mean
values of equivalent areas subtracted from nearby background.
Example 3--Protein Co-Encapsulation of Protein Component with
Polynucleotide Component Enriches Cells with Active RISC for
Enhanced Target Gene Modulation
[0214] Superior efficacy of the instant nanoparticles (Risc RNAi
F7; Formula B) is demonstrated in comparison to guide strand alone
(Formula E) nanoparticles in 3D cultured FL83B hepatocytes
(ATCC).
[0215] 3D hepatocyte cell cultures are prepared by plating about
25,000 murine FL83B murine hepatocyte cells on to ECM-coated spun
polymer scaffolds (Corning, UltraMax) in chamber slides. Formula B
and Formula E nanoparticles are added at various concentrations to
the cultures and harvested for microscopy after about 3 days.
[0216] Dose response for Factor VII protein inhibition are assayed
by confocal fluorescence microscopy for Factor VII immunosignal
(GeneTex) and qPCR.
[0217] Formula B administration demonstrates full inhibition of
Factor VII protein at about 1 pmol (about 4 nM) continuing through
about 2.0 nM with recovery beginning around about 0.32 nM
demonstrating about a 3 log response range. In contrast, Formula E
bearing the guide strand only shows full inhibition by microscopy
at about 5 nmol (about 20 .mu.M) recovering by about 1.25 nmol
(about 5 .mu.M) for about a 0.4 log response range and approximate
increase in activity of about 3.5 logs (about 2 nM vs. about 12.5
.mu.M) for encapsulated RISC complex vs encapsulated guide RNA
alone. Results are illustrated in FIG. 3.
Example 4--In Vivo Demonstration of Superior Results of Instant
RISC Nanoparticles
[0218] Systemic in vivo delivery is demonstrated in C57BL/6NCrSim
mice in about a 3 day acute study. Mice are administered equal
particle number doses (about 10e13) of 1) ASOR RISC RNAiF7 (Formula
B), ASOR RNAiF7 (Formula E), ASOR siF7 (Formula F); control animals
treated with about standard 200 .mu.l of about 1 mg/ml ASOR
erythritol (Formula G). For G1, Formula B, additional regimens
using about 10e12 particle (about 136 .mu.g/kg) doses are
tested.
[0219] For RISC nanoparticles, about 10e13 particles are
approximately equal to about 250 pmol or about 1.36 mg/kg of
protein complex while this particle dose is approximately equal to
about 1 nmol or about 0.75 mg/kg of polynucleotide component.
Particle numbers are estimated assuming closest theoretical packing
into final particle size measured by DLS.
[0220] At study termination, citrated plasma is collected for
Factor VII ELISA by terminal cardiac puncture. By visual
observation during necropsy, blood from RISC nanoparticle-treated
animals is bright red and of very low viscosity, while blood from
control animals is dark with a purple hue and of much higher
viscosity in the collection syringe. Blood from animals treated
with nanoparticles made to contain F7 RNA or siRNA without Ago2
appear to be of an intermediate nature. A Factor VII ELISA of
citrated plasma samples shows that only nanoencapsulated RISC has a
significant (about 83%) reduction in Factor VII plasma protein at
Day3. Lower doses of nanoencapsulated RISC or oligo or siRNA show
no change or in some cases elevations suggesting that biological
modulation is occurring.
[0221] We conclude that the instant RISC nanoparticle demonstrates
superior effective systemic delivery and efficacy and retention of
enzymatic activity. Supramolecular crystalline RISC shows
significant improvement in inhibition of phenotypic activity and
transcript levels confirming in vitro results observed in 3D cell
culture.
Example--5 Superior Efficacy of Instant CRISPR Complex
Nanoparticles Demonstrated In Vivo
[0222] We assay for functional in vivo activity of the CRISPR
complex nanoparticles i.e. bacterial endonuclease Cas9 reacted with
about a 105 nt anti-Factor VII guide RNA (sgRNA, described as
Formula H in Example 1) in Balb/C immunocompetent mice. In this
study, we compare two formulas, a shorter dissolution capsule
(which is prepared using a shorter crystallization period amenable
to in vitro studies, Formula Ha) and a longer dissolution capsule
(generally used for longer in vivo circulation, Formula Hb).
