Compositions and methods for in vivo genetic engineering of lymphocyte precursors

Abstract

Disclosed are compositions and methods for in vivo genetic engineering of lymphocyte precursors. A lipid nanoparticle (LNP), surface-functionalized for lymphocyte precursor targeting, encapsulates a DNA payload comprising a therapeutic gene flanked by engineered Recombination Signal Sequences (RSS). The RSS spacer incorporates at least one non-natural nucleotide, reducing integration risks. Because RAG1 and RAG2 are expressed only after commitment to the lymphoid lineage, integration is restricted to immune precursors and cannot occur in stem cells or reproductive cells. Upon systemic delivery, the endogenous RAG1 / RAG2 complex integrates the payload into the precursor genome. Modified precursors undergo clonal expansion in vivo, amplifying the therapeutic effect from relatively low doses. In certain embodiments, the DNA payload encodes receptors or antibodies that specifically recognize aggregated amyloid proteins, including transthyretin (TTR) fibrils and immunoglobulin light- chain aggregates, enabling therapeutic treatment of amyloidosis. However, the platform is not limited to amyloidosis and can be applied to oncology, infectious diseases, autoimmune disorders, and other protein misfolding conditions.

Description

[0001] Specification

[0002] Technical Field

[0003] The present invention relates to compositions and methods for the in vivo genetic engineering of lymphocyte precursors, with emphasis on lipid nanoparticle (LNP) delivery systems and engineered Recombination Signal Sequences (RSS). In particular, the invention provides therapeutic applications for diseases involving aggregated amyloid proteins, while also encompassing broader uses in oncology, infectious diseases, autoimmunity, and other diseases.

[0004] Background of the Invention

[0005] Adoptive cell therapies such as Chimeric Antigen Receptor (CAR)-T cells have demonstrated remarkable clinical efficacy in hematologic malignancies (Porter et al., 2011). However, these therapies require ex vivo manipulation of autologous cells, which involves harvesting, shipment to centralized facilities, genetic modification, expansion, quality control, and reinfusion. This complex workflow increases cost, introduces logistical delays, and limits scalability (CAR-T Manufacturing Landscape Review, 2022).

[0006] Alternative delivery approaches:

[0007] • Viral vectors (AAV, retroviral, lentiviral): While historically effective, these platforms are hindered by insertional mutagenesis risks (Hacein-Bey-Abina et al., 2003), long production timelines, and manufacturing bottlenecks. AAV vectors are widely studied for in vivo gene delivery, but they are limited by payload size, immune responses, and scalability challenges.

[0008] • Physical methods (electroporation, microinjection): Suitable for ex vivo modification but poorly suited for systemic delivery due to toxicity, limited targeting, and inefficiency.

[0009] Lipid nanoparticles (LNPs): By contrast, LNPs have emerged as a safe, non- viral, and scalable delivery platform. Clinical studies have demonstrated systemic nucleic acid delivery via LNPs, including CRISPR-mediated transthyretin amyloidosis therapy (Gillmore et al., 2021). LNPs offer tunable chemistry for selective targeting of hematopoietic and lymphoid populations (Sago et al., 2018), favorable safety profiles (Hou et al., 2021), and reproducible large-scale manufacturing consistent with vaccine precedents. RAG recombinase as an endogenous editing system: The RAG1 / RAG2 recombinase mediates precise V(D)J recombination at Recombination Signal Sequences (RSS) during lymphocyte development (Schatz & Ji, 2011). Importantly, RAG1 and RAG2 are not expressed in pluripotent stem cells or reproductive cells. They are only produced once a progenitor cell has committed to the lymphoid lineage, ensuring that integration cannot occur in stem cell compartments or germline cells. Leveraging RAG recognition of engineered RSS offers an endogenous, lineage- restricted alternative to exogenous nucleases, further enhancing safety.

[0010] Non-natural nucleotides as a safety feature: Incorporation of non-natural nucleotides into RSS spacer regions can alter recognition or repair kinetics, reducing the likelihood of unintended genomic integration and providing an additional safety layer for clinical application (Henry & Romesberg, 2003).

