B cell compositions and methods of use

Abstract

The present disclosure relates to a method of reducing urine glycosaminoglycans (GAG) or cerebral spinal fluid (CSF) glycosaminoglycans (GAG) in a subject, comprising: administering a single dose of a population of modified B cells to the subject.

Description

PCT / US25 / 50889 14 October 2025 (14.10.2025)Attorney Docket No. 43632-02150 (IMCO-015 / 01 WO)B CELL COMPOSITIONS AND METHODS OF USERELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 707,106, filed on October 14, 2024, the contents of which are incorporated by reference herein in their entirety.INCOPORATION-BY-REFERENCE OF SEQUENCE LISTING

[0002] The Sequence Listing XML associated with this application is provided electronically in XML file format and is hereby incorporated by reference into the specification. The name of the XML file containing the Sequence Listing XML is “IMCO- 015_01WO_SeqList_ST26.xml”. The XML file is 16,792 bytes, created on October 13, 2025, and is being submitted electronically.TECHNICAL FIELD

[0003] The present disclosure relates to the use of B cells for long term in vivo delivery of a therapeutic agent, such as an antigen-specific antibody or protein (e.g., an enzyme), and in particular to administering single and multiple dosages of the B cells.BACKGROUND

[0004] Current methods for treating chronic diseases and disorders include direct infusion of a therapeutic agent (e.g., enzyme replacement therapy), gene therapy via a viral vector, and adoptive transfer of stem cells (e.g., hematopoietic stem cell transfer). However, each of these methods have disadvantages. Injection of a recombinant therapeutic protein suffers from the finite half-life of the protein, and all three methods provide sub-optimal tissue penetration by the therapeutic agent. Altering endogenous tissues to produce a therapeutic agent, such as via injection of recombinant adeno-associated virus (AAV) and lentiviral vectors, generally results in the therapeutic agent being produced from a centralized location. Production of the therapeutic agent from one location increases the chances for localized toxicity in the producing tissues. Additionally, as recombinant viruses are viewed as foreign, it is unlikely viral vectors can be administered multiple times without causing an adverse reaction, meaning that there is a single injection opportunity to achieve the correct dosage of the therapeutic agent. Given the biological variation inherent in a procedure such as in vivo introduction of nucleic acids intoPCT / US25 / 50889 14 October 2025 (14.10.2025) cells using a virus, it would be very tenuous to achieve a desired dosage under the constraints of a single injection.

[0005] Recently, the use of differentiated B cell compositions for long term in vivo expression of a transgene has been identified as a promising strategy for the treatment of various diseases and disorders. However, methods for administering modified B cells for delivery of therapeutic agents have not yet been described in order to achieve therapeutically effective levels of the agents in vivo.

[0006] Accordingly, there still remains a need in the art for the long-term treatment for many chronic diseases and disorders. The present disclosure provides methods for administering and dosing genetically modified B cell compositions for treating chronic diseases and disorders. The present disclosure provides these and other advantages as described in the detailed description.SUMMARY

[0007] In some aspects, disclosed herein is a method of reducing the level of urine glycosaminoglycans (GAG), comprising: administering a single dose of a population of modified B cells to a subject, wherein the dose is 2.5 x 107cells / Kg of body weight of the subject. In some embodiments, six months after administration of the single dose of a population of modified B cells the urine GAG level in the subject administered the single dose of a population of modified B cells is below the upper limit of normal for the patient .

[0008] In some aspects, disclosed herein is a method of reducing the level of cerebral spinal fluid (CSF) heparan sulfate (HS), comprising: administering a single dose of a population of modified B cells to a subject wherein the dose is 2.5 x 107cells / kg of body weight of the subject. In some embodiments, six months after administration of the single dose of a population of modified B cells the CSF HS level in the subject administered the single dose of a population of modified B cells is 75% the CSF HS level compared to a control subject that is not administered the single dose of a population of modified B cells after 6 months of treatment. In some embodiments, six months after administration of the single dose of a population of modified B cells the CSF HS level in the subject administered the single dose of a population of modified B cells is at or below the upper limit of normal for the patient CSF / mL of CSF. In some embodiments, the population of modified B cells express iduronidase (IDUA). In some embodiments, the subject is suffering from Hurler-Scheie or Scheie forms of Mucopolysaccharidosis type I (MPS I). In some embodiments, the subject is an adult. In some embodiments, the subject has previously been treated with enzyme replacement therapy. InPCT / US25 / 50889 14 October 2025 (14.10.2025) some embodiments, the subject was previously treated with a-L-iduronidase. In some embodiments, six months after administration of the single dose of a population of modified B cells the subject administered the single dose of a population of modified B cells has improved flexibility in the shoulder flexion as compared to a control subject that is not administered the single dose of a population of modified B cells. In some embodiments, six months after administration of the single dose of a population of modified B cells the flexibility in the shoulder flexion of the subject administered the single dose of a population of modified B cells has an increase of 25% in degree of flexibility as compared to a control subject that is not administered the single dose of a population of modified B cells. In some embodiments, six months after administration of the single dose of a population of modified B cells the subject administered the single dose of a population of modified B cells can walk a longer distance in the six-minute walk test (6MWT) as compared to a control subject that is not administered the single dose of a population of modified B cells. In some embodiments, six months after administration of the single dose the subject administered the single dose of a population of modified B cells can walk 25% longer distance in the 6MWT as compared to a control subject that is not administered the single dose of a population of modified B cells. In some embodiments, the subject: a) is diagnosed with MPS I Hurler-Scheie or Scheie syndrome; b) is currently receiving an approved IDUA therapy; c) has two or more of the following disease related complications: i) liver volume greater than 3% of body weight; ii) has <80% decreased respiratory function by forced vital capacity (FVC) of predicted normal value; iii) walks <400 meters in 6-minute walk test (6MWT); or iv) joint restriction of greater than 200 in shoulder flexion; d) has evidence of disease-related complications including at least one of: i) evidence of moderate / severe corneal clouding; ii) history of hydrocephalus or increased ventricular size on MRI; iii) documented thickening of the mitral valve on echocardiogram; iv) evidence of dysostosis multiplex, including:(1) prior or ongoing carpal tunnel issues, or affected digits;(2) significant restriction of range of motion at the mandible, shoulder, elbow, or hip;(3) prior or continued moderate kyphosis (>20 degrees of angulation);PCT / US25 / 50889 14 October 2025 (14.10.2025)(4) lower extremity skeletal disease requiring prior surgical intervention at the knee (valgus deformity) or hip (dysplasia including the femur and / or acetabulum); or(5) history of impaired growth, as defined as height below 5thpercentile for age; e) is > 18 years of age at time of study registration; f) has a creatinine clearance, calculated or measured directly (Cockroft-Gualt), that is >60ml creatine / min / 1.73m2; g) has an absolute neutrophil count >2,500 cells / mm3; h) has an IgG count of > 610mg IgG protein / dl of blood; i) has CD3, CD4, CD8, and CD19 counts above lower limit of normal; j) has a platelet count >150,000 cells / pl of blood; k) has a hemoglobin count >11.7 gm / dl of blood for females or >13.3 gm / dl of blood for males; l) has an alanine aminotransferase (ALT) count <200, or bilirubin <2 (unless there is a history of Gilberts syndrome, or a similar diagnosis); and m) has an ejection fraction > 40% by echocardiogram.

[0009] In some embodiments, the population of modified B cells comprises a polynucleotide that comprises the pKT2 / EEK-IDUA-DHFR transposon. In some embodiments, the population of modified B cells comprises a polynucleotide that is 95% identical to SEQ ID NO: 1. In some embodiments, the population of modified B cells comprises a polynucleotide that is 100% identical to SEQ ID NO: 1. In some embodiments, the subject is selected for treatment with the dose comprising determining the expression of one or more biomarkers.

[0010] Any of the above aspects, or any other aspect described herein, can be combined with any other aspect.

[0011] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In the Specification, the singular forms also include the plural unless the context clearly dictates otherwise; as examples, the terms “a,” “an,” and “the” are understood to be singular or plural and the term “or” is understood to be inclusive. By way of example, “an element” means one or more element. Throughout the specification the word “comprising,” or variations such as “comprises” or “comprising,” will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, orPCT / US25 / 50889 14 October 2025 (14.10.2025)0.01% of the stated value. Unless otherwise clear from the context, all numerical values provided herein are modified by the term “about.”

[0012] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The references cited herein are not admitted to be prior art to the claimed invention. In the case of conflict, the present Specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting. Other features and advantages of the disclosure will be apparent from the following detailed description and claim.BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIG. 1 is a diagram of Sleeping Beauty (SB) transposon and transposase constructs / map for transposition and expression of human IDUA. IDUA is regulated by the EEK promoter (see Example 1). A bidirectional promoter that incorporates an EFla promoter upstream of EEK regulates transcription of a drug resistant human L22Y-F31S dihydrofolate reductase (DHFR) in the opposite direction. A CMV-regulated SBIOOx provides SB transposase activity. Capped and polyadenylated SBIOOx-encoding mRNA was generated by in vitro transcription, provided from TriLink. Arrows: direction of transcription. The boxes with dark triangles are T2 SB inverted repeat / direct repeat (IR / DR) sequences. pA, polyadenylation signal.

[0014] FIG. 2 shows the measurement of glycosaminoglycan (GAG) amounts in urine samples from Subject #1, who was administered ISP-001 cells, as described in Example 4.

[0015] FIG. 3 shows measurements of heparan sulfate in CSF samples obtained from Subject #1, who was administered ISP-001 cells, as described in Example 4.

[0016] FIG. 4A-FIG. 4C shows functional endpoint measurements from Subject #1, who was administered ISP-001 cells, as described in Example 4.DETAILED DESCRIPTION

[0017] The present invention utilizes autologous and / or allogeneic B cells that have been altered through introduction of nucleic acids to produce a therapeutic agent and relates to methods of administering the modified B cells to subjects in need thereof. In some embodiments, the terms “engineered B cell,” “genetically engineered B cell,” “modified B cell,” and “genetically modified B cell” are used interchangeably herein to refer to such altered B cells that comprise one or more nucleic acids (e.g., a transgene) to produce a therapeuticPCT / US25 / 50889 14 October 2025 (14.10.2025) agent (e.g., a transgene that enables expression of a polypeptide such as a therapeutic polypeptide). Specifically, the modified B cells can be administered as a single dosage or multiple dosages. Unexpectedly, it was found that certain B cell dosages produce greater than expected levels of therapeutic agent in comparison to other dosages.

[0018] Mucopolysaccharidosis Type I (MPS I)

[0019] Mucopolysaccharidosis Type I (MPS I) results from the enzymatic deficiency of a- L-iduronidase which causes lysosomal accumulation of dermatan sulfate (DS) and heparan sulfate (HS) in various tissues, resulting in multisystem disease. The estimated prevalence is 1 / 88,000. MPS I is clinically divided into 3 categories: the severe form of MPS I Hurler (MPS IH), and intermediate phenotypes which have been described as Hurler-Scheie (MPS IH / S), and Scheie (MPS IS).

