# US 2025/0375508 A1 · Methods and compositions for cas immune tolerance induction to support crispr-cas in vivo gene editing

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- Number: US 2025/0375508 A1
- Document ID: US20250375508A1
- Status: Published application
- Kind: A1
- Application number: 18/877,079
- Filed: 2023-06-28
- Published: 2025-12-11
- Earliest claimed: 2022-06-29
- Assignees: University of Pittsburgh—Of the Commonwealth System of Higher Education; The Regents of the University of California
- Applicants: University of Pittsburgh - Of the Commonwealth System of Higher Education; The Regents of the University of California
- Inventors: Stephen C. Balmert; Louis D. Falo; Steven R. Little; David H. Raulet; Gregory M. Barton; Emrullah Korkmaz
- Web page: https://patentgrep.com/patent/US20250375508A1

## CPC

- A61K 39/001134: {Transforming growth factor [TGF]}
- A61K 9/0019: {Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner (non-active ingredients are additionally classified in A61K47/00)}
- A61K 9/0021: {Intradermal administration, e.g. through microneedle arrays or needleless injectors}
- A61K 9/5036: {Polysaccharides, e.g. gums, alginate; Cyclodextrin}
- A61K 31/203: Retinoic acids {; Salts thereof}
- A61K 31/436: the heterocyclic ring system containing a six-membered ring having oxygen as a ring hetero atom, e.g. rapamycin
- A61K 31/593: 9,10-Secocholestane derivatives, e.g. cholecalciferol, i.e. vitamin D3
- A61K 35/17: Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes (when activated by a specific antigen A61K39/00)
- A61K 38/1841: {Transforming growth factor [TGF]}
- A61K 38/2013: {IL-2}
- A61K 39/001: {Preparations to induce tolerance to non-self, e.g. prior to transplantation}
- A61K 39/00114: {Interleukins [IL]}
- A61K 39/001154: {Enzymes}
- A61K 39/39: characterised by the immunostimulating additives, e.g. chemical adjuvants
- A61K 45/06: Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
- A61K 48/0025: {wherein the non-active part clearly interacts with the delivered nucleic acid}
- A61K 48/005: {characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered}
- A61P 37/06: Immunosuppressants, e.g. drugs for graft rejection
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein}
- A61K 2039/54: {characterised by the route of administration}
- A61K 2039/55511: {Organic adjuvants}
- A61K 2039/55522: {Cytokines; Lymphokines; Interferons}
- A61K 2039/55533: {IL-2}
- A61K 2039/55555: {Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers}
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Tolerogenic compositions are disclosed that are of use for inducing a tolerogenic immune response to a CRISPR-Cas effector polypeptide in a subject. In some aspects, the tolerogenic composition includes: a) one or more microparticles; b) one or more regulatory T cell (Treg) stimulating agents encapsulated within each microparticle; and c) a CRISPR-Cas effector polypeptide or immunogenic fragment thereof, or a fusion polypeptide comprising a CRISPR-Cas effector polypeptide or immunogenic fragment thereof. In other aspects, the tolerogenic composition includes a) a dissolvable microneedle array; b) one or more agents that promote differentiation of tolerogenic DCs in the dissolvable microneedle array; and c) a CRISPR-Cas effector polypeptide or immunogenic fragment thereof, or a fusion polypeptide comprising a CRISPR-Cas effector polypeptide or immunogenic fragment thereof.

## Claims

### Claim 1

A tolerogenic composition comprising:
a1) one or more microparticles;
b1) one or more regulatory T cell (Treg) stimulating agents encapsulated within the one or more microparticles; and
c1) a CRISPR-Cas effector polypeptide or immunogenic fragment thereof, or a fusion polypeptide comprising a CRISPR-Cas effector polypeptide or an immunogenic fragment thereof; or
a2) a dissolvable microneedle array;
b2) one or more agents that promote differentiation of tolerogenic DCs in the dissolvable microneedle array; and
c2) a CRISPR-Cas effector polypeptide or immunogenic fragment thereof, or a fusion polypeptide comprising a CRISPR-Cas effector polypeptide or an immunogenic fragment thereof.

### Claim 2 (depends on 1)

The tolerogenic composition of claim 1, comprising:
a1) the one or more microparticles;
b1) the one or more regulatory T cell (Treg) stimulating agents encapsulated within the one or more microparticles; and
c1) the CRISPR-Cas effector polypeptide or immunogenic fragment thereof, or a fusion polypeptide comprising a CRISPR-Cas effector polypeptide.

### Claim 3 (depends on 2)

The tolerogenic composition of claim 2, wherein the one or more Treg stimulating agents comprise CCL22, retinoic acid, or vasoactive intestinal peptide.

### Claim 4 (depends on 2)

The tolerogenic composition of claim 2, wherein the one more Treg stimulating agents comprise IL-2, TGF-β, rapamycin, a rapamycin derivative, or a CCR8 ligand.

### Claim 5 (depends on 2)

The tolerogenic composition of claim 2, wherein the one or more Treg stimulating agents comprise TGFβ, IL2, and rapamycin.

### Claim 6 (depends on 2)

The tolerogenic composition of claim 2, wherein the tolerogenic composition comprises i) a microparticle comprising TGFβ; ii) a microparticle comprising IL-2, and iii) a microparticle comprising rapamycin.

