Next-Generation CRISPR Approaches for Therapeutic Development

Next-generation CRISPR Approaches Explore strategies to advance therapeutic programs supporting current regulatory guidance WHITEPAPER CRISPR knock-in methods are dependent upon DNA repair mechanisms and cell cycle status The knock-in process depends on eukaryotic cells’ preferred DNA repair pathways responding to a double-stranded DNA break (DSB) within its genome. Such preference is heavily influenced by the cell’s current cell cycle status. Non-homologous end joining (NHEJ) is often the preferred method of DSB repair for a cell, given that its activity is allowed in all cell cycle phases. While NHEJ has high efficiency, it is error-prone. Therefore, this pathway has historically been the preferred method to knockout a target gene via natural introduction of a missense or nonsense mutation.1 The homology-directed repair (HDR) pathway is an alternative repair pathway that can occur after the DSB. This repair method is considered less efficient and more precise than NHEJ. Still, it is only available during the synthesis and G2 phase of the cell cycle, given that its machinery depends upon a sister chromatid to serve as the homology donor template for repair. HDR remains one of the preferred methods for gene knock-in, with the creation of DNA donor templates typically delivered as a plasmid containing homology-directed arms for insertion via HDR.2 In recent years, homology-independent targeted insertion (HITI), which uses the NHEJ pathway, has become more popular. This method is achieved by first using two RNPs flanking the target sites, resulting in DSBs in both the target cell genome and the donor vector. HITI is completed by ligating the vector donor template into the target site and is considered more efficient than HDR.3 Another editing pathway called microhomologymediated end joining (MMEJ) uses NHEJ and is achieved by using donor vectors with 5-25bp of homology on either end of the DSBs created by an RNP. The end result with MMEJ is a knock-in by the annealing of two ends flanking the DSB site, and this method is reported to be 2-3 times more efficient than HDR.1,4 Common CRISPR Delivery Methods How CRISPR nucleases and donor templates were first deployed CRISPR was originally delivered with plasmids expressing the Cas nuclease and gRNA in the target cells, with HDR donor Next-generation CRISPR Approaches CRISPR Overview The discovery of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Cas9 gene editing system has revolutionized the cell and gene therapy field. This system was discovered from a prokaryotic adaptive immune response against bacteriophage infections that allowed targeted double-stranded DNA (dsDNA) breaks within its genome directed by guide RNA (gRNA) to remove the bacteriophage insertions.1 Researchers soon applied their findings to knock-out and knock-in target genes within eukaryotic cells by creating specific gRNAs to the target site complexed with a nuclease — termed ribonucleoprotein (RNP). Common applications include ex vivo cell gene editing of inducible pluripotent stem cells (iPSCs), hematopoietic stem cells, and primary immune cells for chimeric antigen receptor T-cells (CAR-T), natural killer cells (NK), and natural killer T cells (NKT). NEXT-GENERATION 2 CRISPR APPROACHES Mitosis G0 G1 G2 Synthesis NHEJ (HITI) & MMEJ NHEJ (HITI) & HDR Cell Cycle Pathway and DNA Repair Mechanisms used for CRISPR Cells responding to double-stranded DNA breaks (dsDNA) from a CRISPR Cas9 endonuclease will have different dsDNA repair mechanisms available depending on their cell cycle status. Gap 0 (G0) Gap 1 (G1), Gap 2 (G2) NEXT-GENERATION 3 CRISPR APPROACHES templates amplified via PCR and gel extracted. Later, researchers discovered that plasmids could be delivered in a linear or supercoiled fashion as a donor template. As the field has evolved, so have the methods for CRISPR delivery both in terms of their Cas nucleases and donor templates.5 Commonly used viral vectors for CRISPR delivery Adeno-associated virus (AAV) is a non-enveloped DNA virus from the Parvovirdae family. Wild-type AAV was discovered as an