[0011] Therapeutic potential: By targeting lymphocyte precursors in vivo rather than mature cells, the invention exploits the natural process of V(D)J Recombination and the potential for clonal expansion. Once genetically modified in vivo, these precursor cells differentiate and can proliferate into large populations of effector lymphocytes. This provides amplified and durable therapeutic effects from relatively low systemic doses, reducing toxicity and improving practicality compared to conventional viral gene therapies.

[0012] Amyloidosis as a target: Amyloidosis diseases such as transthyretin amyloidosis (ATTR) and light-chain amyloidosis (AL) are characterized by the deposition of aggregated protein fibrils. These aggregated species are structurally distinct from monomeric proteins and present significant challenges for conventional antibody generation. The present invention provides methods for engineering lymphocyte precursors in vivo to recognize aggregated amyloid proteins, enabling therapeutic strategies for amyloidosis and related diseases.

[0013] Prior Art and Distinction

[0014] U.S. Patent No. 8,617,845 B2 (Gallo et al.): Discloses engineered RSS for immunoglobulin diversity in vitro, but does not teach systemic in vivo LNP delivery, targeting lymphocyte precursors, or non-natural nucleotide incorporation. • EP3612631A4 (Brazel et al.) : Describes RAG-mediated V(D) J constructs but omits systemic LNP delivery and safety-enhancing modifications such as non-natural nucleotides.

[0015] The present invention uniquely combines (i) systemic LNP delivery targeted to lymphocyte precursors, (ii) engineered RSS sites recognized by endogenous RAG recombinase, (iii) lineage restriction via RAG1 / 2 activity, (iv) non-natural nucleotides that reduce off-target risks, and (v) application to aggregated amyloid proteins that are difficult to target with conventional antibodies. Together, these features distinguish the invention from prior approaches and provide clinically relevant therapeutic advantages.

[0016] Summary of the Invention

[0017] The invention provides compositions and methods for in vivo genetic engineering of lymphocyte precursors. The composition features a lipid nanoparticle (LNP) encapsulating a DNA payload flanked by engineered RSS, with at least one non-natural nucleotide in the spacer. Upon delivery, endogenous RAG1 / RAG2 integrates the payload into the genome, enabling targeted, nuclease- free genetic reprogramming. Because lymphocyte precursors naturally undergo clonal expansion and differentiation, the therapeutic effect is amplified over time, achieving durable immune reprogramming with lower systemic doses compared to viral vector-based approaches. In particular embodiments, the invention is used to generate immune cells that recognize aggregated amyloid proteins, including transthyretin (TTR) fibrils and immunoglobulin lightchain aggregates, providing therapeutic benefit in amyloidosis. However, the invention is not limited to amyloidosis. The same platform can be applied to oncology indications, infectious diseases, autoimmune disorders, and other conditions involving misfolded or pathogenic proteins.

[0018] Detailed Description

[0019] Delivery Vehicle: LNPs are surface-functionalized with targeting moieties that selectively bind lymphocyte precursor surface markers, such as Surrogate Light Chain (SLC), CD 10, IL-7, CD 19, c-KIT (CD 117), CXCR4, or CD90 (Thy-1).

[0020] Genetic Payload: The encapsulated DNA encodes therapeutic sequences (e.g., TCRs, BCRs, CARs, cytokines, or antibodies), flanked by engineered RSS. Spacer regions incorporate non- natural nucleotides to enhance specificity and reduce risks of unintended integration. Since RAG1 / 2 are only expressed in committed lymphoid precursors, RAG mediated integration risk is minimized in pluripotent stem or reproductive cells, reducing germline risks.

[0021] Amyloidosis targeting: In certain embodiments, the DNA payload encodes a B-cell receptor (BCR), T-cell receptor (TCR), or antibody variable region that specifically binds to aggregated amyloid proteins. These include, but are not limited to:

[0022] • Transthyretin (TTR) fibrils associated with ATTR amyloidosis.

[0023] • Immunoglobulin light-chain fibrils associated with AL amyloidosis.

[0024] • Other amyloidogenic aggregates associated with systemic or localized amyloidosis.

[0025] The encoded receptors can be designed to recognize conformational epitopes unique to aggregated proteins, enabling discrimination from monomeric precursors.

[0026] Broader therapeutic applications: Beyond amyloidosis, the invention can be applied to:

[0027] • Oncology: generating TCRs, CARs, or BCRs that recognize tumor-associated antigens.

