WO2022216977A1 - Rapid design, build, test, and learn technologies for identifying and using non-viral carriers - Google Patents

Rapid design, build, test, and learn technologies for identifying and using non-viral carriers Download PDF

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WO2022216977A1
WO2022216977A1 PCT/US2022/023902 US2022023902W WO2022216977A1 WO 2022216977 A1 WO2022216977 A1 WO 2022216977A1 US 2022023902 W US2022023902 W US 2022023902W WO 2022216977 A1 WO2022216977 A1 WO 2022216977A1
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nucleic acid
composition
acid construct
base pairs
methacrylate
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French (fr)
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Anthony D. DUONG
Danielle J. HUK
Cherry GUPTA
Kenneth R. SIMS JR.
Michael S. Koeris
Zachary R. SHANK
Ashlee J. COLBERT
Andrea D. MCCUE
Emma K. SCHMITZ
Caleb T. HILLRICH
Shannon D. MILLER
Joanna L. HOY
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Battelle Memorial Institute Inc
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Priority to EP22785473.4A priority patent/EP4320233A4/en
Priority to JP2023562184A priority patent/JP2024516108A/ja
Priority to IL326440A priority patent/IL326440A/en
Priority to CA3216359A priority patent/CA3216359A1/en
Priority to AU2022253899A priority patent/AU2022253899A1/en
Publication of WO2022216977A1 publication Critical patent/WO2022216977A1/en
Priority to IL307454A priority patent/IL307454B1/en
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    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1065—Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
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    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6804—Nucleic acid analysis using immunogens
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
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    • C12Q2525/00—Reactions involving modified oligonucleotides, nucleic acids, or nucleotides
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    • C12Q2525/00—Reactions involving modified oligonucleotides, nucleic acids, or nucleotides
    • C12Q2525/30—Oligonucleotides characterised by their secondary structure
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites

Definitions

  • CRISPR/Cas9 Therapeutics based on the CRISPR/Cas9 system have an exceptional potential to treat a number of genetic diseases due to the capability of this system for precise and programmable gene editing.
  • Gene editing and repair using the CRISPR/Cas9 system has two main mechanisms, including non-homologous end joining (NHEJ) which repairs the site of cut by inducing random indel mutation, and homology-directed repair (HDR), which repairs the cut site based on a pre-existing template. Because a pre-designed template can be used for HDR- directed repair, therapies based on this mechanism can be tailored to cure a large number of different genetic diseases.
  • NHEJ non-homologous end joining
  • HDR-directed repair HDR
  • nucleic acid construct further comprises from about 6 to about 12 random bases at the 3’ end of the polynucleotide barcode.
  • a a first terminus comprising a first capping unit derived from a first chain transfer agent in a RAFT copolymerization process
  • a method of in vivo screening for a desired polymer nanoparticle for use as a delivery vehicle comprising (a) preparing a library comprising two or more types of polymer nanoparticles, wherein each polymer nanoparticle is associated with a nucleic acid construct comprising a different polynucleotide barcode, (b) administering the library to an animal, (c) removing cells or tissues from the animal, (d) isolating the nucleic acid constructs from the cells or the tissues of the animal, (e) detecting the nucleic acid constructs in the cells or the tissues of the animal, and (f) identifying the desired polymer nanoparticle for use as a delivery vehicle.
  • nucleic acid construct is separated from material in the cells or tissues by binding the nucleic acid construct with a molecule with a binding affinity to the nucleic acid construct greater than the binding affinity to the cell or tissue material.
  • Fig. l is a schematic diagram showing a simplified flow diagram illustrating a DBTL cycle for non-viral gene delivery development based on automated synthesis, high throughput testing, and machine learning design.
  • Figs. 5(a)-5(c) are a schematic drawing of nucleic acid construct labeling reaction methods using electrostatic loading reaction (Fig. 5(a)), avidin-streptavidin conjugation (Fig. 5(b)), and direct amidification (Fig. 5(c)).
  • Fig. 6 is an e-gel showing amplification of nucleic acid constructs electrostatically bound to polymer nanoparticles.
  • the presence of the double band in the samples with nucleic acid construct confirms that the barcodes were attached to the PNP.
  • the absence of the double band in the not test control (NTC) validates the positive result.
  • Fig. 13 is an electrophoresis gel showing a band corresponding to barcode (BC) amplicons produced from PCR performed on samples of PNPs with barcodes attached at various molar ratios of PNP to BC (i.e. moles of polymer divided by moles of barcode).
  • BC barcode
  • the presence of the band for all barcoded PNP samples confirms that the barcode can be detected via PCR on PNPs labeled with barcodes at ratios of anywhere from 20: 1 to 10,000: 1 (moles PNP:moles BC).
  • tissue or cell samples include brain tissue or cells, muscle tissue or cells, skin tissue or cells, heart tissue or cells, kidney tissue or cells, stomach tissue or cells, liver tissue or cells, urinary tract tissue or cells, gastrointestinal tract tissue or cells, head or neck tissue or cells, lung tissue or cells, reproductive tract tissue or cells, pancreatic tissue or cells, or any other tissue or cell type from an animal.
  • the nucleic acid constructs are removed from cells or tissues by treating with a mixture of an organic phase (e.g., phenol chloroform) and an aqueous phase (e.g., water).
  • the organic phase e.g., phenol chloroform
  • the organic phase e.g., phenol chloroform
  • the organic phase can be evaporated and the nucleic acid constructs can be suspended in water and diluted to appropriate concentrations for PCR and/or sequencing.
  • the isolated nucleic acid constructs are suspended in either water or a buffer after sufficient washing.
  • the nucleic acid constructs can be sequenced, to detect the polynucleotide barcodes using any suitable sequencing method including Next Generation Sequencing (e.g., using Illumina, ThermoFisher, or PacBio or Oxford Nanopore Technologies sequencing platforms), sequencing by synthesis, pyrosequencing, nanopore sequencing, or modifications or combinations thereof can be used.
  • the sequencing can be amplicon sequencing.
  • the sequencing can be whole genome sequencing.
  • the sequencing can be exome/targeted hybridization sequencing. Methods for sequencing nucleic acids are also well-known in the art and are described in Sambrook et ah, “Molecular Cloning: A Laboratory Manual”, Cold Spring Harbor Laboratory Press, incorporated herein by reference.
  • the nucleic acid construct can comprise a polynucleotide barcode and the barcode comprises a unique sequence not present in any known genome for identification of the polynucleotide barcode.
  • a set of different nucleic acid constructs with different polynucleotide barcodes can be used to allow for multiplexing of samples on one sequencing run.
  • the polynucleotide barcodes can be from about 5 to about 35 base pairs in length, about 5 to about 34 base pairs in length, about 5 to about 33 base pairs in length, about 5 to about 32 base pairs in length, about 5 to about 31 base pairs in length, about 5 to about 30 base pairs in length, about 5 to about 29 base pairs in length, about 5 to about 28 base pairs in length, about 5 to about 27 base pairs in length, about 5 to about 26 base pairs in length, about 5 to about 25 base pairs in length, about 5 to about 24 base pairs in length, about 5 to about 23 base pairs in length, about 5 to about 22 base pairs in length, about 5 to about 21 base pairs in length, about 5 to about 20 base pairs in length, about 5 to about 19 base pairs in length, about 5 to about 18 base pairs in length, about 5 to about 17 base pairs in length, about 5 to about 16 base pairs in length, about 5 to about 15 base pairs in length, about 5 to 14 base pairs in length, about 5 to 13 base pairs in length, about 5 to 12
  • first primer binding segment can be linked at its 3’ end to the 5’ end of a random sequence fragment and the second primer binding segment can be linked at its 5’ end to the 3’ end of the polynucleotide barcode where the polynucleotide barcode is linked at its 5’ end to the 3’ end of the random sequence fragment.
  • first primer binding segment can be linked at its 3’ end to the 5’ end of the polynucleotide barcode and the second primer binding segment can be linked at its 5’ end to the 3’ end of the polynucleotide barcode.
  • the identity of the monomer units is not particularly limited so long as the monomer units being used are compatible with a controlled living/radical polymerization, such as reversible-deactivation radical polymerization, atom transfer radical polymerization (ATRP), reversible addition fragmentation chain transfer polymerization (RAFT), iodine-transfer polymerization (ITP), selenium-centered radical- mediated polymerization, telluride-mediated polymerization (TERP), stibine-mediated polymerization, ring-opening polymerization, or like polymerization processes.
  • a controlled living/radical polymerization such as reversible-deactivation radical polymerization, atom transfer radical polymerization (ATRP), reversible addition fragmentation chain transfer polymerization (RAFT), iodine-transfer polymerization (ITP), selenium-centered radical- mediated polymerization, telluride-mediated polymerization (TERP), stibine-mediated polymerization, ring-opening polymerization, or
  • RAFT polymerization is generally known in the art. Suitable reagents, monomers, and conditions for RAFT polymerization previously investigated can be used in the copolymers, methods, and compositions described herein, such as those described in United States Patent Nos. 9,006,193, 9,464,300, and 9,476,063, the disclosures of each of which are incorporated by reference in their entirety.