integrating virus that targets human chromosome 19, but it was still considered non-pathogenic, given that humans are approximately 80% seropositive. The AAV format commonly used as a delivery vehicle for gene therapies is a modified recombinant AAV virus lacking the DNA replication proteins Rep78/68, resulting in episomal expression of DNA in infected cells, which still confers therapeutic benefits without the risk of genome integration.6,7 One of the shortcomings of this vector is its transgene delivery limit only being <4.7kb. AAV can be used to deliver the nuclease, gRNA, or the donor template. However, the common nucleases used are 4.2kb expressing the Streptococcus pyrogenes (Cas9) and ~3.15kb for the Staphylococcus aureus (SaCas9). SaCas9’s smaller size improves the feasibility of delivery via AAV but has limitations in the Protospacer Adjacent Motif (PAM) sequence for targeting. While the larger and more common Cas9 is still within the technical cloning limit of AAV, its transduction still has shown some technical difficulties in practice. Given the difficulties of expressing a nuclease for CRISPR gene editing within an AAV vector, many groups have attempted approaches where multiple AAV vectors deliver the gRNA, nuclease, and donor template on separate AAV constructs or by different mechanisms.8,9 Although the 4.7kb transgene size limit is still a technical hurdle, methods have been reported allowing the fusion of two transgenes in tandem expressed on two different AAV vectors, alleviating this limitation for HDR donor template delivery.1,7 Despite these creative approaches and advances to overcome some of this vector’s limitations, the most common format of CRISPR knock-in seen using AAV to date is with an AAV vector serving only as an HDR donor template, producing an episomal donor product along with an RNP delivered via electroporation for HDR knock-in.10 Lentivirus (LV) — an enveloped RNA retrovirus vector with a cloning capacity of <8kb capable of transducing both dividing and non-dividing cells — has a rich history in the field. It randomly integrates into regions within a genome with a propensity for insertion at frequently transcribed sites. While a target transgene can be strongly expressed using both vectors, the seemingly random integration pattern raises concerns about insertional mutagenesis and the potential for creating a neoplastic condition when used in clinical applications.7,11 While both LV and gamma retrovirus (γ-retrovirus ) are members of the Retroviridae family, LV has shown a greater safety profile than γ-retrovirus. Early clinical trials where γ-retrovirus was used to target X-linked severe combined immunodeficiency (X-Linked SCID) and Wiskott-Aldrich syndrome (WAS) resulted in a large number of patients developing acute T lymphocytic leukemia due to insertional mutagenesis.12,13 Further clinical research has since been conducted using LV to treat X-linked SCID, WAS, and blood cancers using chimeric antigen receptor (CAR) T-cells. These studies have shown improved safety profiles compared to γ-retrovirus.11,14 However, the FDA recently released a black box warning on approved CAR-T cell therapies using LV for manufacturing because insertional mutagenesis was observed in a small number of patients receiving CAR-T cell therapies.15 Developing knock-in methods for all cell cycle phases expands CRISPR’s therapeutic potential. NEXT-GENERATION 4 CRISPR APPROACHES Given its larger cloning capacity, LV can deliver the traditional Cas9 and a single gRNA in one vector with the donor template or knock-in gene in another vector. While LV delivery is efficient, stable expression due to integration poses other risks when used for CRISPR. Therefore, researchers have created a non-integrating transient expressing LV already used in a few clinical applications, including a cancer vaccine targeting dendric cells.7,14,16,17 The field is continuing to evolve along this vector, but its application in the context of CRISPR trails in popularity compared to other approaches at this moment. Commonly used non-viral methods for CRISPR delivery Electroporation is a very popular approach