[0020] Patients with Hurler-Scheie and Scheie syndrome suffer from significant and life-long morbidity and an impaired ability to carry out daily activities. Over 80% of Scheie patients suffer from at least one of the following symptoms / findings: cardiac valve abnormalities, joint contractures, and corneal clouding. Roughly half suffer from all three. Other symptoms reported in this population include carpal tunnel syndrome, hernia, coarse facial features, hepatomegaly, chronic ear, nose, and throat and respiratory problems, including sleep apnea and recurrent otitis media. Scheie patients also require a number of surgeries in their lifetime with 40% of children reporting two or more surgeries by 10 years of age. Despite low to no cognitive impairment, Hurler-Scheie patients have relatively severe somatic symptoms and a limited life expectancy to the 2nd or 5th decade in the absence of treatment with enzyme replacement therapy (ERT).

[0021] ERT provides clinical benefit over untreated disease progression, but it has significant limitations. ERT has a very short half-life and is therefore clinically delivered with a pattern of episodic overall exposure. This results in limited penetration into a number of tissues. Due to the short half-life, treatment with ERT requires weekly infusions lasting from 3 to 4 hours leading to a substantial burden on patients and their families.

[0022] Patients suffering from an MPS 1 disorder typically have one or more of the following clinical features: short stature; macrocephaly; coarse face; full cheeks; hearing loss; recurrent ear infections; cloudy corneas; glaucoma; retinal degeneration; low nasal bridge; anteverted nostrils; broad nasal tip; full lips; gum hypertrophy; enlarged tongue; hypertrophy of alveolar ridge; small teeth; misaligned teeth; short neck; endocardial fibroelastosis; cardiomyopathy; aortic regurgitation; mitral regurgitation; mitral valve thickening; aortic valve thickening; narrow coronary arteries; thickened coronary arteries; frequent upper and lower respiratoryPCT / US25 / 50889 14 October 2025 (14.10.2025) tract infections; enlarged tonsils; enlarged adenoids; enlarged vocal cords; narrow trachea; thickened mainstem bronchi; chronic obstructive airway disease; oar-shaped ribs (narrow at vertebral end, broad at sternal end); short, thick, irregular clavicles; Umbilical hernia; hepatomegaly; splenomegaly; inguinal hernia; joint stiffness; joint contractures; dysostosis multiplex; Premature closure of the metopic suture; premature closure of the sagittal suture; j- shaped sella turcica; hydrocephalus; odontoid hypoplasia; dysplastic vertebral bodies; gibbus; coxa valga; flared iliac wings; small femoral heads; bullet-shaped phalanges; carpal tunnel syndrome; claw-hand deformity; dermal melanocytosis; hirsutism; developmental delay evident by 12-24 months of age; progressive mental deterioration; mental retardation; neurodegeneration; excretion of dermatan sulfate and heparan sulfate in urine; or alpha-L- iduronidase activity is <1%.

[0023] Definitions

[0024] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used in the specification and appended claims, unless specified to the contrary, the following terms have the meaning indicated. With regard to this specification, any time a definition of a term as defined herein, differs from a definition given for that same term in an incorporated reference, the definition explicitly defined herein is the correct definition of the term.

[0025] The words “a” and “an” denote one or more, unless specifically noted.

[0026] “About” is meant to refer to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. In any embodiment discussed in the context of a numerical value used in conjunction with the term “about,” it is specifically contemplated that the term about can be omitted.

[0027] Unless the context requires otherwise, throughout the present specification and claims, the word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be construed in an open and inclusive sense, that is, as “including, but not limited to”.

[0028] By “consisting of’ is meant including, and limited to, whatever follows the phrase “consisting of.” Thus, the phrase “consisting of’ indicates that the listed elements are required or mandatory and that no other elements may be present. By “consisting essentially of’ is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements.PCT / US25 / 50889 14 October 2025 (14.10.2025)Thus, the phrase “consisting essentially of’ indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.

[0029] Reference throughout this specification to “biological activity” or “bioactivity” refers to any response induced in an in vitro assay or in a cell, tissue, organ, or organism, (e.g., an animal, or a mammal, or a human) as the result of administering any compound, agent, polypeptide, conjugate, pharmaceutical composition contemplated herein. Biological activity may refer to agonistic actions or antagonistic actions. The biological activity may be a beneficial effect; or the biological activity may not be beneficial, i.e. a toxicity. In some embodiments, biological activity will refer to the positive or negative effects that a drug or pharmaceutical composition has on a living subject, e.g., a mammal such as a human. Accordingly, the term “biologically active” is meant to describe any compound (i.e. ISP-001) possessing biological activity, as herein described. Biological activity may be assessed by any appropriate means currently known to the skilled artisan. Such assays may be qualitative or quantitative. The skilled artisan will readily appreciate the need to employ different assays to assess the activity of different polypeptides; a task that is routine for the average researcher. Such assays are often easily implemented in a laboratory setting with little optimization requirements, and more often than not, commercial kits are available that provide simple, reliable, and reproducible readouts of biological activity for a wide range of polypeptides using various technologies common to most labs. When no such kits are available, ordinarily skilled researchers can easily design and optimize in-house bioactivity assays for target polypeptides without undue experimentation; as this is a routine aspect of the scientific process.

[0030] An “increased” or “enhanced” amount is typically a “statistically significant” amount, and may include an increase that is 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, or 50 or more times (e.g., 100, 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 2.1, 2.2, 2.3, 2.4, etc.) an amount or level described herein. Similarly, a “decreased” or “reduced” or “lesser” amount is typically a “statistically significant” amount, and may include a decrease that is about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, or 50 or more times (e.g., 100, 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) an amount or level described herein.

[0031] A “subject,” as used herein, includes any animal that exhibits a disease or symptom, or is at risk for exhibiting a disease or symptom, which can be treated with an agent of the invention. Suitable subjects include laboratory animals (such as mouse, rat, rabbit, or guineaPCT / US25 / 50889 14 October 2025 (14.10.2025) pig), farm animals, and domestic animals or pets (such as a cat or dog). Non-human primates and, preferably, human patients, are included.

[0032] “Pharmaceutical composition” refers to a formulation of a modified compound (e.g. a ISP-001) and a medium generally accepted in the art for the delivery of the compound to an animal, e.g., humans. Such a medium may include any pharmaceutically acceptable carriers, diluents, or excipients, therefore.

[0033] “Pharmaceutically effective excipients” and “pharmaceutically effective carriers” are well known to those of skill in the art, and methods for their preparation are also readily apparent to the skilled artisan. Such compositions, and methods for their preparation, may be found, e.g., in Remington’s Pharmaceutical Sciences, 19th Edition (Mack Publishing Company, 1995, incorporated herein).

[0034] The terms “polynucleotide”, “nucleotide”, “nucleotide sequence”, and “nucleic acid” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three dimensional structure, and may perform any function known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may include non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.

[0035] “Substantially” or “essentially” means of ample or considerable amount, quantity, size; nearly totally or completely; for instance, 95% or greater of some given quantity.

[0036] “Therapeutic agent” refers to any compound that, when administered to a subject, (e.g., preferably a mammal, more preferably a human), in a therapeutically effective amount is capable of effecting treatment of a disease or condition as defined below. In some embodiments, the compound is a modified B cell. In some embodiments, the compound is ISP- 001.

[0037] “Therapeutically effective amount” or “Therapeutically effective dose” refers to an amount of a compound (i.e. ISP-001) of the invention that, when administered to a subject, (e.g., preferably a mammal, more preferably a human), is sufficient to effect treatment, asPCT / US25 / 50889 14 October 2025 (14.10.2025) defined below, of a disease or condition in the animal. The amount of a compound of the invention that constitutes a “therapeutically effective amount” will vary depending on the compound, the condition and its severity, the manner of administration, and the age of the animal to be treated, but can be determined routinely by one of ordinary skill in the art having regard to his own knowledge and to this disclosure.

[0038] “ Treatment” or “treating” refers to the administration of an effective amount of a compound (i.e. ISP-001) according to the present disclosure with the purpose of easing, ameliorating, arresting, eradicating (curing) or preventing symptoms or disease states.

[0039] As used herein, the terms “disease,” “disorder,” and “condition” may be used interchangeably or may be different in that the particular malady, injury or condition may not have a known causative agent (so that etiology has not yet been worked out), and it is, therefore, not yet recognized as an injury or disease but only as an undesirable condition or syndrome, wherein a more or less specific set of symptoms have been identified by clinicians.

[0040] “MPS I” as used herein, refers to a group of diseases that result in the buildup of glycosaminoglycans (GAGs), or mucopolysaccharides due to a deficiency of alpha-L iduronidase. In some embodiments, MPS I can include Hurler syndrome (MPS IH) (OMIM: 607014). In some embodiments, MPS I can include Scheie Syndrome (MPS IS) (OMIM: 607016). In some embodiments, MPS I can include Hurler-Scheie Syndrome (MPS IH / S)(0MIM: 607015).

[0041] The practice of the present invention will employ, unless indicated specifically to the contrary, conventional methods of molecular biology, recombinant DNA techniques, protein expression, and protein / peptide / carbohydrate chemistry within the skill of the art, many of which are described below for the purpose of illustration. Such techniques are explained fully in the literature. See, e.g., Sambrook, et al., Molecular Cloning: A Laboratory Manual (3rd Edition, 2000); DNA Cloning: A Practical Approach, vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., 1984); Oligonucleotide Synthesis: Methods and Applications (P. Herdewijn, ed., 2004); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985); Nucleic Acid Hybridization: Modern Applications (Buzdin and Lukyanov, eds., 2009); Transcription and Translation (B. Hames & S. Higgins, eds., 1984); Animal Cell Culture (R. Freshney, ed., 1986); Freshney, R.I. (2005) Culture of Animal Cells, a Manual of Basic Technique, 5th Ed. Hoboken NJ, John Wiley & Sons; B. Perbal, A Practical Guide to Molecular Cloning (3rd Edition 2010); Farrell, R., RNA Methodologies: A Laboratory Guide for Isolation and Characterization (3rd Edition 2005). The publications discussed above are provided solely for their disclosure before the filing date of the present application. Nothing herein is to bePCT / US25 / 50889 14 October 2025 (14.10.2025) construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0042] Modified B cells of the present disclosure

[0043] In some aspects, the modified B cells of the present disclosure are allogeneic.

[0044] In some aspects, the modified B cells of the present disclosure are autologous.

[0045] In some aspects, the modified B cells of the present disclosure have been modified such that the B cells express and secrete iduronidase (IDUA), or a biologically active fragment thereof.