[0223] Three dosing levels are examined with a two dose regimen; 1)
about 3e12 particles at about 1.6 mg/kg; 2) about 3e11 particles at
about 160 .mu.g/kg q3D; and 3) about 3e10 particles at about 16
.mu.g/kg q3D of RISC nanoparticles. Particle numbers are estimated
assuming closest theoretical packing into final particle size
measured by DLS. A fractional factorial design is used with groups
of three assigned to each dissolution profile design, and mice are
sacrificed to collect livers for microscopy, western blotting, qPCR
and mutational analysis at about 7 days post initiation of
treatment.
[0224] We investigate changes in Factor VII and Cas9 protein
component immunosignal by confocal immunofluorescence microscopy
for the groups over the dose range. Cas9 immunosignal is used for
capsule localization and trafficking while Factor VII protein
immunosignal demonstrated the phenotypic effect of Factor VII
mutation and/or CRISPR-based silencing. Results are illustrated in
FIG. 4.
[0225] We find that Cas9 immunosignal (the nanoparticle cargo)
shows a punctate cytoskeletal pattern at mg/kg dose levels (highest
dose, about 3e12 particles) in individual hepatocytes that
decreases with dose level to not detectable at about 16 .mu.g/kg
(lowest dose level, 3e10 particles).
[0226] At the same time, Factor VII immunosignal is maximally
decreased as detectable Cas9 signal disappears suggesting a
sequence of events where capsule processing to "release" Cas9 for
action is hindered in the case of potentially too many capsules
entering the cell. This means that the highest dose tested is not
the optimal biological dose (OPD). This pattern of persistent Cas9
immunosignal indicating saturation corresponding with poorer Factor
VII inhibition is confirmed in 3D hepatocyte cell culture.
[0227] For Factor VII immunosignal, we observe reduction in signal
in 3D mouse hepatocytes that appears maximal at about 1 pmol (about
4 nM) and is nearly recovered by about 0.1 pmol (about 0.4 nM). At
the highest dose level of about 5 nmol, no reduction in Factor VII
is observed consistent with the existence of an optimal biological
dose.
[0228] We additionally perform western blotting of liver lysates
from mice under denaturing conditions confirming our observations
by microscopy. Under denaturing conditions, Factor VII separates
into three fragments of about 73, 50 and 43 kDa. Densitometry of
Factor VII bands (Genetex) normalized to Lactate dehydrogenase
(Cell Signaling) levels indicated about 40-85% knockdown of the
three fragments at the peak effective middle dose of about
2.times.3e12 q3D particles or about 2.times.160 .mu.g/kg. Results
are summarized below in Table 4.
TABLE-US-00004 TABLE 4 Densitometry for Factor VII western blotting
of 7 day liver lysates 70 kDa 50 kDa 43 kDa Treatment fragment
.DELTA. % fragment .DELTA. % fragment .DELTA. % PBS 35 .+-. 3 5
.+-. 0.6 26.3 .+-. 2.2 2 .times. 1.6 mg/kg 29 .+-. 2 -17.3 2.5 .+-.
0.12 -49.4* 18.4 .+-. 1.3 -29.8* 2 .times. 160 .mu.g/kg .sup. 20
.+-. 3.7 -42.24* 0.72 .+-. 0.23 -85.5* 11.63 .+-. 2 -55.7* 2
.times. 16 .mu.g/kg 20.8 .+-. 6.4 -40.4 2.8 .+-. 0.25 -44.7* .sup.
16 .+-. 7.7 -39.2 Notes: *= p < 0.5
[0229] With respect to mutation, amplicon sequencing using an
Illumina Miseq approach detects no double strand breaks as having
occurred at the target site indicating no cutting activity of the
Cas9 enzyme. We examine mRNA levels for the best responding group
(middle dose) using primers that either bind upstream of the Exon 2
binding site (Exon 1) or downstream of the Exon 2 binding site
(Exon 4). qPCR executed using Exon 1 primer indicates no change in
Factor VII transcript had occurred while qPCR executed using the
downstream Exon 4 primer indicates an approximately 39% decrease in
transcript measured as (.DELTA.cycle time, (2 -(.DELTA..DELTA.Ct)),
for treated vs. control, about 0.62.+-.0.16 vs. about 1.01.+-.0.12,
mean.+-.Std Err) using TBP+RPLPO as normalizing variables for
Formula Ha. mRNA knockdown is confirmed by in situ hybridization
using a third probe targeted to downstream of the binding site for
the high and middle dose but not the low dose groups confirming
microscopy and western blotting. Inhibition appears similar by in
situ hybridization for at least the high dose of Formula Hb which
was examined.