  • RAFT copolymerization may be achieved using chain transfer agents (CTAs) containing one or more terminal carboxyl groups in order to obtain carboxy terminated polymers with ends available for bonding to the payload via the methods described above.
  • CTAs chain transfer agents
  • the first or second chain transfer agent can be selected from the group consisting of bis(carboxymethyl)trithiocarbonate, bis(2-amino-2- oxoethyl) trithiocarbonate, bis[4-(2-hydroxyethoxycarbonyl)benzyl] trithiocarbonate, 4-cyano- 4-(ethylsulfany!thiocarbonyl) sulfanylvpentanoic acid, 4-cyano-4- ((phenylcarbonothioyl)thio)pentanoic acid, and 4-cyano-4-
  • the RAFT copolymer can be associated with a DNA molecule, in particular a nucleic acid construct of the present disclosure, via several methods including, electrostatic interaction, high affinity, non-covalent bond, avidin-streptavidin conjugation, or by direct covalent attachment through, for example, an amide bond.
  • the RAFT copolymer can be associated with a DNA molecule, in particular a nucleic acid construct of the present disclosure, via electrostatic interaction complexed with a biological molecule.
  • the RAFT copolymer can be associated with a DNA molecule, in particular a nucleic acid construct of the present disclosure, via electrostatic interaction complexed with a biological molecule.
  • the polymer nanoparticle composition can be coated with one or more polymers to protect the compositions from immune responses or to enhance endosomal escape.
  • the one or more polymers comprise polyethylene glycol.
  • the one or more polymers comprise polyethylene glycol poly-L-lysine.
  • the one or more polymers comprise polyethylenimine.
  • the one or more polymers comprise polyethylene glycol poly-L-lysine and polyethylenimine.
  • the second block can be prepared from one or more monomer units, and can have a molecular weight (M n )in the range of about 1 kDa to about 500 kDa, or about 10 kDa to about 200 kDa, or about 10 kDa to about 160 kDa, or about 1 kDa to about 80 kDa, and a degree of polymerization in the range of about 10 to about 3500, or about 10 to about 2500, or about 20 to about 2000, or about 50 to about 1200, or about 50 to about 1000.
  • M n molecular weight
  • the second block degree of polymerization is in the range of about 3 to about 2500; or about 20 to about 2000, or about 30 to about 1500, or about 40 to about 1200, or about 10 to about 500, or about 12 to about 450, or about 20 to about 400, or about 25 to about 350, or about 50 to about 300, or about 100 to about 250, or about 150 to about 200, or about 5 to about 50, or about 5 to about 20, and the like.
  • the third, fourth, or subsequent block molecular weight (Mn) is in the range of about 10 kDa to about 70 kDa, about 15 kDa to about 65 kDa, about 20 kDa to about 60 kDa, about 25 kDa to about 55 kDa, about 30 kDa to about 50 kDa, about 35 kDa to about 45 kDa, about 5 kDa to about 15 kDa, and the like.
  • a single chain transfer agent can be used in the RAFT polymerization process in connection with the present disclosure.
  • one or more single chain transfer agents can be used in the RAFT polymerization process in connection with the present disclosure.
  • a first chain transfer agent and a second chain transfer agent (which can be the same or different) can be used at each step of the RAFT polymerization process in connection with the present disclosure.
  • a third block prepared from one or more monomer units covalently attached to the first and/or second blocks, and having a molecular weight (M n )in the range of about 1 kDa to about 500 kDa and a degree of polymerization in the range of about 10 to about 2500;
  • Illustrative payloads for the polymer nanoparticle described herein can include any one or a combination of compositions selected from the group comprising: nucleic acids (e.g., DNA or RNA), pDNA, oligodeoxyribonucleic acids (ODNs), dsDNA, ssDNA, antisense oligonucleotides, antisense RNA, siRNA, messenger RNA, guide RNA (e.g., small guide RNA), ribonucleoproteins, donor DNA strands used in the CRISPR/Cas9 system, and enzymes, such as CRISPR-associated enzymes, e.g., Cas9, enzymes used in other gene editing systems, such as ZFNs, custom designed homing endonucleases, TALENS systems, other gene editing endonucleases, and reverse transcriptase.
  • nucleic acids e.g., DNA or RNA
  • ODNs oligodeoxyribonucleic acids
  • a modified Transformer model (rather than a GNN) may be used to predict polymer properties (and, thus, rapidly identify top candidates for non-viral carriers for delivering base editing proteins, among other applications).
  • the modified Transformer model exploits relative positional information of inputs to create numerical embeddings for monomer string inputs. These numerical embeddings can then be used in deep learning and statistical models for polymer property prediction. Additionally, the Transformer model is more computationally efficient compared to many other deep learning architectures that can process sequential data.
  • the original Transformer architecture consists of an encoding and decoding architecture.
  • the encoder takes an input sequence of data and outputs a high dimensional embedding, while the decoder takes the high dimensional embedding as an input and tries to predict the original or similar sequence to the one input into the encoder.
  • the present disclosure does not need to predict a sequential output, so it only uses the encoding portion of the Transformer to predict polymer properties, both physical and in- vitro/in-vivo.
  • the reaction mixture was argon purged before being heated at 60 oC for 24 hours.
  • the reaction product was purified using the same purification process and dried in vacuo.
  • the resulting polymer was dialyzed in deionized water for 4 days with multiple water changes each day. Finally, the dialyzed material was lyophilized for 4 days and stored at room temperature for experimental use.
  • Composition Example 1 Electrostatic attachment of nucleic acid constructs (containing DNA barcodes) to polymer nanoparticles
  • EXAMPLE 11 [00239] Sequence Analysis and Bioinformatics.
  • first generation CRISPR is considered the “cut and paste” of gene editing
  • base editors are considered to be an “eraser and pencil” function, allowing for precise single base edits to a genome, opening new mechanisms for revolutionary ALS treatments.
  • innovations in gene delivery have significantly lagged innovations in technologies for gene editing itself.
  • efficient delivery of base editing systems to the specific cell types involved in driving the progression of ALS represents a key limitation impeding its safe and efficient implementation for treatment of the disorder.
  • Non-viral delivery vehicles will be used to address many of these limitations.
  • microglia non-viral delivery vehicles with base editing payloads is highly innovative because it has the potential to lead to a new therapy for ALS.
  • the DBTL technologies of the present disclosure are generalizable to enable the creation of advanced non-viral delivery vehicles capable of accessing the other cell types involved in ALS.
  • This screen should result in three large data sets including particle physical characteristics, in vitro bioactivity, and in vivo biodistribution and toxicity, which, taken together, will provide the basis for an informed design of a novel non-viral delivery vehicle library which will be synthesized in a second iteration.
  • This novel library can then be tested for functional gene editing tests in a microglial cell line modified to express a mutant SOD1 protein.
  • the PNPs can be loaded with mRNA encoding CBE designed to inactivate GFP and SOD1, detected by fluorescence measurement and sequencing.