[0046] An exemplary amino acid sequence for IDUA (including an N-terminal signal peptide) is shown below:MRPLRPRAALLALLASLLAAPPVAPAEAPHLVQVDAARALWPLRRFWRSTGFCPPLPHSQAD QYVLSWDQQLNLAYVGAVPHRGIKQVRTHWLLELVTTRGSTGRGLSYNFTHLDGYLDLLREN QLLPGFELMGSASGHFTDFEDKQQVFEWKDLVSSLARRYIGRYGLAHVSKWNFETWNEPDHH DFDNVSMTMQGFLNYYDACSEGLRAASPALRLGGPGDSFHTPPRSPLSWGLLRHCHDGTNFF TGEAGVRLDYISLHRKGARSS IS ILEQEKWAQQIRQLFPKFADTPIYNDEADPLVGWSLPQPWRADVTYAAMWKVIAQHQNLLLANTTSAFPYALLSNDNAFLSYHPHPFAQRTLTARFQVN NTRPPHVQLLRKPVLTAMGLLALLDEEQLWAEVSQAGTVLDSNHTVGVLASAHRPQGPADAW RAAVLIYASDDTRAHPNRSVAVTLRLRGVPPGPGLVYVTRYLDNGLCSPDGEWRRLGRPVFP TAEQFRRMRAAEDPVAAAPRPLPAGGRLTLRPALRLPSLLLVHVCARPEKPPGQVTRLRALP LTQGQLVLVWSDEHVGSKCLWTYEIQFSQDGKAYTPVSRKPSTFNLFVFSPDTGAVSGSYRVRALDYWARPGPFSDPVPYLEVPVPRGPPSPGNP ( SEQ ID NO : 2 )

[0047] An exemplary amino acid sequence for IDUA without the N-terminal signal peptide is shown below:APHLVQVDAARALWPLRRFWRSTGFCPPLPHSQADQYVLSWDQQLNLAYVGAVPHRGIKQVR THWLLELVTTRGSTGRGLSYNFTHLDGYLDLLRENQLLPGFELMGSASGHFTDFEDKQQVFE WKDLVSSLARRYIGRYGLAHVSKWNFETWNEPDHHDFDNVSMTMQGFLNYYDACSEGLRAAS PALRLGGPGDSFHTPPRSPLSWGLLRHCHDGTNFFTGEAGVRLDYISLHRKGARSS IS ILEQ EKWAQQIRQLFPKFADTPI YNDEADPLVGWSLPQPWRADVTYAAMWKVIAQHQNLLLANTTSAFPYALLSNDNAFLSYHPHPFAQRTLTARFQVNNTRPPHVQLLRKPVLTAMGLLALLDEE QLWAEVSQAGTVLDSNHTVGVLASAHRPQGPADAWRAAVLIYASDDTRAHPNRSVAVTLRLR GVPPGPGLVYVTRYLDNGLCSPDGEWRRLGRPVFPTAEQFRRMRAAEDPVAAAPRPLPAGGR LTLRPALRLPSLLLVHVCARPEKPPGQVTRLRALPLTQGQLVLVWSDEHVGSKCLWTYEIQF SQDGKAYTPVSRKPSTFNLFVFSPDTGAVSGSYRVRALDYWARPGPFSDPVPYLEVPVPRGPPSPGNP ( SEQ ID NO : 3 )

[0048] In some aspects, the modified B cells have been modified such that the B cells express and secrete IDUA using a Sleeping Beauty (SB) transposon system. As would be appreciated by the skilled artisan, SB is a non-viral vector system for gene therapy, which combines the advantages of an integrating vector with the simplicity of uncomplexed DNA. The SB transposon system consists of two components: a transposon containing a gene expressionPCT / US25 / 50889 14 October 2025 (14.10.2025) cassette flanked by inverted repeat / direct repeats (IR / DRs) and a source of transposase enzyme. Further discussion of the Sleeping Beauty transposon is described in Example 1.

[0049] Accordingly, in some aspects, the modified B cells of the present disclosure have been modified to express and secrete IDUA by introducing into the B cells a transposon comprising a gene expression cassette flanked by inverted repeat / direct repeats (IR / DRs) and mRNA encoding a SB transposase, including, but not limited to, SBIOOx (see Jin Z, Maiti S, Huis H, et al. The hyperactive Sleeping Beauty transposase SBIOOX improves the genetic modification of T cells to express a chimeric antigen receptor. Gene Ther. 2011;18(9):849-856).

[0050] In some aspects, the gene expression cassette comprises an EEK promoter. In some aspects, an EEK promoter comprises, consists essentially of, or consists of a nucleic acid sequence that is at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or at least 100% identical to the nucleic acid sequence put forth below:T AAAC C G G T GAG T T T C AT G G T T AC T T G C C T GAGAAGAT T AAAAAAAG T AAT G C T AC C T T AT G AGGGAGAGTCCCAGGGACCAAGATAGCAACTGTCATAGCAACCGTCACACTGCTTTGGTCAA GGAGAAGACCCTTTGGGGAACTGAAAACAGAACCTTGAGCACATCTGTTGCTTTCGCTCCCA TCCTCCTCCAACAGGGCTGGGTGGAGCACTCCACACCCTTTCACCGGTCGTACGGCTCAGCC AGAGTAAAAATCACACCCATGACCTGGCCACTGAGGGCTTGATCAATTCACTTTGAATTTGG CAT T AAAT AC CAT T AAG G TAT AT T AAC T GAT T T T AAAAT AAGAT AT AT T C G T GAC C AT G T T T T T AAC T T T C AAAAAT G T AG C T G C C AG T G T G T GAT T T T AT T T C AG T T G T AC AAAAT AT C T AAA C C T AT AG C AAT G T GAT T AAT AAAAAC T T AAAC AT AT T T T C C AG TACCTTAATTCTGTGATAG GAAAAT T T T AAT C T GAG TATTTTAATTTCATAATCTC T AAAAT AG TTTAATGATTTGTCATT GTGTTGCTGTCGTTTACCCCAGCTGATCTCAAAAGTGATATTTAAGGAGATTATTTTGGTCT GCAACAACTTGATAGGGCTCAGCCTCTCCCACCCAACGGGTGGAATCCCCCAGAGGGGGATT TCCAAGAGGCCACCTGGCAGTTGCTGAGGGTCAGAAGTGAAGCTAGCCACTTCCTCTTAGGC AGGTGGCCAAGATTACAGTTGACCCGTACGTGCAGCTGTGCCCAGCCTGCCCCATCCCCTGC TCATTTGCATGTTCCCAGAGCACAACCTCCTGCCCTGAAGCCTTATTAATAGGCTGGTCACA CTTTGTGCAGGAGTCAGACTCAGTCAGGACACAGCT ( SEQ ID NO : 4 ) .

[0051] In some aspects, the gene expression cassette comprises a nucleic acid sequence that encodes for IDUA. Accordingly, in some aspects, the gene expression cassette comprises a nucleic acid sequence that encodes for the amino acid sequence of SEQ ID NO: 2.

[0052] In some aspects, a nucleic acid encoding for IDUA comprises, consists essentially of, or consists of a nucleic acid sequence that is at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or at least 100% identical to the nucleic acid sequence put forth below:ATGCGTCCCCTGCGCCCCCGCGCCGCGCTGCTGGCGCTCCTGGCCTCGCTCCTGGCCGCGCC CCCGGTGGCCCCGGCCGAGGCCCCGCACCTGGTGCAGGTGGACGCGGCCCGCGCGCTGTGGC CCCTGCGGCGCTTCTGGAGGAGCACAGGCTTCTGCCCCCCGCTGCCACACAGCCAGGCTGAC CAGTACGTCCTCAGCTGGGACCAGCAGCTCAACCTCGCCTATGTGGGCGCCGTCCCTCACCGPCT / US25 / 50889 14 October 2025 (14.10.2025)CGGCATCAAGCAGGTCCGGACCCACTGGCTGCTGGAGCTTGTCACCACCAGGGGGTCCACTG GACGGGGCCTGAGCTACAACTTCACCCACCTGGACGGGTACCTGGACCTTCTCAGGGAGAAC CAGCTCCTCCCAGGGTTTGAGCTGATGGGCAGCGCCTCGGGCCACTTCACTGACTTTGAGGA CAAGCAGCAGGTGTTTGAGTGGAAGGACTTGGTCTCCAGCCTGGCCAGGAGATACATCGGTA GGTACGGACTGGCGCATGTTTCCAAGTGGAACTTCGAGACGTGGAATGAGCCAGACCACCAC GACTTTGACAACGTCTCCATGACCATGCAAGGCTTCCTGAACTACTACGATGCCTGCTCGGA GGGTCTGCGCGCCGCCAGCCCCGCCCTGCGGCTGGGAGGCCCCGGCGACTCCTTCCACACCC CACCGCGATCCCCGCTGAGCTGGGGCCTCCTGCGCCACTGCCACGACGGTACCAACTTCTTC ACTGGGGAGGCGGGCGTGCGGCTGGACTACATCTCCCTCCACAGGAAGGGTGCGCGCAGCTC CATCTCCATCCTGGAGCAGGAGAAGGTCGTCGCGCAGCAGATCCGGCAGCTCTTCCCCAAGT TCGCGGACACCCCCATTTACAACGACGAGGCGGACCCGCTGGTGGGCTGGTCCCTGCCACAG CCGTGGAGGGCGGACGTGACCTACGCGGCCATGGTGGTGAAGGTCATCGCGCAGCATCAGAA CCTGCTACTGGCCAACACCACCTCCGCCTTCCCCTACGCGCTCCTGAGCAACGACAATGCCT TCCTGAGCTACCACCCGCACCCCTTCGCGCAGCGCACGCTCACCGCGCGCTTCCAGGTCAAC AACACCCGCCCGCCGCACGTGCAGCTGTTGCGCAAGCCGGTGCTCACGGCCATGGGGCTGCT GGCGCTGCTGGATGAGGAGCAGCTCTGGGCCGAAGTGTCGCAGGCCGGGACCGTCCTGGACA GCAACCACACGGTGGGCGTCCTGGCCAGCGCCCACCGCCCCCAGGGCCCGGCCGACGCCTGG CGCGCCGCGGTGCTGATCTACGCGAGCGACGACACCCGCGCCCACCCCAACCGCAGCGTCGC GGTGACCCTGCGGCTGCGCGGGGTGCCCCCCGGCCCGGGCCTGGTCTACGTCACGCGCTACC TGGACAACGGGCTCTGCAGCCCCGACGGCGAGTGGCGGCGCCTGGGCCGGCCCGTCTTCCCC ACGGCAGAGCAGTTCCGGCGCATGCGCGCGGCTGAGGACCCGGTGGCCGCGGCGCCCCGCCC CTTACCCGCCGGCGGCCGCCTGACCCTGCGCCCCGCGCTGCGGCTGCCGTCGCTTTTGCTGG TGCACGTGTGTGCGCGCCCCGAGAAGCCGCCCGGGCAGGTCACGCGGCTCCGCGCCCTGCCC CTGACCCAAGGGCAGCTGGTTCTGGTCTGGTCGGATGAACACGTGGGCTCCAAGTGCCTGTG GACATACGAGATCCAGTTCTCTCAGGACGGTAAGGCGTACACCCCGGTCAGCAGGAAGCCAT CGACCTTCAACCTCTTTGTGTTCAGCCCAGACACAGGTGCTGTCTCTGGCTCCTACCGAGTT CGAGCCCTGGACTACTGGGCCCGACCAGGCCCCTTCTCGGACCCTGTGCCGTACCTGGAGGT CCCTGTGCCAAGAGGGCCCCCATCCCCGGGCAATCCA ( SEQ ID NO : 5 )

[0053] Accordingly, the present disclosure provides a transposon comprising an expression cassette comprising an EEK promoter, a nucleic acid sequence encoding for IDUA, and nucleic acid sequence encoding a poly- A tail. Such a transposon is illustrated in FIG. 1 as pKT2ZEEK- IDUA.