Example 6--Superior Efficacy of Instant CRISPR Complex
Nanoparticles Demonstrated In Vivo
[0230] We assay for functional in vivo activity of the CRISPR
complex nanoparticles i.e. bacterial endonuclease Cas9 reacted with
about a 105 nt anti-Factor VII guide RNA (sgRNA, described as
Formula H in Example 1) in Balb/C immunocompetent mice. In this
study, we compare two formulas, a shorter dissolution capsule
(prepared using a shorter crystallization period amenable to in
vitro studies, Formula Ha) and a longer dissolution capsule
(generally used for longer in vivo circulation, Formula Hb).
[0231] Three dosing levels are examined with a two dose regimen; 1)
about 10e14 particles at about 1.6 mg/kg; 2) about 10e13 particles
at about 160 .mu.g/kg q3D; and 3) about 10e12 particles at about 16
.mu.g/kg q3D of RISC nanoparticles. Particle numbers are estimated
assuming closest theoretical packing into final particle size
measured by DLS. A fractional factorial design is used with groups
of three assigned to each dissolution profile design, and mice are
sacrificed to collect livers for microscopy, western blotting, qPCR
and mutational analysis at 7 days post initiation of treatment.
[0232] We investigate changes in Factor VII and Cas9 protein
component immunosignal by confocal immunofluorescence microscopy
for the groups over the dose range. Cas9 immunosignal is used for
capsule localization and trafficking while Factor VII protein
immunosignal demonstrates the phenotypic effect of Factor VII
mutation and/or CRISPR-based silencing. Results are illustrated in
FIG. 4.
[0233] We find that Cas9 immunosignal (the nanoparticle cargo)
shows a punctate cytoskeletal pattern at mg/kg dose levels (highest
dose, about 10e14 particles) in individual hepatocytes that
decreased with dose level to not detectable at about 16 .mu.g/kg
(lowest dose level, about 10e12 particles).
[0234] At the same time, Factor VII immunosignal is maximally
decreased as detectable Cas9 signal disappears suggesting a
sequence of events where capsule processing to "release" Cas9 for
action is hindered in the case of potentially too many capsules
entering the cell. This means that the highest dose tested is not
the optimal biological dose (OPD). This pattern of persistent Cas9
immunosignal indicating saturation corresponding with poorer Factor
VII inhibition is confirmed in 3D hepatocyte cell culture.
[0235] For Factor VII immunosignal, we observe reduction in signal
in 3D mouse hepatocytes that appear maximal at about 1 pmol (about
4 nM) and is nearly recovered by about 0.1 pmol (about 0.4 nM). At
the highest dose level of about 5 nmol, no reduction in Factor VII
is observed consistent with the existence of an optimal biological
dose.
[0236] We additionally perform western blotting of liver lysates
from mice under denaturing conditions confirming our observations
by microscopy. Under denaturing conditions, Factor VII separates
into three fragments of about 73, 50 and 43 kDa. Densitometry of
Factor VII bands (Genetex) normalized to VDAC (CMillipore) levels
indicate about 40-85% knockdown of the three fragments at the peak
effective middle dose of about 2.times.3e12 q3D particles or about
2.times.160 .mu.g/kg. Results are summarized above in Table 4.
[0237] With respect to mutation, amplicon sequencing using an
Illumina Miseq approach detects no double strand breaks as having
occurred at the target site which indicates no cutting activity of
the Cas9 enzyme. We examine mRNA levels for the best responding
group (middle dose) using primers that either bind upstream of the
Exon 2 binding site (Exon 1) or downstream of the Exon 2 binding
site (Exon 4). qPCR executed using Exon 1 primer indicates no
change in Factor VII transcript had occurred while qPCR executed
using the downstream Exon 4 primer indicated an approximately 30%
decrease in transcript measured as (.DELTA.cycle time, (2
-(.DELTA..DELTA.Ct)), for treated vs. control, about 0.07.+-.0.09
vs. about 1.01.+-.0.07, mean.+-.Std Err) using TBP+RPLPO as
normalizing variables for Formula Ha. Consistent mRNA knockdown is
confirmed by in situ hybridization using a third probe targeted to
downstream of the binding site for the middle dose with some
response seen in the high and low dose groups confirming microscopy
and western blotting. Inhibition appears similar by in situ
hybridization for at least the high dose of Formula Hb which is
examined.