for ex vivo gene editing of cells that are difficult to transfect, such as primary cells. Electroporation is a physical delivery method where small electrical currents open cell membrane channels, allowing for the entry of exogenous materials — in this case, the RNP complex and the various options for DNA donor template delivery used for CRISPR gene editing. Electroporation methods require process development in various stages, including determining the specific voltages used during the transfection, optimization of the ratios of gRNA to nuclease in the RNP complexes, and final concentration of RNP to include in the electroporation buffer. Process optimization is required at multiple steps, including the characterization of RNP complexes after a ratio is determined to meet regulatory agency recommendations.1,18 RNPs have also been successfully delivered both ex vivo and in vivo using lipid nanoparticle (LNP) formulations encapsulating protein-based or nucleic acid-based CRISPR constructs. Furthermore, LNPs are an approved FDA method of drug delivery, making this an attractive delivery method for developers.19,20 The delivery of nucleases in mRNA format is also an increasingly popular approach since mRNA has a faster onset after translation and is only transiently expressed, reducing the chances of offtarget effects.18 LNPs used for the delivery of mRNA are a common approach, with a well-documented safety profile demonstrated via COVID-19 vaccines and various other clinical trials. The LNP formulation protects the mRNA constructs from degradation and aids in tropism, although it has been noted that LNP constructs have an affinity for the liver. While many reports still note this tropism for the liver with LNPs delivered in vivo, much research is being conducted to create formulations that have more targeted tropisms termed selective organ targeting (SORT).21,22 This method has many benefits with its transient expression profile. This is in part due to the degradation of mRNA in the cytoplasm, which requires attention in the process development stage to deliver sufficient mRNA to the cells for the successful production of an RNP that works with a donor template for knock-in.23 Extracellular vesicles (EV) or virus-like particle (VLP) delivery systems are also gaining in popularity. VLPs and EVs are similar in containing the viral envelope and the target protein. However, VLPs contain the addition of the structural proteins, with the target protein fused to the Gag polyprotein, which is cleaved by the Pol, releasing the target protein. EVs function through viral budding meditated by their outer layer of viral envelope proteins and the target protein inside, resulting in endocytosis of the EVs contents into the target cell. EV and VLP delivery approaches have many benefits over viral vectors, with their ability to overcome cargo restrictions and a safety profile, given that the risk for insertional mutagenesis is absent. Developers are using EVs and VLPs to deliver nucleases for gene editing both in vitro and in vivo.1,24 New CRISPR editing approaches for in vivo editing Developers using CRISPR were initially limited to gene knockout after targeting a locus with an RNP if the cell’s DSB repair pathway was limited to NHEJ. Gene knock-ins were only thought possible if cells were locked into the time points of the cell cycle (late S-phase and G2) where HDR mechanisms were available, given the presence of sister chromatids. These limitations have been overcome with the discovery of HITI and MMEJ, allowing for a gene knock-in to occur when RNPs create a DSB in cells that only have NHEJ repair systems available due to their cell cycle status. While methods to target cells that are not mitotically active have been defined, the knock-in rates have been variable and dependent on the targeted cell type. Most of these knock-in approaches have primarily been in the ex vivo setting, like CAR-T cells, CAR NK cells, and inducible pluripotent stem cell (iPSC) editing. With the development of base editors, a new CRISPR-based method suited for in vivo editing approaches has evolved rapidly since its first report