[0054] In some aspects, the B cells of the present disclosure can be cultured with one or more activating factors. In some aspects, the one or more activating factors can comprise CD40L, IL-2, IL-4, IL- 10, IL- 15, IL-21, a CD40L cross-linking agent, or any combination thereof. In some aspects, the one or more activating factors comprise each of CD40L, IL-2, IL-4, IL- 10, IL-15, IL-21, and a CD40L cross-linking agent. In some aspects, the CD40L crosslinking agent can be a CD40L cross-linking antibody. In some aspects, the B cells are cultured with the one or more activating factors prior to the introduction of the transposon system.

[0055] In some aspects, the B cells of the present disclosure can be differentiated by culturing with one or more B cell activating factors. In some aspects, the B cell activating factors that are used to differentiate the B cells comprise CD40L, CpG, IFN-a, IFN-6, IL-2, IL-6, IL- 10, IL- 15, or any combination thereof. In some aspects, the B cell activating factors that are usedPCT / US25 / 50889 14 October 2025 (14.10.2025) to differentiate the B cells comprise each of CD40L, CpG, IFN-a, IFN-6, IL-2, IL-6, IL- 10 and IL-15.

[0056] The B cells of the present disclosure can be prepared using any of the methods described in US Patent Publication No. US20230081163A1 and PCT Application Publication No. W02016100932A1.

[0057] Accordingly, in some aspects, the modified B cells of the present disclosure are produced by a method comprising: a) isolating pan-B cells, memory B cells or switched memory B cells from a sample, thereby obtaining an isolated B cell population; b) culturing the isolated B cell population in vitro with CD40L, IL-2, IL-4, IL- 10, IL- 15, IL-21, and a CD40L crosslinking agent, thereby obtaining an expanded B cell population; c) transfecting the expanded B cell population with a transposon system; and d) differentiating the expanded B cell population in vitro with one or more B cell activating factors, thereby obtaining a modified B cell composition.

[0058] In some aspects, the isolated B cell population comprises B cells that are CD20+, CD27+, and CD 138-. In some aspects, the isolated B cell population comprises B cells that are CD20+ and IgG+. In some aspects, the isolated B cell population comprises B cells that are CD20-, CD38- and CD 138-.

[0059] In some aspects, the sample comprises whole blood or peripheral blood mononuclear cells (PBMCs).

[0060] In some aspects, isolating can comprise: 1) depleting CD3+ and CD56+ cells and 2) enriching for CD27+ cells.

[0061] In some aspects, the CD40L is sCD40L-his.

[0062] In some aspects, the one or more activating factors of the culturing step comprise CD40L, IL-2, IL-4 and IL- 10.

[0063] In some aspects, feeder cells are absent from the culturing steps.

[0064] In some aspects, the expanded B cell population is migratory.

[0065] In some aspects, cells of the expanded B cell population preferably migrate toward CXCL12.

[0066] In some aspects, at least 20% of the cells of the expanded B cell population are migratory.

[0067] In some aspects, the one or more B cell activating factors of the differentiating step comprise one or more of CD40L, CpG, IFN-a, IFN-8, IL-2, IL-6, IL-10 and IL-15. In some aspects, the one or more B cell activating factors of the differentiating step comprise each of CD40L, CpG, IFN-a, IFN-5, IL-2, IL-6, IL- 10 and IL-15.PCT / US25 / 50889 14 October 2025 (14.10.2025)

[0068] In some aspects, the modified B cell populations of the present disclosure comprise B cells that are CD20-, CD38+, and CD 138+ and / or B cells that are CD20-, CD38+, and CD138-.

[0069] Accordingly, in some aspects, ISP-001 cells are modified B cells that are produced using the methods described above such that an expression cassette comprising an EEK promotor and a nucleic acid sequence encoding IDUA has been inserted into the genome of the B cells using a SB transposon system.

[0070] Also provided is the exemplary bifunctional transposon (pKT2ZEEK-IDUA-DHFR), Figure 1) encoding human IDUA along with a human dihydrofolate reductase synthesized to encode a novel variant enzyme (L22Y, F31 S) that is resistant to the folate antagonist methotrexate (MTX: (Mcivor RS. 1996. Bone Marrow Transplantation 18:SS0-54.)) is prepared. The sequence for the plasmid is provided as SEQ ID NO: 1. Cells modified with this plasmid can be referred to as “ISP-001.” In some embodiments, a cell can be modified with a plasmid that has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1 :C C T G GAT C C AGAT C C C TAT AC AG T T GAAG T C G GAAG T T TAG AT AC AC T T AAG T T G GAG T C AT T AAAAC T C G T T T T T C AAC T AC T C C AC AAAT T T C T T G T T AAC AAAC AAT AG T T T T G G C AAG T C AG T TAG GAC AT CTACTTTGTGCAT GAC AC AAG TCATTTTTC C AAC AAT T G T T T AC AGAC AGA T T AT T T C AC T TAT AAT T C AC T G T AT C AC AAT T C C AG T G G G T C AGAAG T T TAG AT AC AC T AAG TTGACTGTGCCTTTAAACAGCTTGGAAGCTGCGCACTAGGCAAGTTAACTAACTCCTCTGAA T G T C AG TATTTCCATCTG T AAGAT GAAC AC AG TGGGGCTC C AAT T C C AT AC C AC AT T T G T AG AG G T T T T AC T T G C T T T AAAAAAC C T C C C AC AC C T C C C C C T GAAC C T GAAAC AT AAAAT GAAT GCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCAT C AC AAAT T T C AC AAAT AAAG C AT T T T T T T C AC T G C AT T C T AG T T G T G G T T T G T C C AAAC T C A TCAATGTATCTTATCATGTCTGGCCAGCTAGAGCGGCCGCTTAATCATTCTTCTCATATACT TCAAATTTGTACTTAATGCCTTTCTCCTCCTGGACATCAGAGAGAACACCTGGGTATTCTGG CAGAAGTTTATATTTCTCCAAATCAATTTCTGGAAAAAACGTGTCACTTTCAAAGTCTTGCA TGATCCTTGTCACAAATAGTTTAAGATGGCCTGGGTGATTCATGGCTTCCTTATAAACAGAA CTGCCACCAACTATCCAGACCATGTCTACTTTATTTGCTAATTCTGGTTGTTCAGTAAGTTT TAAGGCATCATCTAGACTTCTGGAAAGAAAATGAGCTCCTTGTGGAGGTTCCTTGAGTTCTC T G C T GAGAAC T AAAT T AAT T C TAG C C T T T AAAG G T C GAT T C T T C T C AG GAAT G GAGAAC C AG GTCTTCTTACCCATAATCACCAGATTCTGTTTACCTTCTACTGAAGAGGTTGTGGTCATTCT CTGGAAATATCTGGATTCATTCCTGAGCGGTGGCCAGGGATAGTCCCCGTTCTTGCCGATGC CCATGTTCTGGGACACAGCGACGATGCAGTTTAGCGAACCAACCATGATGGAAGCTACTGTA CACCAACCTGTCAGGAGAGGAAAGAGAAGAAGGTTAGTACAATTGTCTAGGGCTGCAGGGTT CATAGTGCCACTTTTCCTGCACTGCCCCATCTCCTGCCCACCCTTTCCCAGGCATAGACAGT CAGT GAC T TACCAAAC T C AC AG G AG G GAG AAG G GAG AAG C T T GAAT GT T CACAGAGAC TAG T GCACTTATATATGGTTCTCCCCCACCCTGGGGAAAAAGGTGGAGCCAGTACACCACATCACT TTCCCAGTTTACCCAAGCCCCACCTTCTCTAGGCACCAGTTCAATTGCCCACCCCTCCCCCC AACTTCTCAGGGACTGTGGGCCATGTGCTCTCTGCCCACTGAGGGGCACTCAGCCCTCAAGC ATGCTCTTCTCCACTAGTCACCCCTATTGACCTTATGTATGTGCCAATAATGGGAAAAACCC ATTGACTCACCCCCTATTGACCTTTTGTACTGGGCAAAACCCAATGGAAAGTCCCTATTGAC TCAGTGTACTTGGCTCCAATGGGACTTTCCTGTTGATTGGCGCGCCCGGGGGATCCAGTTTGPCT / US25 / 50889 14 October 2025 (14.10.2025)G T T AAT T AAAC C G G T GAG T T T C AT G G T T AC T T G C C T GAGAAGAT T AAAAAAAG T AAT G C T AC CTTATGAGGGAGAGTCCCAGGGACCAAGATAGCAACTGTCATAGCAACCGTCACACTGCTTT GGTCAAGGAGAAGACCCTTTGGGGAACTGAAAACAGAACCTTGAGCACATCTGTTGCTTTCG CTCCCATCCTCCTCCAACAGGGCTGGGTGGAGCACTCCACACCCTTTCACCGGTCGTACGGC TCAGCCAGAGTAAAAATCACACCCATGACCTGGCCACTGAGGGCTTGATCAATTCACTTTGA ATTTGGCAT T AAAT AC CAT T AAG G TAT AT T AAC T GAT T T T AAAAT AAGAT AT AT T C G T GAC C ATGTTTTTAACTTTCAAAAATGTAGCTGCCAGTGTGTGATTTTATTTCAGTTGTACAAAATA T C T AAAC C TAT AG C AAT G T GAT T AAT AAAAAC T T AAAC AT AT T T T C C AG TACCTTAATTCTG T GAT AG GAAAAT T T T AAT C T GAG TATTTTAATTTCATAATCTC T AAAAT AG TTTAATGATTT GTCATTGTGTTGCTGTCGTTTACCCCAGCTGATCTCAAAAGTGATATTTAAGGAGATTATTT TGGTCTGCAACAACTTGATAGGGCTCAGCCTCTCCCACCCAACGGGTGGAATCCCCCAGAGG GGGATTTCCAAGAGGCCACCTGGCAGTTGCTGAGGGTCAGAAGTGAAGCTAGCCACTTCCTC TTAGGCAGGTGGCCAAGATTACAGTTGACCCGTACGTGCAGCTGTGCCCAGCCTGCCCCATC CCCTGCTCATTTGCATGTTCCCAGAGCACAACCTCCTGCCCTGAAGCCTTATTAATAGGCTG GTCACACTTTGTGCAGGAGTCAGACTCAGTCAGGACACAGCTCTAGAGTCGAGAATTCGGCC ATGCGTCCCCTGCGCCCCCGCGCCGCGCTGCTGGCGCTCCTGGCCTCGCTCCTGGCCGCGCC CCCGGTGGCCCCGGCCGAGGCCCCGCACCTGGTGCAGGTGGACGCGGCCCGCGCGCTGTGGC CCCTGCGGCGCTTCTGGAGGAGCACAGGCTTCTGCCCCCCGCTGCCACACAGCCAGGCTGAC CAGTACGTCCTCAGCTGGGACCAGCAGCTCAACCTCGCCTATGTGGGCGCCGTCCCTCACCG CGGCATCAAGCAGGTCCGGACCCACTGGCTGCTGGAGCTTGTCACCACCAGGGGGTCCACTG GACGGGGCCTGAGCTACAACTTCACCCACCTGGACGGGTACCTGGACCTTCTCAGGGAGAAC CAGCTCCTCCCAGGGTTTGAGCTGATGGGCAGCGCCTCGGGCCACTTCACTGACTTTGAGGA CAAGCAGCAGGTGTTTGAGTGGAAGGACTTGGTCTCCAGCCTGGCCAGGAGATACATCGGTA GGTACGGACTGGCGCATGTTTCCAAGTGGAACTTCGAGACGTGGAATGAGCCAGACCACCAC GACTTTGACAACGTCTCCATGACCATGCAAGGCTTCCTGAACTACTACGATGCCTGCTCGGA GGGTCTGCGCGCCGCCAGCCCCGCCCTGCGGCTGGGAGGCCCCGGCGACTCCTTCCACACCC CACCGCGATCCCCGCTGAGCTGGGGCCTCCTGCGCCACTGCCACGACGGTACCAACTTCTTC ACTGGGGAGGCGGGCGTGCGGCTGGACTACATCTCCCTCCACAGGAAGGGTGCGCGCAGCTC CATCTCCATCCTGGAGCAGGAGAAGGTCGTCGCGCAGCAGATCCGGCAGCTCTTCCCCAAGT TCGCGGACACCCCCATTTACAACGACGAGGCGGACCCGCTGGTGGGCTGGTCCCTGCCACAG CCGTGGAGGGCGGACGTGACCTACGCGGCCATGGTGGTGAAGGTCATCGCGCAGCATCAGAA CCTGCTACTGGCCAACACCACCTCCGCCTTCCCCTACGCGCTCCTGAGCAACGACAATGCCT TCCTGAGCTACCACCCGCACCCCTTCGCGCAGCGCACGCTCACCGCGCGCTTCCAGGTCAAC AACACCCGCCCGCCGCACGTGCAGCTGTTGCGCAAGCCGGTGCTCACGGCCATGGGGCTGCT GGCGCTGCTGGATGAGGAGCAGCTCTGGGCCGAAGTGTCGCAGGCCGGGACCGTCCTGGACA GCAACCACACGGTGGGCGTCCTGGCCAGCGCCCACCGCCCCCAGGGCCCGGCCGACGCCTGG CGCGCCGCGGTGCTGATCTACGCGAGCGACGACACCCGCGCCCACCCCAACCGCAGCGTCGC GGTGACCCTGCGGCTGCGCGGGGTGCCCCCCGGCCCGGGCCTGGTCTACGTCACGCGCTACC TGGACAACGGGCTCTGCAGCCCCGACGGCGAGTGGCGGCGCCTGGGCCGGCCCGTCTTCCCC ACGGCAGAGCAGTTCCGGCGCATGCGCGCGGCTGAGGACCCGGTGGCCGCGGCGCCCCGCCC CTTACCCGCCGGCGGCCGCCTGACCCTGCGCCCCGCGCTGCGGCTGCCGTCGCTTTTGCTGG TGCACGTGTGTGCGCGCCCCGAGAAGCCGCCCGGGCAGGTCACGCGGCTCCGCGCCCTGCCC CTGACCCAAGGGCAGCTGGTTCTGGTCTGGTCGGATGAACACGTGGGCTCCAAGTGCCTGTG GACATACGAGATCCAGTTCTCTCAGGACGGTAAGGCGTACACCCCGGTCAGCAGGAAGCCAT CGACCTTCAACCTCTTTGTGTTCAGCCCAGACACAGGTGCTGTCTCTGGCTCCTACCGAGTT CGAGCCCTGGACTACTGGGCCCGACCAGGCCCCTTCTCGGACCCTGTGCCGTACCTGGAGGT CCCTGTGCCAAGAGGGCCCCCATCCCCGGGCAATCCATGAGCCTGTGCTGAGCCCCAGTGGG ATCCTCTAGAGTCGAGAATTCACTCCTCAGGTGCAGGCTGCCTATCAGAAGGTGGTGGCTGG TGTGGCCAATGCCCTGGCTCACAAATACCACTGAGATCTTTTTCCCTCTGCCAAAAATTATG GGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATT GCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCAPCT / US25 / 50889 14 October 2025 (14.10.2025)TTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCATATGCTGGCTGC CATGAACAAAGGTGGCTATAAAGAGGTCATCAGTATATGAAACAGCCCCCTGCTGTCCATTC CTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTTTTTATATTTTGTTTTGTGTTAT TTTTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTTACTAGCCAGATTTTTCCTCCTC TCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGAAGATCCCTCGACCTGCATAC CGGTCAAGCTAGCGATATCAATTAACCCTCACTAAAGGGAGACCAAGTTAAACAATTTAAAG G C AAT G C T AC C AAAT AC T AAT T GAG T G T AT G T AAAC T T C T GAG C C AC T G G GAAT G T GAT GAA AGAAAT AAAAGC T GAAAT GAAT CAT T C T C T C TAG TAT TAT T C T GAT AT T T CACAT T C T T AAA ATAAAGTGGTGATCCTAACTGACCTAAGACAGGGAATTTTTACTAGGATTAAATGTCAGGAA TTGTGAAAAAGTGAGTTTAAATGTATTTGGCTAAGGTGTATGTAAACTTCCGACTTCAACTG TATAGGGATCTGGTACCATTTAAATCTGTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCG GTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGA ATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTA AAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAAT CGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCC TGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCT TTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTG TAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGC CTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAG CAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAG TGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCC AGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCG GTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCT TTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGT CAT GAGAT TAT C AAAAAG GAT C T T C AC C T AGAT CCTTTTTGC C AG T G T T AC AAC C AAT T AAC CAAT T C T GAT TAGAAAAAC T CAT CGAGCAT CAAAT GAAAC T GCAAT T TAT T CAT AT CAGGAT TAT CAAT AC CATATTTTT GAAAAAG CCGTTTCTGTAAT GAAG GAGAAAAC T C AC C GAG G C AG TTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACA ACCTATTAATTTCCCCTCGT C AAAAAT AAG G T T AT C AAG T GAGAAAT C AC CAT GAG T GAC GA CTGAATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAG CCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGC CTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCA ACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCT AATACCTGGAATGCTGTTTTTCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGT ACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCA TCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCA TCGGGCTTCCCATACAAGCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCA TTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTCGACGTTTCCCGTT GAAT AT G G C T CAT AAC AC CCCTTGTATTACTGTTTATG T AAG C AGAC AG TTTTATTGTTCAT GATGCA ( SEQ ID NO : 1 )