Example 7--CRISPR Nanoparticles Avoid Immunologic Response
[0238] Animals from Example--5 are examined for immune response to
nanoparticle treatment.
[0239] Lethal inflammatory responses have been a problematic
sequelae of conventional non-viral strategies for systemic delivery
of bacterially-derived NPs. In this study (from Example--5), all
mice survive and are assayed for a panel of inflammatory cytodines,
including GM-CSF, IFNa, IFNg, IL-1b, IL-6, MCP-1, Rantes and TNFa
in the highest dose level group (2.times.1.6 mg/kg, 3e12 particles)
for both nanoparticle designs (Formulas Ha, Hb) vs. control. As
previously mentioned, this top dose level was also found to be
non-optimal coincident with dysregulation in intracellular
trafficking. We find no changes from control in any parameter for
any of the mice in the shorter circulation design capsule (Formula
Ha) and note some significant elevations (IFNa, IL-6, MCP-1, TNFa)
for one mouse in the longer dissolution design.
[0240] We compared Factor VII inhibition between ASOR-ligand
crystalline particles bearing Cas9, non-canonical Cas9 or dead Cas9
protein complexes in 3D hepatocyte cell culture. The canonical
spCas9 endonuclease undergoextensive structural conformation change
upon guide RNA binding. Upon guide RNA recognition and binding, the
REC lobe moves--65 angstoms (about 6.7 nm) towards the Nuc lobe in
a clamshell motion indicating mechanical fragility. Non-canonical
Cas9 enzymes do not contain this particular structure for nuclease
activity. dCas9 is mutated (H840A, D10A) in the HNH and RUVEC
cleavge domains respectively but does not lose substrate
recognition for the guide RNA and joint substrate recognition for
the target chromosome site. When dCas9 enzyme binds its sgRNA
substrate, it physically reconfigures and transforms into a
specific chromosomal binding protein to block mRNA transcription by
the RNA polymerase.
Example 8--CRISPR Nanoparticles Avoid Immunologic Response
[0241] Animals from Example--5 are examined for immune response to
nanoparticle treatment.
[0242] Lethal inflammatory responses have been a problematic
sequelae of conventional non-viral strategies for systemic delivery
of bacterially-derived NPs. In this study (from Example--5), all
mice survive and are assayed for a panel of inflammatory cytodines,
including GM-CSF, IFNs, IFNg, IL-1b, IL-6, MCP-1, Rantes and TNFa
in the highest dose level group (about 2.times.1.6 mg/kg, about
3e12 particles) for both nanoparticle designs (Formulas Ha, Hb) vs.
control. As previously mentioned, this top dose level is also found
to be non-optimal coincident with dysregulation in intracellular
trafficking. We find no changes from control in any parameter for
any of the mice in the shorter circulation design capsule (Formula
Ha) and note some significant elevations (IFNs, IL-6, MCP-1, TNFa)
for one mouse in the longer dissolution design.
[0243] We compare Factor VII inhibition between ASOR-ligand
crystalline particles bearing Cas9, non-canonical Cas9 or dead Cas9
protein complexes in 3D hepatocyte cell culture. dCas9 is mutated
(H840A, D10A) in the HNH and RUVEC cleavge domains respectively but
does not lose substrate recognition for the guide RNA and joint
substrate recognition for the target chromosome site. When dCas9
enzyme binds its sgRNA substrate, it physically reconfigures and
transforms into a specific chromosomal binding protein to block
mRNA transcription by the RNA polymerase.
Example 9--CRISPR Nanoparticles Avoid Immunologic Response
[0244] Animals from Example--5 are examined for immune response to
nanoparticle treatment.