[0028] • Infectious diseases: producing receptors or antibodies targeting viral proteins, bacterial toxins, or fungal antigens.

[0029] • Autoimmunity: engineering regulatory B-cells or T-cells that express tolerogenic factors to suppress pathogenic immune responses.

[0030] • Protein misfolding disorders: targeting aggregated proteins in diseases such as Parkinson’s or Alzheimer’s.

[0031] Method of Use: Administration of the composition results in targeted delivery to lymphocyte precursors in vivo. Endogenous RAG1 / RAG2 recognizes the engineered RSS and integrates the therapeutic sequence into the genome. As the modified precursors commit to the B-cell or T-cell lineage, they undergo clonal expansion and differentiation to generate large populations of effector cells capable of executing the desired therapeutic function. Because this occurs in vivo, the need for ex vivo cell manipulation is eliminated.

[0032] Brief Description of the Drawings FIG. 1 illustrates an exemplary lipid nanoparticle (10) surface-functionalized with targeting antibodies (12), encapsulating a plasmid DNA payload (14, 18). The plasmid includes engineered recombination signal sequences (20, 22, 24) flanking a therapeutic gene (26).

[0033] Detailed Description of the Drawings

[0034] Referring now to FIG. 1, the lipid nanoparticle (10) comprises a bilayer structure formulated from ionizable lipids, cholesterol, DSPC, and PEG-lipids. The nanoparticle is decorated with targeting antibodies (12) to enable selective delivery to lymphocyte precursors expressing corresponding surface markers.

[0035] Encapsulated within the nanoparticle is at least one plasmid DNA payload (14, 18). The plasmid contains engineered recombination signal sequences (20, 22, 24), each flanking a therapeutic gene (26). The therapeutic gene may encode a B-cell receptor, T-cell receptor, chimeric antigen receptor, antibody, cytokine, or tolerogenic factor, depending on the desired application.

[0036] Upon systemic administration, the lipid nanoparticle delivers the plasmid into lymphocyte precursors. Endogenous RAG1 and RAG2 proteins recognize the engineered RSS sequences (20, 22, 24) and mediate targeted integration of the therapeutic gene (26) into the genome. Because RAG1 / 2 expression is restricted to committed lymphoid precursors, integration does not occur in stem cells or reproductive cells. The modified precursors subsequently undergo clonal expansion and differentiation, producing effector lymphocytes with the encoded therapeutic function.

[0037] Sequence Listing

[0038] A sequence listing is submitted herewith in XML format, compliant with WIPO Standard ST.26, and is incorporated by reference in its entirety. The listing provides engineered recombination signal sequences (RSS) that include at least one non-natural nucleotide. In the listing, the nonnatural nucleotide is represented as “n” to indicate a position that is not a standard adenine (A), thymine (T), cytosine (C), or guanine (G).

[0039] The sequences disclosed herein are exemplary only. Although the listing shows embodiments with a single non-natural nucleotide, in certain embodiments the RSS spacer may comprise two or more non-natural nucleotides. The scope of the invention is not limited to the specific sequences disclosed but encompasses all variants incorporating one or more non-natural nucleotides that retain recognition by RAG1 and RAG2 recombinase.

Claims

Claims1. A composition comprising a lipid nanoparticle (LNP) encapsulating a DNA molecule flanked by engineered recombination signal sequences (RSS), wherein the DNA molecule comprises at least one non-natural nucleotide within an RSS spacer.

2. The composition of claim 1, wherein the LNP is surface-functionalized with a targeting moiety that binds a lymphocyte precursor marker.

3. The composition of claim 2, wherein the targeting moiety binds one or more of Surrogate Light Cham (SLC), CD 10, IL-7 receptor, CD 19, c-KIT (CD 117), CXCR4, or CD90 (Thy-1).

4. The composition of claim 1, wherein the DNA molecule encodes a therapeutic polypeptide selected from the group consisting of: a B-cell receptor (BCR), a T-cell receptor (TCR), a chimeric antigen receptor (CAR), a cytokine, an antibody, or a tolerogenic factor.