[0071] Without wishing to be bound by theory, engineered and differentiated B cells (i.e. plasmablasts and plasma cells) are an excellent vehicle for the delivery of therapeutic proteins for the following reasons: i) they continuously secrete very high levels of engineered protein product; ii) they can migrate to and occupy diverse tissues; and iii) they may survive for years in vivo. An important property of the precursors to plasmablasts (e.g. CD19+cells) is their ability to sustain large-scale expansion both in vivo and in vitro - a highly beneficial property for the production of therapeutic B cells at a dosage sufficient for human application. GivenPCT / US25 / 50889 14 October 2025 (14.10.2025) the normal role of B cells in producing proteins (i.e. antibodies) in vivo, they represent an excellent cell type for the prospect of sustained delivery of IDUA that could more effectively treat MPS IH, MPS IH / S, and MPS IS.

[0072] Methods to produce ISP-001 cells are disclosed in the Examples disclosed herein.

[0073] Suitable formulations for ISP-001 cells, or modified B cells, for use in the present disclosure have been described in WO2018 / 201071, incorporated herein in its entirety. In some embodiments, ISP-001 or modified B cells are suspended in phosphate buffered solution (PBS). In some embodiments, ISP-001 or modified B cells are suspended in 0.1-10% human serum albumin (HSA). In some embodiments, ISP-001 or modified B cells are suspended in PBS and 1% HSA.

[0074] Methods of treatment and reducing levels of specific biomarkers

[0075] The present disclosure provides methods of treating MPS I in a subject comprising administering a B cell genetically modified to express IDUA (IDUA+ B cells; (“ISP-001” or “ISP-001 cells”)) to a subject having, or suspected of having, MPS I. In some embodiments, a single, maximally effective dose of ISP-001 cells is administered to the subject. In some embodiments, two or more doses of ISP-001 cells are administered to the subject, thereby maximizing the amount of engrafted ISP-001 cells. In some embodiments, the two or more doses of ISP-001 cells that are administered to the subject comprise less ISP-001 cells than the single, maximally effective dose of ISP-001 cells. In some embodiments, when two or more doses of ISP-001 cells are administered to a subject at a dosage of ISP-001 cells that is below the maximally effective single dose of ISP-001 cells, a resultant synergistic increase in IDUA production occurs.

[0076] In one embodiment, administering ISP-001 cells to a subject results in normal levels of IDUA seen in a healthy, control subject. In one embodiment, administering ISP-001 cells to a subject results in greater than normal levels of IDUA in the subject. In one embodiment, administering ISP-001 cells to a subject reduces levels of GAGs in the subject to a normal level. In one embodiment, administering ISP-001 cells to a subject reduces levels of GAGs in the subject to less than a normal level of GAGs in the subject.

[0077] The present disclosure provides methods of treating MPS I in a subject, the method comprising administering to the subject at least one population of modified B cells of the present disclosure. The present disclosure provides at least one population of modified B cells for use in treating MPS I in a subject. The present disclosure provides the use of at least one population of modified B cells in the manufacture of a medicament for the treatment of MPS I in a subject.PCT / US25 / 50889 14 October 2025 (14.10.2025)

[0078] The present disclosure provides methods of reducing the level of urine glycosaminoglycans (GAGs) in a subject, the method comprising administering to the subject at least one population of modified B cells of the present disclosure. The present disclosure provides at least one population of modified B cells for use in reducing the level of urine glycosaminoglycans (GAGs) in a subject. The present disclosure provides the use of at least one population of modified B cells in the manufacture of a medicament for the reduction in the level of urine glycosaminoglycans (GAGs) in a subject.

[0079] The present disclosure provides methods of reducing the level of heparin sulfate (HS) in the CSF of a subject, the method comprising administering to the subject at least one population of modified B cells of the present disclosure. The present disclosure provides at least one population of modified B cells for use in reducing the level of HS in the CSF of a subject. The present disclosure provides the use of at least one population of modified B cells in the manufacture of a medicament for the reduction in the level of HS in the CSF of a subject.

[0080] The present disclosure provides methods of increasing the mobility of a subject having MPS I, the method comprising administering to the subject at least one population of modified B cells of the present disclosure. The present disclosure provides at least one population of modified B cells for use in increasing the mobility of a subject having MPS I. The present disclosure provides the use of at least one population of modified B cells in the manufacture of a medicament for improving the mobility of a subject having MPS I.