[0245] Lethal inflammatory responses have been a problematic
sequelae of conventional non-viral strategies for systemic delivery
of bacterially-derived NPs. In this study (from Example--5), all
mice survive and are assayed for a panel of inflammatory cytodines,
including GM-CSF, IFNa, IFNg, IL-1b, IL-6, MCP-1, Rantes and TNFa
in the highest dose level group (about 2.times.1.6 mg/kg, about
10e14 particles) for both nanoparticle designs (Formulas Ha, Hb)
vs. control. As previously mentioned, this top dose level is also
found to be non-optimal coincident with dysregulation in
intracellular trafficking. We find no changes from control in any
parameter for any of the mice in the shorter circulation design
capsule (Formula Ha) and note some significant elevations (IFNa,
IL-6, MCP-1, TNFa) for one mouse in the longer dissolution
design.
[0246] We compare Factor VII inhibition between ASOR-ligand
crystalline particles bearing Cas9, non-canonical Cas9 or dead Cas9
protein complexes in 3D hepatocyte cell culture. The canonical
spCas9 endonuclease undergo extensive structural conformation
change upon guide RNA binding. Upon guide RNA recognition and
binding, the REC lobe moves--65 angstoms (about 6.7 nm) towards the
Nuc lobe in a clamshell motion indicating mechanical fragility.
Non-canonical Cas9 enzymes do not contain this particular structure
for nuclease activity. dCas9 is mutated (H840A, D10A) in the HNH
and RUVEC cleavge domains respectively but does not lose substrate
recognition for the guide RNA and joint substrate recognition for
the target chromosome site. When dCas9 enzyme binds its sgRNA
substrate, it physically reconfigures and transforms into a
specific chromosomal binding protein to block mRNA transcription by
the RNA polymerase.
Example 10--In Vitro Analysis of Factor VII Protein Levels
[0247] 3D hepatocyte cell cultures are prepared by plating about
25,000 murine FL3B murine hepatocyte cells on to ECM-coated spun
polymer scaffolds (Corning, UltraMax) in chamber slides. It is
worth noting that cells completely reorganize their internal
geometry in 3D systems, supporting the use of 3D cultures in CRISPR
and intracellular trafficking studies.
[0248] Results for Formulas Ha and Hb are illustrated in FIG. 4.
Neither formulation showed significant mutation activity while
plasmid Cas9 and plasmid sgRNA delivered from separate ASOR
nanoencapsulates did show mutational activity by amplicon deep
sequencing. In a separate 3D experiment, Formula Hc, in contrast to
Formula Ha, induces mutation of the F7 sequence at the level of the
chromosome along with inhibition of F7 protein expression.
[0249] Neither formulation shows significant mutation activity
while plasmid Cas9 and sgRNA delivered from separate ASOR
nanoencapsulates do show mutational activity by amplicon deep
sequencing.
[0250] We conclude that protein co-encapsulation with a
polynucleotide substrate is effective for enhancing incorporation
into crystalline supramolecular complexes for retention of enzyme
bioactivity in non-inflammatory systemic delivery of bacterial
dCas9 protein.
Example 11--Protein Co-Encapsulation of Protein Component with
Polynucleotide Component Enriches Cells with Active RISC for
Enhanced Target Gene Modulation
[0251] Superior efficacy of the instant nanoparticles (Risc RNAi
F7; Formula B) is demonstrated in comparison to guide strand alone
(Formula E) nanoparticles in 3D cultured FL83B hepatocytes
(ATCC).
[0252] 3D hepatocyte cell cultures are prepared by plating about
25,000 murine FL83B murine hepatocyte cells on to ECM-coated spun
polymer scaffolds (Corning, UltraMax) in chamber slides. Formula B
and Formula E nanoparticles are added at various concentrations to
the cultures and harvested for microscopy after about 3 days.
[0253] Dose response for Factor VII protein inhibition is assayed
by confocal fluorescence microscopy for Factor VII immunosignal
(GeneTex).
[0254] Formula B administration demonstrates full inhibition of
Factor VII protein at about 1 pmol (about 4 nM) continuing through
about 2.0 nM with recovery beginning around about 0.32 nM
demonstrating about a 3 log response range. In contrast, Formula E
bearing the guide strand only shows full inhibition by microscopy
at about 5 nmol (about 20 .mu.M) recovering by about 1.25 nmol
(about 5 .mu.M) for about a 0.4 log response range and approximate
increase in activity of about 3.5 logs (about 2 nM vs. 12.5 .mu.M)
for encapsulated RISC complex vs encapsulated guide RNA alone.