in 2016.25 Base editors are comprised of a mutated Cas9 nuclease that is catalytically dead (dCas9), resulting in an enzyme that can precisely NEXT-GENERATION 5 CRISPR APPROACHES target a sequence of DNA but cannot create a DNA break. This dCas9 is fused with a cytosine deaminase or an adenosine deaminase within a narrow window proximal to the dCas9 binding site. Cytidine deaminase allows cytosine (C) to thymine (T) and guanine (G) to adenine (A) transitions with uracil intermediaries. Adenosine deaminase allows A➡G and T➡C transitions with an inosine intermediary. Base editors have undergone multiple generations, improving their base editing efficiencies with each iteration. The first generation consisted of a dCas9 and cytosine or adenine deaminases, while the second generation improved its editing efficiency three-fold with the addition of a uracil DNA glycosylase inhibitor, given that the innate cellular base excision repair mechanisms would reverse the intermediates created by the cytosine deaminase. The third generation of base editors contains a Cas9 nickase (nCas9) designed to induce a nick on the non-edited strand, pushing favorable DNA repair mechanisms that result in a six-fold improvement over the second generation base editors.25 Further improvements on base editors have occurred, including modifications that improve the editing profile (reducing bystander editing), reducing the number of insertions-deletions (indels), the addition of nuclear localization signals to each Cas, and improving the editing of G/C sequences. This has resulted in several more generations of base editors in the subsequent years.26 While base editors have significantly improved gene editing technologies, it was limited to four DNA substitutions, which led to the creation of prime editing. Prime editing uses an nCas9 to create a nick for the attached reverse transcriptase (RT) to extend the target gene from the prime editing guide RNA (pegRNA), which directs the complex to the target site and serves as the template for the intended integration.26,27 The result is a gene editing tool able to create all 12 types of DNA substitutions and the ability for insertions and deletions. This gene-editing tool significantly increases the possible therapeutic landscape of correcting up to 89% of pathogenic genetic diseases documented in ClinVar.27 The most popular delivery format of base editors is LNP-encapsulated mRNA constructs for in vivo delivery, given the benefits of transient expression and the safety profile of mRNA vectors. Many groups are targeting a wide range of disease states with this new technology, but process development for these base editing approaches is rigorous. For example, a base editor RNP must still be characterized, which would require development work for the characterization assays in addition to formulation and complexing of LNP mRNA constructs. Next-generation CRISPR manufacturing methods require next-generation DNA vectors Many of the shortcomings of CRISPR and other gene editing modalities have been due, in part, to the modest number of edited cells generated. Attempts to improve these yields, like delivering CRISPR via viral vectors, have modestly improved knock-in rates (i.e., AAV donor template delivery and proteinbased RNP, etc.). A few new CRISPR approaches have significantly improved ex vivo cell editing yields using a thoughtful implementation of selection or enrichment systems alongside next-generation DNA vectors as donor templates. The first example of this comes from a group at Genentech, where they compared three DNA donor template formats’ efficiency in generating CRISPR HDR knock-in primary CD8 T cells. They compared linear dsDNA, a traditional pUC plasmid, and a next-generation Nanoplasmid™ plasmid DNA vector system. They reported that the Nanoplasmid vector generated twice the number of edited cells compared to the CRISPR-based approaches can now achieve knock-in rates of 80-90%. NEXT-GENERATION 6 CRISPR APPROACHES traditional pUC plasmid and three times as much as the linear dsDNA donor template.28 Their findings supported using Nanoplasmid, a nextgeneration plasmid