[0081] In some aspects, the methods and uses comprising the populations of modified B cells of the present disclosure further comprise the administration of at least one enzyme replacement therapy to the subject. In some aspects, the administration of a population of modified B cells of the present disclosure and at least one enzyme replacement therapy is performed in temporal proximity.

[0082] Accordingly, the present disclosure provides methods of treating MPS I in a subject, the method comprising administering to the subject a combination of at least one population of modified B cells of the present disclosure and at least one enzyme therapy. The present disclosure provides a combination of at least one population of modified B cells of the present disclosure and at least one enzyme therapy for use in treating MPS I in a subject. The present disclosure provides the use of a combination of at least one population of modified B cells of the present disclosure and at least one enzyme therapy in the manufacture of a medicament for the treatment of MPS I in a subject.

[0083] The present disclosure provides at least one population of modified B cells for use in a method of treating MPS I in a subject, wherein the method further comprises administeringPCT / US25 / 50889 14 October 2025 (14.10.2025) to the subject at least one enzyme replacement therapy. The present disclosure provides the use of at least one population of modified B cells in the manufacture of a medicament for the treatment of MPS I in a subject, wherein the treatment further comprises administering to the subject at least one enzyme replacement therapy.

[0084] The present disclosure provides at least one enzyme replacement therapy for use in a method of treating MPS I in a subject, wherein the method further comprises administering to the subject at least one population of modified B cells of the present disclosure. The present disclosure provides the use of at least one enzyme replacement therapy in the manufacture of a medicament for the treatment of MPS I in a subject, wherein the treatment further comprises administering to the subject at least one population of modified B cells of the present disclosure.

[0085] In some aspects, the population of modified B cells of the present disclosure can be ISP-001 cells.

[0086] Dosages

[0087] ISP-001 may be administered with any suitable dosing interval, for example a dosing interval of about 1 day to 1 month, for example a dosing interval of about 1 to 21 days, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 days or a dose interval of between about 7 and 21 days, such as 7, 10 or 14 days. In some embodiment, ISP-001 is dosed only once.

[0088] In some embodiments, the ISP-001 cells are administered at a dose between about IxlO7cells / kg and 9xl07cells / kg per administration, such as between about 2.5 xlO7cells / kg and 5 xlO7cells / kg per administration. In some embodiments, the does is 2.5 xlO7cells / kg.

[0089] Any and all sub-ranges within the foregoing ranges are also envisaged and form part of the invention.

[0090] For the following dosages, cells / kg refers to the number of modified B cells per kg of bodyweight of the subject to which the B cells are being administered.

[0091] In some aspects, a population of modified B cells of the present disclosure comprises about 2.0 x 107cells / kg to about 3.0 x 107cells / kg. In some aspects, a population of modified B cells of the present disclosure comprises about 2.5 x 107cells / kg. That is, the modified B cells of the present disclosure can be administered at a dose of about 2.0 x 107cells / kg to about 3.0 x 107cells / kg, or at a dose of about 2.5 x 107cells / kg.

[0092] In some aspects, a population of modified B cells of the present disclosure comprises about 4.0 x 107cells / kg to about 6.0 x 107cells / kg. In some aspects, a population of modified B cells of the present disclosure comprises about 5.0 x 107cells / kg. That is, the modified BPCT / US25 / 50889 14 October 2025 (14.10.2025) cells of the present disclosure can be administered at a dose of about 4.0 x 107cells / kg to about 6.0 x 107cells / kg, or at a dose of about 5.0 x 107cells / kg.

[0093] In some aspects, a population of modified B cells of the present disclosure comprises about 7.0 x 107cells / kg to about 8.0 x 107cells / kg. In some aspects, a population of modified B cells of the present disclosure comprises about 7.5 x 107cells / kg. That is, the modified B cells of the present disclosure can be administered at a dose of about 7.0 x 107cells / kg to about 8.0 x 107cells, or at a dose of about 7.5 x 107cells / kg.

[0094] In some aspects, a population of modified B cells of the present disclosure comprises about 9.0 x 107cells / kg to about 1.1 x 108cells / kg. In some aspects, a population of modified B cells of the present disclosure comprises about 1.0 x 108cells / kg. That is, the modified B cells of the present disclosure can be administered at a dose of about 9.0 x 107cells / kg to about 1.1 x 108cells / kg, or at a dose of about 1.0 x 108cells / kg.

[0095] In some aspects, a population of modified B cells of the present is administered intravenously.

[0096] In some aspects, a subject can receive one or more populations of modified B cells of the present disclosure (i.e. the subject can receive multiple doses of the modified B cells of the present disclosure).

[0097] In aspects, wherein a subject receives more than one population of modified B cells of the present disclosure (i.e. more than one dose of modified B cells of the present disclosure), the administration of two populations can be separated by at least about one month, at least about two months, at least about three months, at least about four months, at least about five months, at least about six months, at least about seven months, at least about eight months, at least about nine months, at least about 10 months, at least about 11 months, at least about 12 months, at least about 13 months, at least about 14 months, at least about 15 months, at least about 16 months, at least about 17 months, at least about 18 months, at least about 19 months, at least about 20 months, at least about 21 months, at least about 22 months, at least about 23 months, or at least about 24 months.

[0098] In some aspects, the administration of two populations (i.e. doses) can be separated by about one month, about two months, about three months, about four months, about five months, about six months, about seven months, about eight months, about nine months, about 10 months, about 11 months, about 12 months, about 13 months, about 14 months, about 15 months, about 16 months, about 17 months, about 18 months, about 19 months, about 20 months, about 21 months, about 22 months, about 23 months, or about 24 months. In some aspects, the administration of two populations (i.e. doses) can be separated by about 12 months.PCT / US25 / 50889 14 October 2025 (14.10.2025)In some aspects, the administration of two populations (i.e. doses) can be separated by about 18 months.

[0099] In aspects wherein the subject receives more than one population of modified B cells of the present disclosure (i.e. more than one dose of modified B cells of the present disclosure), the individual doses administered to the subject can comprise different amounts of cells.

[0100] In a non-limiting example, a subject may receive an initial dose of modified B cells of the present disclosure, and then any subsequent doses of the modified B cells of the present disclosure will comprise less cells than the initial dose. In some aspects, a subject may receive an initial dose of about 7.5 x 107cells / kg and then receive at least one subsequent dose of about 5.0 x 107cells / kg. In some aspects, a subject may receive an initial dose of about 7.5 x 107cells / kg and then receive at least one subsequent dose of about 2.5 x 107cells / kg. In some aspects, a subject may receive an initial dose of about 5.0 x 107cells / kg and then receive at least one subsequent dose of about 2.5 x 107cells / kg.

[0101] In a non-limiting example, a subject may receive an initial dose of modified B cells of the present disclosure, and then any subsequent doses of the modified B cells of the present disclosure will comprise more cells than the initial dose. In some aspects, a subject may receive an initial dose of about 2.5 x 107cells / kg and then receive at least one subsequent dose of about 5.0 x 107cells / kg. In some aspects, a subject may receive an initial dose of about 2.5 x 107cells / kg and then receive at least one subsequent dose of about 7.5 x 107cells / kg. In some aspects, a subject may receive an initial dose of about 5.0 x 107cells / kg and then receive at least one subsequent dose of about 7.5 x 107cells / kg.

[0102] Enzyme Replacement Therapy

[0103] In some aspects, the at least one enzyme replacement therapy can be laronidase (sold under the tradename Aldurazyme ®).

[0104] In some aspects, laronidase can be administered to the subject intravenously.

[0105] In some aspects, laronidase can be administered to the subject in an amount of about 0.58 mg / kg of body weight. In some aspects, laronidase can be administered to the subject about once weekly.

[0106] In some aspects wherein a subject is less than about 20 kg in body weight, laronidase should be injected with a total volume of about 100 ml. In some aspects wherein is greater than about 20 kg in body weight, laronidase should be injected with a total volume of about 250 ml. In some aspects, the laronidase should be administered to the subject over the course of about 3 hours to about 4 hours.PCT / US25 / 50889 14 October 2025 (14.10.2025)

[0107] As used herein, the term “temporal proximity” refers to that administration of one therapeutic agent (e.g., a population of modified B cells of the present disclosure) occurs within a time period before or after the administration of another therapeutic agent (e.g., an enzyme replacement therapy), such that the therapeutic effect of the one therapeutic agent overlaps with the therapeutic effect of the other therapeutic agent. In some embodiments, the therapeutic effect of the one therapeutic agent completely overlaps with the therapeutic effect of the other therapeutic agent. In some embodiments, “temporal proximity” means that administration of one therapeutic agent occurs within a time period before or after the administration of another therapeutic agent, such that there is a synergistic effect between the one therapeutic agent and the other therapeutic agent. “Temporal proximity” may vary according to various factors, including but not limited to, the age, gender, weight, genetic background, medical condition, disease history, and treatment history of the subject to which the therapeutic agents are to be administered; the disease or condition to be treated or ameliorated; the therapeutic outcome to be achieved; the dosage, dosing frequency, and dosing duration of the therapeutic agents; the pharmacokinetics and pharmacodynamics of the therapeutic agents; and the route(s) through which the therapeutic agents are administered. In some embodiments, “temporal proximity” means within 15 minutes, within 30 minutes, within an hour, within two hours, within four hours, within six hours, within eight hours, within 12 hours, within 18 hours, within 24 hours, within 36 hours, within 2 days, within 3 days, within 4 days, within 5 days, within 6 days, within a week, within 2 weeks, within 3 weeks, within 4 weeks, with 6 weeks, or within 8 weeks. In some embodiments, multiple administration of one therapeutic agent can occur in temporal proximity to a single administration of another therapeutic agent. In some embodiments, temporal proximity may change during a treatment cycle or within a dosing regimen.

[0108] During the course of treatment in the methods and uses of the present disclosure, administration of the at least one enzyme replacement therapy can be discontinued and / or resumed at one or more time points. In some aspects, the discontinuation or resumption of the at least one enzyme replacement therapy can be based on one or more clinical measurements obtained from the subject, including but not limited to, functional measurements (e.g. mobility measurements such as the 6 minute walk test) and clinical biomarkers (IDUA levels, glycosaminoglycan amounts, etc.).

[0109] Indications and Subjects

[0110] In some embodiments, the present disclosure provides methods of treating MPS I in a subject in need thereof. In some embodiments, the present disclosure provides methods ofPCT / US25 / 50889 14 October 2025 (14.10.2025) treating MPS IS in a subject in need thereof. In some embodiments, the present disclosure provides methods of treating MPS IH in a subject in need thereof. In some embodiments, the present disclosure provides methods of treating MPS IH / S in a subject in need thereof.[OHl] In some embodiments, treating refers to the treatment of a MPS I or an associated disorder (e.g., MPS IS, MPS IH, or MPS IH / S) in a subject, e.g., in a human, including (a) inhibiting the progression of the MPS I or associated disorder; or (b) relieving the one or more symptoms of the MPS I or an associated disorder. In some embodiments, treatment may refer to a short-term (e.g., temporary and / or acute) and / or a long-term (e.g., sustained) reduction in one or more symptoms. In some embodiments, treatment results in an improvement or remediation of the symptoms. The improvement is an observable or measurable improvement or may be an improvement in the general feeling of well-being of the subject.