Results are illustrated in FIG. 3.
Example 12--In Vitro Analysis of Factor VII Protein Levels
[0255] 3D hepatocyte cell cultures are prepared by plating about
25,000 murine FL3B murine hepatocyte cells on to ECM-coated spun
polymer scaffolds (Corning, UltraMax) in chamber slides. It is
worth noting that cells completely reorganize their internal
geometry in 3D systems, supporting the use of 3D cultures in CRISPR
and intracellular trafficking studies.
[0256] Formula Ha (ASOR Cas9 crF7) and Formula I (ASOR dCas9 crF7)
are analyzed for functional activity by treating 3D mouse
hepatocyte cultures at two dose levels (about 2 nM and 0.4-0.6 nM)
and then are assayed for Factor VII protein levels after about 3
days by confocal immunofluorescence microscopy. We observed a
similar pattern for both formulations with significant inhibition
for both formulations at about 2 nM and partial inhibition at about
0.4-0.6 nM of Cas9 protein. Results are illustrated in FIG. 5.
[0257] Neither formulation shows significant mutation activity
while plasmid Cas9 and sgRNA deliver from separate ASOR
nanoencapsulates show mutational activity by amplicon deep
sequencing.
[0258] We conclude that protein co-encapsulation with a
polynucleotide substrate is effective for enhancing incorporation
into crystalline supramolecular complexes for retention of enzyme
bioactivity in non-inflammatory systemic delivery of bacterial
dCas9 protein.
Example 13--In Vitro Analysis of Factor VII Protein Levels
[0259] 00209 3D hepatocyte cell cultures are prepared by plating
about 25,000 murine FL3B murine hepatocyte cells on to ECM-coated
spun polymer scaffolds (Corning, UltraMax) in chamber slides. It is
worth noting that cells completely reorganize their internal
geometry in 3D systems, supporting the use of 3D cultures in CRISPR
and intracellular trafficking studies.
[0260] 00210 Results for Formulas Ha and I are illustrated in FIG.
4. Neither formulation shows significant mutation activity while
plasmid Cas9 and plasmid sgRNA delivered from separate ASOR
nanoencapsulates show mutational activity by amplicon deep
sequencing. In a separate 3D experiment, Formula He and Formula Hd,
in contrast to Formula Ha, induce mutation of the F7 sequence at
the level of the chromosome along with inhibition of F7 protein
expression.
[0261] 00211 Neither formulation shows significant mutation
activity while plasmid Cas9 and sgRNA delivered from separate ASOR
nanoencapsulates show mutational activity by amplicon deep
sequencing.
[0262] 00212 We conclude that protein co-encapsulation with a
polynucleotide substrate is effective for enhancing incorporation
into crystalline supramolecular complexes for retention of enzyme
bioactivity in non-inflammatory systemic delivery of bacterial
dCas9 protein.
Example 14--Spectral Characterization of Instant Crystalline
Nanoparticles
[0263] To further elucidate a basis for the surprising
effectiveness for effective systemic delivery of nanoparticles with
co-encapsulated protein and polynucleotide components, we analyze
thermal spectra by differential scanning calorimetry (DSC) from
dried and crushed powders and FTIR spectra in hydrated and
partially dehydrated powders prepared either by drying under vacuum
with mild heat or buffer exchange with 10 mM ammonium acetate
(NH4Oac) followed by similar drying optionally with sample freezing
at -80.degree. C. preceding drying. For FTIR spectra, final
nanoparticle preparations are compared to ligand-coated micelle
intermediates prepared up until cocrystallization in a salt mixture
of primarily Cs-modified lithium.
TABLE-US-00005 TABLE 5 Spectral characteristics of nanoparticles
and uncrystallized intermediates. FTIR spectrum, FTIR spectrum, DSC
Transitions.sup.1, hydrated.sup.3 dehydrated.sup.3 Particle/
.degree. C., gt midpoints, et Delta Cp (wavenumber, (wavenumber,
Cargo Formula nadirs (J/(g * .degree. C.)) cm -1) cm -1) Milli-Q
water ND ND FIG. 6 ND Hepes + 10% et's 301.1, 315.5, 335.3 10.828,
1.493, 5.175 FIG. 7 FIG. 8 Lactitol diluent Cs-modified ND ND FIG.