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PCT/US2022/023902 2021-04-07 2022-04-07 Rapid design, build, test, and learn technologies for identifying and using non-viral carriers Ceased WO2022216977A1 (en)

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CN202280040638.0A CN117500923A (zh) 2021-04-07 2022-04-07 用于鉴定和使用非病毒载体的快速设计、构建、测试和学习技术
EP22785473.4A EP4320233A4 (en) 2021-04-07 2022-04-07 RAPID DESIGN, BUILD, TEST, AND LEARNING TECHNOLOGIES TO IDENTIFY AND USE NON-VIRAL VECTORS
JP2023562184A JP2024516108A (ja) 2021-04-07 2022-04-07 非ウイルス性担体を同定および使用するための迅速な設計、構築、試験、および学習技術
IL326440A IL326440A (en) 2021-04-07 2022-04-07 Technologies for rapid design, construction, testing, learning, identification, and use of non-viral vectors
CA3216359A CA3216359A1 (en) 2021-04-07 2022-04-07 Rapid design, build, test, and learn technologies for identifying and using non-viral carriers
AU2022253899A AU2022253899A1 (en) 2021-04-07 2022-04-07 Rapid design, build, test, and learn technologies for identifying and using non-viral carriers
IL307454A IL307454B1 (en) 2021-04-07 2023-10-03 Technologies for rapid design, construction, testing, learning, identification, and use of non-viral vectors

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Cited By (5)

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