[0112] In some embodiments, the subject may be a neonate, a juvenile, or an adult.

[0113] Administration of ISP-001 cells described herein can occur by injection, irrigation, inhalation, consumption, electro-osmosis, hemodialysis, iontophoresis, and other methods known in the art. In some embodiments, administration route is local or systemic. In some embodiments administration route is intraarterial, intracranial, intradermal, intraduodenal, intrammamary, intrameningeal, intraperitoneal, intrathecal, intramuscular, intratumoral, intravenous, intravitreal, ophthalmic, parenteral, spinal, subcutaneous, ureteral, urethral, vaginal, or intrauterine.

[0114] In some embodiments, the administration route is by infusion (e.g., continuous or bolus). Examples of methods for local administration, that is, delivery to the site of injury or disease, include through an Ommaya reservoir, e.g. for intrathecal delivery (See e.g., US Patent Nos. 5,222,982 and 5,385,582, incorporated herein by reference); by bolus injection, e.g. by a syringe, e.g. into a joint; by continuous infusion, e.g. by cannulation, such as with convection (See e.g., US Patent Application Publication No. 2007-0254842, incorporated herein by reference); or by implanting a device upon which the agents have been reversibly affixed (see e.g. US Patent Application Publication Nos. 2008-0081064 and 2009-0196903, incorporated herein by reference). In some embodiments, the administration route is by topical administration or direct injection.

[0115] In some embodiments, subjects are selected for treatment according to the methods described herein by one or more exclusion and / or inclusion criteria. Inclusion / exclusion criteria may include: age, sex, prior treatment regimens (e.g., a-L-aronidase (AEDURAZYME)), presence of malignancies other than MPS I or an associated disorder, and general health status.PCT / US25 / 50889 14 October 2025 (14.10.2025)

[0116] In some embodiments, subjects are selected for treatment with the ISP-001 cells if they meet all of the following inclusion criteria (that is to say, the subject has the recited biomarker, the subject has been selected to have the recited biomarker or the method comprises the additional step of determining whether the subject has the recited biomarker and treating the subject with ISP-001 cells if so):• Diagnosis of Mucopolysaccharidosis type I Hurler-Scheie or Scheie syndrome.• Currently receiving an approved IDUA therapy• Two or more of the following disease related complications:. Abnormal liver size by CT (liver volume greater than 3% of body weight), or Decreased Respiratory Function by FVC (<80% of predicted normal value), or. Decreased mobility as shown by 6MWT (<400 meters), or. Decreased mobility as shown by shoulder flexion (joint restriction of greater than 20)• Evidence of disease-related complications. These include one or more of the following:. Evidence of moderate / severe corneal clouding, or. History of hydrocephalus or increased ventricular size on MRI, or. Documented thickening of the mitral valve on echocardiogram, or. Evidence of dysostosis multiplex, includingPrior or ongoing carpal tunnel issues, or affected digits, or Significant restriction of range of motion at the mandible, shoulder, elbow, or hip, orPrior or continued moderate kyphosis (>20 degrees of angulation), orLower extremity skeletal disease requiring prior surgical intervention at the knee (valgus deformity) or hip (dysplasia including the femur and / or acetabulum, orHistory of impaired growth, as defined as height below 5thpercentile for age• Willing and able to provide written, signed informed consent after the nature of the study has been explained and prior to any study-related procedures. Ability to provide written consent will be determined by a score of >15 as wellPCT / US25 / 50889 14 October 2025 (14.10.2025) as correct responses on questions #6, 7, and 9 on the University of California, San Diego Brief Assessment of Capacity to Consent (UBACC).• Age > 18 years at time of study registration.• Creatinine clearance: calculated or measured directly (Cockroft-Gualt), that is >60ml / min / l ,73m2.• Absolute Neutrophil Count >2,500 cells / mm3.• IgG > 610mg / dl.• CD3, CD4, CD8, and CD 19 above lower limit of normal.• Platelet count >150,000 cells / pl.• Hemoglobin >11.7 gm / dl for females and >13.3 gm / dl for males.• ALT<200, or bilirubin <2 (unless there is a history of Gilberts syndrome, or a similar diagnosis).• Ejection fraction > 40% by echocardiogram.• Must commit to traveling to the study site for the necessary follow-up evaluations.• Must agree to stay <45-minute drive from the study site for a minimum of 5 days after each cell infusion.• Females of childbearing potential must have a negative serum pregnancy test at the screening visit and have a negative pregnancy result on Day -1. If the Day -1 urine result is positive, repeat testing with a serum pregnancy test will be conducted.• All sexually active subj ects must be willing to use a medically accepted method of barrier contraception from the screening visit until 24 weeks after ISP-001 administration.

[0117] In some embodiments, subjects are excluded from treatment with ISP-001 cells if they meet any one of the following exclusion criteria (that is to say, the subject has the recited biomarker, the subject has been selected to have the recited biomarker or the method comprises the additional step of determining whether the subject has the recited biomarker and excluding the subject from treatment with ISP-001 cells if so):• Known familial inherited cancer syndrome. Suspected cases will be investigated, per the treating physician’ s discretion, using relevant genetic tests to determine presence of germline mutations.PCT / US25 / 50889 14 October 2025 (14.10.2025)• Known history of B cell related cancer, EBV lymphoproliferative disease or autoimmune disorders.• Concern regarding a potential malignancy at time of screening based on a CT scan of neck, chest, and abdomen.• Requirement for systemic immune suppression >1 year of immunosuppression within the past 5 years or >1 month of immunosuppression within the past year at time of study entry.• Requirement for continuous supplemental oxygen.• Any medical condition likely to interfere with assessment of safety or efficacy of the study treatment in the opinion of the investigator.• In the investigator’ s judgement, the subj ect is unlikely to complete all protocol required study visits or procedures, including follow up visits, or comply with the study requirements for participation.• Individuals of childbearing potential who are pregnant or unwilling to use effective contraception for the duration of the study.• Has received any prior disease specific investigational product (excluding a- L- iduronidase).• Has received a-L-iduronidase at any time and experienced a significant AE considered related to IV administration of a-L-iduronidase that, in the opinion of the PI, would put the subject at undue risk.• Underwent a previous hematopoietic stem cell transplant.

[0118] In some aspects, the MPS I is MPS IH (also written as “MPS-I-H”).

[0119] In some aspects, the MPS I is MPS IS (also written as “MPS-I-H”).

[0120] In some aspects, the MPS I is MPS IH / S (also written as “MPS-I-H / S”)

[0121] In some aspects, the subject has a creatinine clearance that is greater than about 60ml / min / 1.73m2.

[0122] In some aspects, the subject has previously administered at least one IDUA-based therapy. In some aspects, the IDUA-based therapy is an enzyme replacement therapy.

[0123] In some aspects, the subject has an abnormal liver size as determined by computed tomography (CT). In some aspects, an abnormal liver size is defined as greater than 3% body weight.

[0124] In some aspects, the subject has decreased respiratory function as measured by forced vital capacity (FVC). In some aspects, decreased respiratory function is defined as a subjectPCT / US25 / 50889 14 October 2025 (14.10.2025) exhibit an FVC that is less than or equal to about 80% of the predicted normal value for the subject.

[0125] In some aspects, the subject exhibits decreased mobility. In some aspects, decreased mobility is defined as a six-minute walk test performance of less than or equal to about 400 meters. In some aspects, decreased mobility is defined as a joint restriction of greater than 20° in a shoulder flexion.

[0126] In some aspects, the subject exhibits evidence of moderate to severe corneal clouding.

[0127] In some aspects, the subject has a history of hydrocephalus or increased ventricular size on MRI.

[0128] In some aspects, the subject has thickening of the mitral valve as measured by echocardiogram.

[0129] In some aspects, the subject exhibits evidence of dysostosis multiplex. In some aspects, the evidence of dysostosis comprises prior or ongoing carpal tunnel issues or affected digits; significant restriction of range of motion at the mandible, shoulder, elbow, and / or hip; prior or continued moderate kyphosis (e.g. great than about 20° angulation); lower extremity skeletal disease; history of impaired growth; or any combination thereof.

[0130] In some aspects, lower extremity skeletal disease is defined as disease that required prior surgical intervention at the knee (valgus deformity) or hip (dysplasia including the femur and / or acetabulum).

[0131] In some aspects, a history of impaired growth is defined as a heigh below the 5thpercentile for the subject’s age.

[0132] Efficacy Evaluations

[0133] In some embodiments, a subject treated with ISP-001 cells has an improved 6-minute walk (6MWT) as compared to a subject not treated with ISP-001 cells. In some embodiments, a subject treated with ISP-001 cells after six months can walk an additional 10 meters in the 6MWT as compared to a subj ect not treated with ISP-001 cells. In some embodiments, a subj ect treated with ISP-001 cells after six months can walk an additional 20 meters in the 6MWT as compared to a subject not treated with ISP-001 cells. In some embodiments, a subject treated with ISP-001 cells after six months can walk an additional 30 meters in the 6MWT as compared to a subject not treated with ISP-001 cells. In some embodiments, a subject treated with ISP- 001 cells after six months can walk an additional 40 meters in the 6MWT as compared to a subject not treated with ISP-001 cells. In some embodiments, a subject treated with ISP-001 cells after six months can walk an additional 50 meters in the 6MWT as compared to a subject not treated with ISP-001 cells. In some embodiments, a subject treated with ISP-001 cells afterPCT / US25 / 50889 14 October 2025 (14.10.2025) six months can walk an additional 60 meters in the 6MWT as compared to a subject not treated with ISP-001 cells. In some embodiments, a subject treated with ISP-001 cells after six months can walk an additional 70 meters in the 6MWT as compared to a subject not treated with ISP- 001 cells. In some embodiments, a subject treated with ISP-001 cells after six months can walk an additional 80 meters in the 6MWT as compared to a subject not treated with ISP-001 cells. In some embodiments, a subject treated with ISP-001 cells after six months can walk an additional 90 meters in the 6MWT as compared to a subject not treated with ISP-001 cells. In some embodiments, a subject treated with ISP-001 cells after six months can walk an additional 100 meters in the 6MWT as compared to a subject not treated with ISP-001 cells. In some embodiments, a subject treated with ISP-001 cells after six months can walk an additional 125 meters in the 6MWT as compared to a subject not treated with ISP-001 cells.

[0134] In some embodiments, a subject treated with ISP-001 cells after six months can walk a distance that is at least 10% further than a subj ect not treated with ISP-001 cells in the 6MWT. In some embodiments, a subject treated with ISP-001 cells after six months can walk a distance that is at least 15% further than a subject not treated with ISP-001 cells in the 6MWT. In some embodiments, a subject treated with ISP-001 cells after six months can walk a distance that is at least 20% further than a subject not treated with ISP-001 cells in the 6MWT. In some embodiments, a subject treated with ISP-001 cells after six months can walk a distance that is at least 25% further than a subject not treated with ISP-001 cells in the 6MWT. In some embodiments, a subject treated with ISP-001 cells after six months can walk a distance that is at least 30% further than a subject not treated with ISP-001 cells in the 6MWT. In some embodiments, a subject treated with ISP-001 cells after six months can walk a distance that is at least 35% further than a subject not treated with ISP-001 cells in the 6MWT. In some embodiments, a subject treated with ISP-001 cells after six months can walk a distance that is at least 40% further than a subject not treated with ISP-001 cells in the 6MWT. In some embodiments, a subject treated with ISP-001 cells after six months can walk a distance that is at least 45% further than a subject not treated with ISP-001 cells in the 6MWT. In some embodiments, a subject treated with ISP-001 cells after six months can walk a distance that is at least 50% further than a subject not treated with ISP-001 cells in the 6MWT.