9 ND lithium ASOR ligand ND ND FIG. 10 ND DMSO ND ND FIG. 11 ND
NH4Oac ND ND FIG. 12 ND ASOR Erythritol G et's, 44.8, 145.6, 0.744,
0.422, FIG. 13 FIG. 14 nanoparticle 159.8, 183.0, 195.7, 1.277,
0.173, 288.7, 312.8 0.168, 0.279, 0.686 ASOR Erythritol G ND ND ND
FIG. 15 micelle.sup.2 ASOR RISC B et's, 40.0, 136.1, 1.008, 1.185,
ND FIG. 16 RNAi F7 150.2, 157.0, 167.6, 0.972, 0.190, nanoparticle7
285.4, 289.0, 314.2 0.746, 0.029, 0.652, 5.185 ASOR RISC C ND ND ND
FIG. 17 RNAi F7 micelle.sup.2 ASOR RISC Da ND ND ND FIG. 18 2RF7
micelle.sup.2 ASOR RNAi F7 E et's, 41.9, 307.8, 2.383, 6.672, ND
FIG. 19 nanoparticle 329.4 1.400 ASOR RNAi F7 E ND ND ND FIG. 20
micelle.sup.2 ASOR Cas9 F7 Ha et's, 46.5, 136.0, 3.381, 0.447, FIG.
21 FIG. 22 nanoparticle 243.3, 305.8 0.899, 1.183 ASOR Cas9 F7 Ha
ND ND ND FIG. 23 micelle.sup.2 ASOR dCas9 F7 I et's, 48.2, 312.6,
0.680, 2.697, ND FIG. 24 nanoparticle 329.8 0.494 ASOR dCas9 F7 I
ND ND ND FIG. 25 micelle.sup.2 *fn1 references paragraph [00249];
fn 2 references paragraph [00250]; and fn 3 references paragraph
[00251]
[0264] Referring to Table 5, Thermal transitions and transition
energies are identified from thermograms generated by differential
scanning calorimetry (DSC) on a STA 449 F3-Jupiter thermal
analyzer. Suspensions are dried to produce powder for analysis, and
about 1-2 mg were scanned at about 20.degree. C./min from room
temperature to about 400.degree. C. in uncrimped aluminum pans.
Abbreviations used here are: gt, glass transition; et, endotherm;
vs. very small.
[0265] Ligand-coated micelles are micelles formulated according to
respective formulation but do not undergo incubation in salt
receiving solution.
[0266] The FTIR spectra are recorded from about 400 to 4000
cm.sup.-1 using an Agilent Cary 670 spectrophotometer, equipped
with a Pile MIRacle ATR accessory a mid-infrared source as the
excitation source. Liquid samples are .mu.laced directly on the
crystal, and the high pressure clamp is used for solid samples.
Prior to analysis, the entire instrument is purged with dry,
CO.sub.2-free air for at least 30 minutes and until background
scans indicated no or negligible change in atmospheric moisture or
CO.sub.2 levels. The ATR crystal and the high pressure clamp are
both cleaned with mQ water (18.OMEGA.) between measurements and
dried with a cotton cloth until the processed spectrum indicated
that no residual sample remained on the crystal. The spectra were
acquired in about 32 scans at a resolution of about 4 cm.sup.-1 at
ambient conditions. Abbreviations, v, very; s, small; md, moderate;
str, strong; brd, broad.
[0267] In these analyses, DSC shows only endotherms in final
lithium-treated particles supporting crystallinity for ASOR
Cs-modified lithium polymorphs.
[0268] Compound differences upon supramolecular assemblies bearing
diverse cargos are further investigated by FTIR spectroscopy.
[0269] FTIR of hydrated nanoparticle powders identify a peak
attributable to the ASOR ligand at about 1035 (about 1010-1050)
cm.sup.-1. Upon partial dehydration, a peak attributable to the
Cs-modified lithium is readily apparent at about 620-650 cm.sup.-1
while at the same time the ASOR ligand peak was greatly diminished.
