# US 12,214,055 B2 · Systems and methods for treating hyper-igm syndrome

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- Number: US 12,214,055 B2
- Document ID: US12214055B2
- Status: Granted
- Kind: B2
- Application number: 16/758,752
- Filed: 2018-10-24
- Published: 2020-10-29
- Granted: 2025-02-04
- Earliest claimed: 2017-10-24
- Applicants: Editas Medicine, Inc.; Fondazione Telethon Ets; Ospedale San Raffaele
- Inventors: Cecillia Cotta-Ramusino; Carrie M. Margulies; Luigi Naldini; Pietro Genovese
- Web page: https://patentgrep.com/patent/US12214055B2

## CPC

- A61K 48/005: {characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered}
- A61K 40/10: characterised by the cell type used
- A61K 40/11: T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
- A61K 40/421: {Immunoglobulin superfamily}
- A61P 37/02: Immunomodulators
- C07K 14/475: Growth factors; Growth regulators
- C07K 14/70575: {NGF/TNF-superfamily, e.g. CD70, CD95L, CD153, CD154 (NGF C07K14/48, TNF C07K14/525)}
- C07K 14/70578: {NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95 (NGF-receptor C07K14/71, TNF-receptor C07K14/7151)}
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein}
- C12N 15/102: {Mutagenizing nucleic acids}
- C12N 15/113: Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)}
- C12N 15/86: Viral vectors
- C12N 15/907: {in mammalian cells}
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2750/14143: viral genome or elements thereof as genetic vector
- C12N 2830/50: regulating RNA stability, not being an intron, e.g. poly A signal
- C12N 2840/203: having an IRES
- 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
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Disclosed herein are genome editing systems and related methods which allow for treatment of Hyper IgM Syndrome, a group of disorders characterized by defective CD40 signaling. The compositions and methods described herein rely on the use of donor templates comprising a CD40L exons to restore proper CD40 signaling and B cell class switch recombination.

## Claims

### Claim 1

An isolated oligonucleotide donor template which comprises, from 5′ to 3′, A1-N-UTR-pA-A2, wherein
A1 is a homology arm that is substantially identical to a first homology arm of a target CD40L nucleic acid;
N is a cargo comprising one or more of exons 2-5 of a CD40L gene;
UTR is a CD40L 3′ untranslated region (UTR);
pA is a polyA tail; and
A2 is a homology arm that is substantially identical to a second homology arm of the target CD40L nucleic acid,
and wherein the cargo does not comprise exon 1 of CD40L.

### Claim 2 (depends on 1)

The isolated oligonucleotide donor template of claim 1, wherein the isolated oligonucleotide donor template comprises:
(a) from 5′ to 3′, A1-S1-N-UTR-pA-S2-A2,
wherein S1 is a first stuffer, wherein S2 is a second stuffer;
(b) from 5′ to 3′, A1-N-UTR-pA-R-A2, wherein R is a reporter; or
(c) from 5′ to 3′, A1-N-IRES-Sel/R-UTR-pA-A2, wherein IRES is an internal ribosome entry site, and wherein Sel/R is a selector gene or a reporter.

### Claim 3 (depends on 2)

The isolated oligonucleotide donor template of claim 2, wherein the isolated oligonucleotide donor template comprises, from 5′ to 3′, A1-S1-N-UTR-pA-R-S2-A2, wherein R is a reporter.

### Claim 4 (depends on 1)

The isolated oligonucleotide donor template of claim 1, wherein the cargo comprises: exon 2 of the CD40L gene; exon 3 of the CD40L gene; exon 4 of the CD40L gene; exon 5 of the CD40L gene; exons 2-5 of the CD40L gene; exons 2-4 of the CD40L gene; exons 2 and 3 of the CD40L gene; exons 3-5 of the CD40L gene; exons 3 and 4 of the CD40L gene; or exons 4 and 5 of the CD40L gene; optionally wherein the cargo comprises a codon optimized CD40L sequence.

### Claim 5 (depends on 2)

The isolated oligonucleotide donor template of claim 2, wherein the reporter is a green fluorescence protein (GFP); a yellow fluorescence protein (YFB); DS-Red; luciferase; a low affinity nerve growth factor receptor (NGFR); truncated CD19; truncated epidermal growth factor receptor (EGFR); neomycin resistance protein; or puromycin resistance protein.