vector designed for cell and gene therapy applications. Nanoplasmid is a purposefully designed plasmid with a small backbone (<500bp) devoid of antibiotic-resistance selection genes and bacterial encoding proteins. Its development was in response to reports in the field providing evidence that large backbone plasmids and the bacterial coding proteins embedded with the backbone were causal factors of increased post-transfection toxicity and transgene silencing while delivering gene payloads to cells.29 Genentech’s study reported knock-in rates of 36.6-46.6% using the Nanoplasmid vector and provided the CAR-T field with a formidable new approach for CRISPR knock-ins.28 The following year, Editas Medicines published an article reporting its SeLection by Essential-gene Exon Knock-in (SLEEK) method. The SLEEK CRISPR HDR knock-in method works by using RNPs to target essential genes like GAPDH along with the target locus for knock-in. The theory behind the approach is that most cells will respond with NHEJ, resulting in indels at the site of an essential gene, which will result in cell death. This negative selection method results in a highly purified cell population that has successfully completed HDR of both the essential and target genes, leading to survival. During the testing of the SLEEK approach, the authors tested their HDR knock-in method with various donor templates, including Nanoplasmid, and reported tremendous knock-in efficiencies of 90% or more.30 In the same year, another group published an alternative knock-in and enrichment process using HITI, called CRISPR enrichMENT (CEMENT). In this knock-in process, an RNP targets the TRAC locus of a primary human T cell and a Nanoplasmid knock-in donor template. The donor template carries the target gene (CAR construct) and dihydrofolate reductase enzyme, which is known to confer resistance to methotrexate, an FDA-approved chemotherapy agent. Cells that successfully underwent HITI were then enriched with a round of methotrexate selection that killed all the cells that did not successfully integrate the dihydrofolate reductase gene. The results were functionally active cells of 80% purity after a 14-day protocol tested using healthy and nonhealthy donor peripheral blood mononuclear cells (PBMCs).31 To read additional studies on Nanoplasmid as an HDR template, please refer to the Nanoplasmid Bibliography. CRISPR-based manufacturing— improving manufacturing efficiencies and access to treatments The recent FDA approval of CRISPR-based therapeutics has created hopeful excitement in the field. The possibility of improved manufacturing timelines, yields, and safety profiles are factors that will support the growth and feasibility of the increased adoption of cell and gene therapies in the clinic. Developers now feel more confident using CRISPR approaches in their pipelines destined for rapid clinical development, but they still face difficulties and uncertainty surrounding manufacturing and regulatory requirements. In 2022, the FDA released draft guidance on Investigational New Drug (IND) applications for Human Gene Therapy Drug Products Incorporating Human Genome Editing technology to edit human somatic cells. For developers applying gene editing technology such as CRISPR, this guidance helps clarify some key requirements around Chemistry, Manufacturing, and Control (CMC) for the geneediting reagents as well as the gene-modified cellular Drug Product. For the first time, the FDA has defined key control considerations for CRISPR reagents. Method of delivery for the genome editing reagent is key: • When delivered in vivo (e.g., by viral vector or nanoparticle), the final formulation is a drug product and the gene editing technology itself is considered as a drug substance. • When a cellular drug product is administered ex vivo, the gene modification technology (e.g., CRISPR reagents) are considered critical raw materials that require a full Drug Substance section in IND filings. The guidance also touches on cGMP manufacturing considerations for these drug substances. While Phase I trials require components to adhere to the FDA’s Guidance for Industry: cGMP for Phase 1 Investigational Drugs, later phase and approved therapeutics will require cGMP components per 21 CFR Parts 210 and 211. Streamlining life-altering therapeutic development Ushering a treatment based on an emerging technology, such as a CRISPR-based therapy, NEXT-GENERATION 7 CRISPR APPROACHES through the clinical process is a complex and lengthy endeavor. Partnering with the right CDMO can help you move more smoothly from discovery through clinical trials. Aldevron supports the transition of CRISPR-based therapies from research to clinical use: • In-stock or custom manufactured nucleases at research and cGMP/clinical grades • Guide RNA (gRNA) sourced from Integrated DNA Technologies (IDT) for research use or guides produced at IDT’s Therapeutic Oligonucleotide Manufacturing Facility for successful completion of preclinical studies and clinical trials • HDR donor templates using next-generation Nanoplasmid™ vectors • First-in-class RNP complexing and analytic services** • Compliant, proprietary QC test panels for RNP Drug Substance* • Multiple delivery approaches including RNP, mRNA, or viral vector • Regulatory experience supporting partners through 1,000+ clinical trials Working with a trusted partner dedicated to clinical applications of CRISPR technologies, like Aldevron, to source your nucleases, gRNAs (in partnership with IDT), HDR templates, and other key material provides a smoother transition from research to clinical trials and beyond. Aldevron provides 21CFR210-211 compliant manufacturing and analytical characterization services, plasmid vector systems, and mRNA services, giving you the flexibility to pursue the approach that works best for your application. Gene editing by CRISPR-Cas9 Adeno-associated virus We support the following common formats of CRISPR donor template approaches: Protein Delivery FOUR DIFFERENT WAYS INTO YOUR TARGET CELL mRNA Delivery 5' G P P P AAA 3' O€-the-shelf Cas Nuclease and custom manufacturing mRNA Cas9 delivery and gRNA complexing in the cell Oering o-the-shelf helper and Rep/Caps for AAV delivery Nanoplasmid™ next-generation plasmid vector systems as a CRISPR donor template mRNA drug substance mRNA drug product LNP †ll/†nish RNP analytical services for GMP RNP complexing Transgene Rep/Cap Gene of interest Expression Plasmid Packaging Plasmids Co-transfection rAAV Vector Common to all rAAV production Helper Plasmid donor templates guideRNA generation guideRNA generation YOUR TARGET CELL Contact Aldevron to speak with a CRISPR expert and explore how we can help you today. *As of May 2024, Free gRNA and Free Cas9 assays are available for characterization. Validation of these assays is in progress. ** Aldevron provides RNPs only to customers who are duly licensed, including to make and have made RNPs, for their intended use. NEXT-GENERATION 8 CRISPR APPROACHES References 1. B. H. Yip, “Recent Advances in CRISPR/Cas9 Delivery Strategies,” Biomolecules, vol. 10, no. 6, Jun. 2020, DOI: 10.3390/BIOM10060839. 2. R. Ceccaldi, B. Rondinelli, and A. D. D’Andrea, “Repair Pathway Choices and Consequences at the Double-Strand Break,” Trends Cell Biol, vol. 26, no. 1, pp. 52–64, Jan. 2016, DOI: 10.1016/J.TCB.2015.07.009. 3. K. Suzuki et al., “In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration,” Nature, vol. 540, no. 7631, p. 144, Dec. 2016, DOI: 10.1038/ NATURE20565. 4. S. Nakade et al., “Microhomology-mediated end-joiningdependent integration of donor DNA in cells and animals using TALENs and CRISPR/Cas9,” Nat Commun, vol. 5, 2014, DOI: 10.1038/NCOMMS6560. 5. C. E. Denes, A. J. Cole, Y. A. Aksoy, G. Li, G. G. 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Eyquem et al., “Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances tumour rejection,” Nature, vol. 543, no. 7643, p. 113, Mar. 2017, DOI: 10.1038/NATURE21405. 11. [11] W. Dong and B. Kantor, “Lentiviral Vectors for Delivery of Gene-Editing Systems Based on CRISPR/Cas: Current State and Perspectives,” Viruses, vol. 13, no. 7, Jul. 2021, DOI: 10.3390/V13071288. 12. [12] S. Hacein-Bey-Abina et al., “Insertional oncogenesis in 4 patients after retrovirus-mediated gene therapy of SCID-X1,” J Clin Invest, vol. 118, no. 9, p. 3132, Sep. 2008, DOI: 10.1172/JCI35700. 