5. The composition of claim 1, wherein the LNP comprises an ionizable lipid, cholesterol, DSPC, and a PEG-lipid.

6. The composition of claim 1, wherein the LNP further comprises a biodegradable ionizable lipid to reduce tissue accumulation.

7. The composition of claim 1, wherein the DNA molecule further comprises a promoter operably linked to the therapeutic sequence.

8. The composition of claim 1, wherein the DNA molecule further comprises a self-cleaving or excisable cassette for controlled removal of the integrated sequence.

9. The composition of claim 1 , wherein the DNA molecule further comprises a molecular barcoding sequence to enable in vivo tracking.

10. The composition of claim 1 , wherein the DNA molecule further comprises redundant RSS spacer variants to reduce off-target integration.I L A method of modifying lymphocyte precursors in vivo, comprising administering the composition of claim 1 to a subject in need thereof.

12. The method of claim 11, wherein the administration is intravenous.

13. The method of claim 11, wherein the administration is intranodal, intramedullary, or intraperitoneal.

14. The method of claim 11, wherein the modified lymphocyte precursors undergo clonal expansion and differentiation in vivo.

15. The method of claim 11, wherein therapeutic effect is achieved at a lower systemic dose compared to viral vector-based therapies.

16. The composition of claim 1, wherein the non-natural nucleotide in the RSS spacer is configured to reduce off-target integration relative to a natural RSS spacer.

17. The composition of claim 1 , wherein the DNA molecule further comprises a genetic insulator element to prevent activation of adjacent endogenous genes.

18. The composition of claim 1, wherein the DNA pay load includes a self-deletion cassette for controlled removal of the integrated sequence upon administration of a secondary stimulus.

19. The composition of claim 1, wherein the non-natural nucleotide is selected to minimize recognition by human DNA repair enzymes, thereby reducing off-target recombination.

20. The composition of claim 1, wherein the targeting moiety is a humanized antibody fragment to reduce immunogenicity.

21. The composition of claim 1, wherein the DNA pay load includes a suicide gene operably linked to an inducible promoter to allow selective elimination of modified cells if adverse effects occur.

22. The method of claim 11, wherein administration is monitored by a companion diagnostic assay detecting successful integration events.

23. The method of claim 11, wherein the genetic modification is reversible using a predefined secondary treatment.

24. The method of claim 11, wherein the subject is pre-screened for pre-existing immunity to LNP excipients to minimize adverse events.

25. The method of claim 11, wherein systemic biodistribution is monitored using an integrated molecular barcoding sequence.

26. The composition of claim 1 , wherein integration occurs only in lymphoid lineage cells due to the requirement of RAG1 and RAG2 expression, thereby preventing integration in stem cells or reproductive cells.

27. The composition of claim 1, wherein the DNA molecule encodes a B-cell receptor (BCR) or antibody variable region that specifically binds to an aggregated amyloid protein.

28. The composition of claim 27, wherein the aggregated amyloid protein is a transthyretin (TTR) fibril.

29. The composition of claim 27, wherein the aggregated amyloid protein is an immunoglobulin light-chain fibril associated with AL amyloidosis.

30. The composition of claim 27, wherein the encoded receptor or antibody binds conformational epitopes unique to aggregated amyloid fibrils and not the corresponding monomeric protein.

31. A method of treating amyloidosis in a subject, comprising administering the composition of claim 27, wherein the modified lymphocyte precursors, once committed to the B-cell lineage, undergo clonal expansion in vivo to produce populations of B-cells that secrete antibodies specific for aggregated amyloid deposits and mediate clearance of amyloid fibrils.

32. A method of treating cancer in a subject, comprising administering the composition of claim 1, wherein the DNA payload encodes a receptor that specifically binds to a tumor- associated antigen.

33. A method of treating a viral infection in a subject, comprising administering the composition of claim 1, wherein the DNA pay load encodes a receptor or antibody that specifically binds to a viral protein.

34. A method of treating an autoimmune disorder in a subject, comprising administering the composition of claim 1, wherein the DNA pay load encodes a tolerogenic gene circuit that suppresses pathogenic immune responses.

35. A method of treating a protein misfolding disorder in a subject, comprising administering the composition of claim 1, wherein the DNA payload encodes a receptor that recognizes aggregated proteins associated with the disorder.

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