### Claim 7 (depends on 2)

The tolerogenic composition of claim 2, wherein the one or more microparticles comprises at least one polymer.

### Claim 8 (depends on 7)

The tolerogenic composition of claim 7, wherein the at least one polymer comprises polyethylene glycol (PEG), a poly(amino acid), polylactate, polylactic acid, polyglutamic acid, polyglycolic acid (PGA), polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), polyvinyl acetate (PVA), poly(ethylene glycol-b-(DL-lactic acid-co-glycolic acid)-b-ethylene glycol) (PEG-PLGA-PEG), Poly (ethylene glycol)-b-poly(D,L-lactide-co-glycolide) (PEG-b-PLGA), polycaprolactone-PEG (PCL-PEG), poly(vinylidene fluoride)-PEG (PVDF-PEG), poly(lactic acid-co-PEG) (PLA-PEG), poly(methyl methacrylate)-PEG (PMMA-PEG), and combinations thereof.

### Claim 9 (depends on 2)

The tolerogenic composition of claim 2, wherein the one or more microparticles comprise alginate.

### Claim 10 (depends on 1)

The tolerogenic composition of claim 1, wherein one or more microparticles are formulated for sustained release of the one or more regulatory T cell (Treg) stimulating agents.

### Claim 11 (depends on 1)

The tolerogenic composition of claim 1, wherein the CRISPR-Cas effector polypeptide is a type II CRISPR-Cas effector polypeptide, a type V CRISPR-Cas effector polypeptide, or a type VI CRISPR-Cas effector polypeptide.

### Claim 12 (depends on 1)

The tolerogenic composition of claim 1, wherein the CRISPR-Cas effector fusion polypeptide comprises: i) a CRISPR-Cas effector polypeptide; and ii) one or more heterologous effector polypeptides.

### Claim 13 (depends on 12)

The tolerogenic composition of claim 12, wherein at least one of the one or more heterologous effector polypeptides is a single stranded nuclease, a double strand nuclease, a helicase, a methylase, a demethylase, an acetylase, a deacetylase, a deaminase, an integrase, a recombinase, a base editor, or a prime editor.

### Claim 14 (depends on 2)

A method of inducing tolerance to a CRISPR-Cas effector polypeptide in a mammalian subject, the method comprising administering to the subject an effective amount of a tolerogenic composition of claim 2, thereby inducing tolerance to the CRISPR-Cas effector polypeptide.

### Claim 15 (depends on 14)

The method of claim 14, wherein the composition is administered intradermally, subdermally, subcutaneously, or intramuscularly.

### Claim 16 (depends on 1)

The tolerogenic composition of claim 1, wherein the composition comprises:
a2) the dissolvable microneedle array;
b2) the one or more agents that promote differentiation of tolerogenic DCs in the dissolvable microneedle array;
c2) a CRISPR-Cas effector polypeptide or immunogenic fragment thereof, or a fusion polypeptide comprising a CRISPR-Cas effector polypeptide or the immunogenic fragment.

### Claim 17 (depends on 16)

The tolerogenic composition of claim 16, wherein the dissolvable microneedle array comprises: i) a substrate comprising a biocompatible material that forms base portion; and ii) a plurality of microneedles extending from the base portion.

### Claim 18 (depends on 17)

The tolerogenic composition of claim 17, wherein the biocompatible material comprises carboxymethylcellulose, polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), hyaluronic acid (HA), or gelatin.

### Claim 19 (depends on 18)

The tolerogenic composition of claim 18, wherein the biocompatible material comprises carboxymethylcellulose.

### Claim 20 (depends on 16)

The tolerogenic composition of claim 16, wherein the one or more agents that promote differentiation of tolerogenic DCs comprise vitamin D3 or a vitamin D3 analog.

### Claim 21 (depends on 17)

A method of inducing tolerance to a CRISPR-Cas effector polypeptide in a mammalian subject, comprising:
administering to the subject an effective amount of a tolerogenic composition of claim 17,
thereby inducing tolerance to the CRISPR-Cas effector polypeptide.

### Claim 22 (depends on 21)

The method of claim 21, wherein the composition is administered intradermally or subdermally.

### Claim 23 (depends on 14)

The method of claim 14, wherein the tolerogenic composition is administered repeatedly to the subject.

### Claim 24 (depends on 23)

The method of claim 23, wherein the tolerogenic composition is administered in a prime-boost strategy to the subject.

### Claim 25 (depends on 14)

The method of claim 14, comprising:
administering to the subject a gene-editing composition comprising the CRISPR-Cas effector polypeptide following administering the effective amount of the tolerogenic composition, wherein the tolerogenic composition induces tolerance to the CRISPR-Cas effector polypeptide present in the gene-editing composition.

### Claim 26 (depends on 25)

The method of claim 25, wherein the gene-editing composition is administered to the subject within about 6 months of administration of the tolerogenic composition.

### Claim 27 (depends on 14)

The method of claim 14, wherein the tolerogenic composition is administered to a subject that previously was administered a gene editing composition.

### Claim 28 (depends on 14)

The method of claim 14, further comprising:
performing a gene editing procedure on the subject.

### Claim 29 (depends on 14)

The method of claim 14, wherein the subject is a human.

### Claim 30 (depends on 1)

The method of claim 1, wherein the subject is a non-human mammal.