13. [13] C. J. Braun et al., “Gene therapy for Wiskott-Aldrich Syndrome—Long-term reconstitution and clinical benefits, but increased risk for leukemogenesis,” Rare Diseases, vol. 2, no. 1, p. e947749, Jan. 2014, DOI: 10.4161/21675511.2014.947749. 14. N. Gurumoorthy, F. Nordin, G. J. Tye, W. S. Wan Kamarul Zaman, and M. H. 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Berglund, “ZVexTM, a dendritic-cell-tropic lentivector, primes protective antitumor T cell responses that are significantly boosted using heterologous vaccine modalities,” Vaccine, vol. 38, no. 41, pp. 6367–6373, Sep. 2020, DOI: 10.1016/J.VACCINE.2020.08.003. 18. J. Huang, Y. Zhou, J. Li, A. Lu, and C. Liang, “CRISPR/Cas systems: Delivery and application in gene therapy,” Front Bioeng Biotechnol, vol. 10, p. 942325, Nov. 2022, DOI: 10.3389/FBIOE.2022.942325/BIBTEX. 19. J. A. Zuris et al., “Efficient Delivery of Genome-Editing Proteins In Vitro and In Vivo,” Nat Biotechnol, vol. 33, no. 1, p. 73, Jan. 2015, DOI: 10.1038/NBT.3081. 20. T. M. Allen and P. R. Cullis, “Liposomal drug delivery systems: from concept to clinical applications,” Adv Drug Deliv Rev, vol. 65, no. 1, pp. 36–48, Jan. 2013, DOI: 10.1016/J.ADDR.2012.09.037. 21. E. A. Taha, J. Lee, and A. Hotta, “Delivery of CRISPRCas tools for in vivo genome editing therapy: Trends and challenges,” Journal of Controlled Release, vol. 342, pp. 345–361, Feb. 2022, DOI: 10.1016/J.JCONREL.2022.01.013. 22. X. Wang et al., “Preparation of selective organ-targeting (SORT) lipid nanoparticles (LNPs) using multiple technical methods for tissue-specific mRNA delivery,” Nature Protocols 2022 18:1, vol. 18, no. 1, pp. 265–291, Oct. 2022, DOI: 10.1038/s41596-022-00755-x. 23. F. Sinclair, A. A. Begum, C. C. Dai, I. Toth, and P. M. Moyle, “Recent advances in the delivery and applications of nonviral CRISPR/Cas9 gene editing,” Drug Delivery and Translational Research 2023 13:5, vol. 13, no. 5, pp. 1500–1519, Mar. 2023, DOI: 10.1007/S13346-023-01320-Z. 24. M. An et al., “Engineered virus-like particles for transient delivery of prime editor ribonucleoprotein complexes in vivo,” Nature Biotechnology 2024, pp. 1–12, Jan. 2024, DOI: 10.1038/s41587-023-02078-y. 25. A. C. Komor, Y. B. Kim, M. S. Packer, J. A. Zuris, and D. R. Liu, “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage,” Nature, vol. 533, no. 7603, p. 420, May 2016, DOI: 10.1038/NATURE17946. 26. A. Kantor, M. E. McClements, and R. E. Maclaren, “CRISPRCas9 DNA Base-Editing and Prime-Editing,” Int J Mol Sci, vol. 21, no. 17, pp. 1–22, Sep. 2020, DOI: 10.3390/ IJMS21176240. 27. A. V. Anzalone et al., “Search-and-replace genome editing without double-strand breaks or donor DNA,” Nature, vol. 576, no. 7785, pp. 149–157, Dec. 2019, DOI: 10.1038/ S41586-019-1711-4. 28. S. A. Oh et al., “High-efficiency nonviral CRISPR/Cas9- mediated gene editing of human T cells using plasmid donor DNA,” J Exp Med, vol. 219, no. 5, May 2022, DOI: 10.1084/ JEM.20211530. 29. J. A. Williams and P. A. Paez, “Improving cell and gene therapy safety and performance using next-generation Nanoplasmid vectors,” Mol Ther Nucleic Acids, vol. 32, pp. 494–503, Jun. 2023, DOI: 10.1016/j.omtn.2023.04.003. 30. A. G. Allen et al., “A highly efficient transgene knockin technology in clinically relevant cell types,” Nature Biotechnology 2023 42:3, vol. 42, no. 3, pp. 458–469, May 2023, DOI: 10.1038/s41587-023-01779-8. 31. H. Balke-Want et al., “Homology-independent targeted insertion (HITI) enables guided CAR knock-in and efficient clinical scale CAR-T cell manufacturing,” Mol Cancer, vol. 22, no. 1, Dec. 2023, DOI: 10.1186/S12943-023-01799-7. PRO-1348-v2 © 2025 Aldevron LLC. All rights reserved. Aldevron, the stylized logo, and the Aldevron product and service marks mentioned herein are trademarks or registered trademarks of Aldevron LLC in the United States and other countries. All other trademarks are the property of their respective owners. Contact us to speak with a custom protein expert to explore how we can help you today. 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