### Claim 6 (depends on 1)

A composition comprising the isolated oligonucleotide donor template of claim 1 and, optionally, a pharmaceutically acceptable carrier.

### Claim 7 (depends on 1)

A vector comprising the isolated oligonucleotide donor template of claim 1.

### Claim 8 (depends on 7)

The vector of claim 7, wherein the vector is an AAV vector, a lentivirus vector, a naked DNA vector, or a lipid nanoparticle.

### Claim 9 (depends on 1)

A genome editing system comprising the isolated oligonucleotide donor template of claim 1.

### Claim 10 (depends on 9)

The genome editing system of claim 9, further comprising a RNA-guided nuclease and at least one guide RNA (gRNA) molecule.

### Claim 11 (depends on 9)

The genome editing system of claim 9, further comprising an isolated gRNA molecule, comprising any one of SEQ ID NOs: 18-25.

### Claim 12 (depends on 9)

A method of altering a cell comprising contacting the cell with the genome editing system of claim 9, thereby altering the cell.

### Claim 13 (depends on 9)

A kit comprising the genome editing system of claim 9.

### Claim 14

A method of altering a cell, comprising the steps of:
forming, in a target CD40L nucleic acid of the cell, at least one single- or double-strand break at a cleavage site, wherein the target CD40L nucleic acid comprises:
a first homology arm 5′ to the cleavage site and a second homology arm 3′ to the cleavage site, and
recombining an exogenous oligonucleotide donor template with the target CD40L nucleic acid by homologous recombination to produce an altered CD40L nucleic acid, wherein the exogenous oligonucleotide donor template comprises a first donor homology arm that is substantially identical to the first homology arm, a cargo comprising one or more of exons 2-5 of a CD40L gene, a 3′ CD40L untranslated region (UTR), a polyA tail, and a second donor homology arm that is substantially identical to the second homology arm, and wherein the cargo does not comprise exon 1 of CD40L, thereby altering the cell.

### Claim 15 (depends on 14)

The method of claim 14, wherein the exogenous oligonucleotide donor template comprises:
(a) the first donor homology arm that is substantially identical to the first homology arm, a first stuffer, the cargo comprising one or more of exons 2-5 of the CD40L gene, the 3′ CD40L untranslated region (UTR), the polyA tail, a second stuffer, and the second donor homology arm that is substantially identical to the second homology arm; or
(b) the first donor homology arm that is substantially identical to the first homology arm, the cargo comprising one or more of exons 2-5 of the CD40L gene, the 3′ CD40L untranslated region (UTR), the polyA tail, a reporter, and the second donor homology arm that is substantially identical to the second homology arm.

### Claim 16 (depends on 15)

The method of claim 15, wherein the exogenous oligonucleotide donor template comprises the first donor homology arm that is substantially identical to the first homology arm, the first stuffer, the cargo comprising one or more of exons 2-5 of the CD40L gene, the 3′ CD40L untranslated region (UTR), the polyA tail, a reporter, the second stuffer, and the second donor homology arm that is substantially identical to the second homology arm.

### Claim 17 (depends on 14)

The method of claim 14, wherein the cargo comprises exon 2 of the CD40L gene; exon 3 of the CD40L gene; exon 4 of the CD40L gene; exon 5 of the CD40L gene; exons 2-5 of the CD40L gene; exons 2-4 of the CD40L gene; exons 2 and 3 of the CD40L gene; exons 3-5 of the CD40L gene; exons 3 and 4 of the CD40L gene; or exons 4 and 5 of the CD40L gene.

### Claim 18 (depends on 14)

The method of claim 14, wherein the step of forming the at least one single- or double-strand break comprises contacting the cell with an RNA-guided nuclease.

### Claim 19 (depends on 14)

The method of claim 14, further comprising introducing the cell into a subject after the recombining step or after the cleavage event is repaired by the at least one DNA repair pathway.

### Claim 20 (depends on 19)

The method of claim 19, wherein:
(a) class switching is restored in the subject;
(b) levels of IgM are decreased in a subject; and/or
(c) levels of IgG are increased in a subject.