This broad doublet at about 655, 620 cm.sup.-1 is not visible in
powders from paired ligand-coated intermediates before lithium
exposure. Generally, the spectral pattern of ASOR-liganded
crystalline capsules is similar despite diverse cargos. However,
spectra from ASOR ligand-coated intermediates does show differences
between diverse cargos that are not apparent in spectra from final
product. An approximately about 875 cm.sup.-1 peak attributable to
the TM-diol surfactant along with the peak attributable to the ASOR
ligand is dampened considerably coincident with the processes in
effect in the crystallization step. Consistent with hypothesis of
considerable surface rearrangement upon lithium exposure, a peak at
about 1690 (about 1685-1715) cm.sup.-1 attributable to carbonyl
(C.dbd.O) stretching is also greatly decreased. Without wishing to
be bound by theory, it is believed that the lithium ion is able to
coordinate a water-stable bond between carboxyl anions,
particularly carboxylate anions, to create a stabilizing network of
bonds and thus innumerable unique supramolecular structures.
[0270] Significant changes in thermal transitions and IR spectra
are important indicators of polymorphic change in crystalline
compounds, pharmaceutical compounds, and nanoscale supramolecular
assemblies.
Example 15--Non-Crystallized Particles for Short-Release
Applications
[0271] Formula B (ASOR RISC RNAiF7) and Formula E (ASOR RNAi F7),
are prepared as ASOR-coated micelles and ligand-coated micelles,
terminating synthesis just before addition to the lithium receiving
bath for crystallization and hardening (referred to herein as
"non-crystallized nanoparticles"). 3D cultures of FL83B are treated
at six dose levels of about 1 pmol, about 0.5 pmol, about 0.1 pmol,
about 0.05 pmol and about 0.01 pmol for Formula B and about 5 nmol,
about 2.5, about 1.25, about 0.75, about 0.5 and about 0.1 nmol for
Formula E to show increased activity at lower doses than observed
with fully crystallized particles. This example shows as one
embodiment the instant non-crystallized nanoparticles are useful to
deliver protein-substrate combinations in applications where a drug
is desirably released faster than it is with a crystallized
particle. In one embodiment, the applications for the instant
non-crystallized nanoparticles are used over the short time course
of a cell culture experiment.
[0272] Significant changes in thermal transitions and IR spectra
are important indicators of polymorphic change in crystalline
compounds, pharmaceutical compounds, and nanoscale supramolecular
assemblies.
Example 16--Effective Formulation and Delivery of Protein Enzyme
Therapies Comprising Protein-Protein Combinations
[0273] VIII. Formula J (Tenfibgen Chymotrypsin-trypsin) is prepared
as ASOR-coated micelles and ligand-coated micelles, terminating
synthesis just before addition to the lithium receiving bath for
crystallization and hardening. Formula K is prepared as a
suspension of Tenfibgen crystalline nanoparticles containing
erythritol for a comparator nanoparticle. Organ cultures of dermal
explants representing normal and wound-activated conditions, eg.
post-radiation, are treated at about 3 dose levels of
Chymotrpsin-Trypsin; eg. about 1 ug, about 5 ug and about 10 ug
among others by either intradermal injection of naked enzyme or by
enzyme delivered topically as a non-ionic micelle, ligand-coated
micelle or crystalline ligand-coated nanoparticle. Erythritol
capsules are applied similarly as a comparator. Organ cultures are
held either overnight for localization of Chymotrypsin (or Syrian
Hamster IgG incorporated as capsule label) in tissue by microscopy
or for about three days for examination of protease activity by
immunohistochemistry and Massons Trichrome staining. More uniform
action of the enzyme upon the tissue is observed to be facilitated
by inventive formulations relative to injection of naked enzyme.
Incorporation by REFERENCE
[0274] Any and all patents, patent applications, patent application
publications and PCT publications, publications, including any and
all references, articles, website articles and abstracts,
referenced or identified herein, included their entire contents,
are incorporated herein by reference in their entireties as if each
has been fully set forth herein.
IX. EQUIVALENTS
[0275] Various modifications and variations of the described
methods, compositions, processes and systems of the invention will
be apparent to those of skill in the art without departing from the
scope and spirit of the invention. Although the invention has been
described in connection with specific exemplary preferred
embodiments, it should be understood that the invention as claimed
should not be unduly limited to such specific exemplary
embodiments. Indeed, various modifications of the described modes
for carrying out and/or practicing the invention are intended to be
within the scope of the claims.
* * * * *