[0135] In some embodiments, a subject treated with ISP-001 cells after six months has an increase in their shoulder flexibility in the shoulder flexion test as compared to a subject not treated with ISP-001 cells. In some embodiments, a subj ect treated with ISP-001 cells after six months has an increase range of motion as compared to a subject not treated with ISP-001 cells. In some embodiments, the subject treated with ISP-001 cells after six months has an increasePCT / US25 / 50889 14 October 2025 (14.10.2025) in the range of motion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 degrees as compared to a subject not treated with ISP-001. In some embodiments, the subject treated with ISP-001 cells after six months has an increase in the range of motion of at least 15 degrees as compared to a subject not treated with ISP-001. In some embodiments, the subject treated with ISP-001 cells after six months has an increase in the range of motion of at least 20 degrees as compared to a subject not treated with ISP-001. In some embodiments, the subject treated with ISP-001 cells after six months has an increase in the range of motion of at least 25 degrees as compared to a subject not treated with ISP-001. In some embodiments, the subject treated with ISP-001 cells after six months has an increase in the range of motion of at least 30 degrees as compared to a subject not treated with ISP-001. In some embodiments, the increase range of motion is in the right arm of the subject treated with ISP-001 cells. In some embodiments, the increase range of motion is in the left arm of the subject treated with ISP-001 cells.

[0136] In some embodiments, a subject treated with ISP-001 cells after 6 months has a 5% increase in the shoulder flexibility the shoulder flexion test as compared to a subject not treated with ISP-001 cells. In some embodiments, a subject treated with ISP-001 cells after 6 months has a 10% increase in shoulder flexibility in the shoulder flexion test as compared to a subject not treated with ISP-001 cells. In some embodiments, a subject treated with ISP-001 cells after 6 months has a 15% increase in shoulder flexibility in the shoulder flexion test as compared to a subject not treated with ISP-001 cells. In some embodiments, a subject treated with ISP-001 cells after 6 months has a 20% increase in shoulder flexibility in the shoulder flexion test as compared to a subject not treated with ISP-001 cells. In some embodiments, a subject treated with ISP-001 cells after 6 months has a 25% increase in shoulder flexibility in the shoulder flexion test as compared to a subject not treated with ISP-001 cells. In some embodiments, a subject treated with ISP-001 cells after 6 months has a 30% increase in shoulder flexibility in the shoulder flexion test as compared to a subject not treated with ISP-001 cells. In some embodiments, a subject treated with ISP-001 cells after 6 months has a 35% increase in shoulder flexibility in the shoulder flexion test as compared to a subject not treated with ISP- 001 cells. In some embodiments, a subject treated with ISP-001 cells after 6 months has a 40% increase in shoulder flexibility in the shoulder flexion test as compared to a subject not treated with ISP-001 cells.

[0137] In some embodiments, a subject treated with ISP-001 cells after six months has a decrease in heparan sulfate levels in CSF from baseline. In some embodiments, the reduction in heparan sulfate levels in CSF is 5% from baseline. In some embodiments, the reduction in heparan sulfate levels is 10% from baseline. In some embodiments, the reduction in heparanPCT / US25 / 50889 14 October 2025 (14.10.2025) sulfate levels is 15% from baseline. In some embodiments, the reduction in heparan sulfate levels is 20% from baseline. In some embodiments, the reduction in heparan sulfate levels is 25% from baseline. In some embodiments, the reduction in heparan sulfate levels is 30% from baseline. In some embodiments, the reduction in heparan sulfate levels is 35% from baseline. In some embodiments the heparan sulfate level is reduced to below 120 ng / ml.

[0138] In some embodiments, a subject treated with ISP-001 cells after three months has a reduced the total urine glycosaminoglycans (GAG) (mg) level (as measured per mmol of creatinine) as compared to baseline. In some embodiments, the urine GAG level is reduced to below 6.5 urine GAG mg / mmol creatinine.

[0139] In some embodiments, a subject treated with ISP-001 cells after six months has a decrease in the levels of non-reduced ends of heparan sulfate as compared to a subject that was not treated with ISP-001. In some embodiments, the reduction in the non-reduced ends of heparan sulfate is seen in IOSO. In some embodiments, the reduction in the non-reduced ends of heparan sulfate is seen in IOS6.

[0140] One skilled in the art would realize that the normal limit of any of the disclosed biomarkers would depend on many factors such as age, sex, weight of the individual, and the lab and / or methodology used to measure the biomarker. In some embodiments, a subj ect treated with ISP-001 cells after six months has a decrease of non-reduced ends of heparan sulfate to the upper limit of normal. In some embodiments, a subject treated with ISP-001 cells after six months has a decrease of urine glycosaminoglycans (GAG) (mg) level to the upper limit of normal. In some embodiments, a subject treated with ISP-001 cells after six months reduces the heparan sulfate level in CSF to the upper limit of normal.

[0141] In some aspects, the administration of the populations of the modified B cells of the present disclosure in the amounts described above result in an increase in the distance walked in six minutes (known in the art as the 6-minute walk test or “6MWT”). In some aspects, the increase in the distance walked is at least about 5 meters, at least about 10 meters, at least about 15 meters, at least about 20 meters, at least about 25 meters, at least about 30 meters, at least about 35 meters, at least about 40 meters, at least about 45 meters, at least about 50 meters, at least about 55 meters, at least about 60 meters, at least about 65 meters, at least about 70 meters, at least about 75 meters, at least about 80 meters, at least about 85 meters, at least about 90 meters, at least about 95 meters, at least about 100 meters, at least about 105 meters, at least about 110 meters, at least about 115 meters, at least about 120 meters, at least about 125 meters, at least about 130 meters, at least about 135 meters, at least about 140 meters, at least about 145 meters, at least about 150 meters, at least about 155 meters, at least about 160 meters, atPCT / US25 / 50889 14 October 2025 (14.10.2025) least about 165 meters, at least about 170 meters, at least about 175 meters, at least about 180 meters, at least about 185 meters, at least about 190 meters, at least about 195 meters, at least about 200 meters, at least about 205 meters, at least about 210 meters, at least about 215 meters, at least about 220 meters, at least about 225 meters, at least about 230 meters, at least about 235 meters, at least about 240 meters, at least about 245 meters, at least about 250 meters, at least about 255 meters, at least about 260 meters, at least about 265 meters, at least about 270 meters, at least about 275 meters, at least about 280 meters, at least about 285 meters, at least about 290 meters, at least about 295 meters, or at least about 300 meters. In some aspects, the increase in the distance walked is about 5 meters, about 10 meters, about 15 meters, about 20 meters, about 25 meters, about 30 meters, about 35 meters, about 40 meters, about 45 meters, about 50 meters, about 55 meters, about 60 meters, about 65 meters, about 70 meters, about 75 meters, about 80 meters, about 85 meters, about 90 meters, about 95 meters, about 100 meters, about 105 meters, about 110 meters, about 115 meters, about 120 meters, about 125 meters, about 130 meters, about 135 meters, about 140 meters, about 145 meters, about 150 meters, about 155 meters, about 160 meters, about 165 meters, about 170 meters, about 175 meters, about 180 meters, about 185 meters, about 190 meters, about 195 meters, about 200 meters, about 205 meters, about 210 meters, about 215 meters, about 220 meters, about 225 meters, about 230 meters, about 235 meters, about 240 meters, about 245 meters, about 250 meters, about 255 meters, about 260 meters, about 265 meters, about 270 meters, about 275 meters, about 280 meters, about 285 meters, about 290 meters, about 295 meters, or about 300 meters. In some aspects, the increase in the distanced walked in six minutes is at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%. In some aspects, the decrease is about 5%, or about 10%, or about 15%, or about 20%, or about 25%, or about 30%, or about 35%, or about 40%, or about 45%, or about 50%, or about 55%, or about 60%, or about 65%, or about 70%, or about 75%. In some aspects, these increases are measured about 3 months, or about 6 months, or about 12 months, or about 15 months, or about 18 months, or about 21 months, or about 24 months after the administration of the first population of modified B cells to the subject. In some aspects, these increases are measured about 6 months after the administration of the first population of modified B cells to the subject. In some aspects, these increases are measured about 12 months after the administration of the first population of modified B cells to the subject.PCT / US25 / 50889 14 October 2025 (14.10.2025)

[0142] In some aspects, the administration of the populations of the modified B cells of the present disclosure in the amounts described above result in an increase in shoulder flexion (see e.g. FIG. 4B showing the measurement of restriction of shoulder flexion). In some aspects, the increase in shoulder flexion is at least about 1°, at least about 2°, at least about 3°, at least about 4°, at least about 5°, at least about 6°, at least about 7°, at least about 8°, at least about 9°, at least about 10°, at least about 11°, at least about 12°, at least about 13°, at least about 14°, at least about 15°, at least about 16°, at least about 17°, at least about 18°, at least about 19°, at least about 20°, at least about 21°, at least about 22°, at least about 23°, at least about 24°, at least about 25°, at least about 26°, at least about 27°, at least about 28°, at least about 29°, at least about 30°, at least about 31°, at least about 32°, at least about 33°, at least about 34°, at least about 35°, at least about 36°, at least about 37°, at least about 38°, at least about 39°, at least about 40°, at least about 41°, at least about 42°, at least about 43°, at least about 44°, at least about 45°, at least about 46°, at least about 47°, at least about 48°, at least about 49°, at least about 50°, at least about 51°, at least about 52°, at least about 53°, at least about 54°, at least about 55°, at least about 56°, at least about 57°, at least about 58°, at least about 59°, at least about 60°, at least about 61°, at least about 62°, at least about 63°, at least about 64°, at least about 65°, at least about 66°, at least about 67°, at least about 68°, at least about 69°, at least about 70°, at least about 71°, at least about 72°, at least about 73°, at least about 74°, at least about 75°, at least about 76°, at least about 77°, at least about 78°, at least about 79°, at least about 80°, at least about 81°, at least about 82°, at least about 83°, at least about 84°, at least about 85°, at least about 86°, at least about 87°, at least about 88°, at least about 89°, at least about 90°, at least about 91°, at least about 92°, at least about 93°, at least about 94°, at least about 95°, at least about 96°, at least about 97°, at least about 98°, at least about 99°, or at least about 100°. In some aspects, the increase in shoulder flexion is about 1°, about 2°, about 3°, about 4°, about 5°, about 6°, about 7°, about 8°, about 9°, about 10°, about 11°, about 12°, about 13°, about 14°, about 15°, about 16°, about 17°, about 18°, about 19°, about 20°, about 21°, about 22°, about 23°, about 24°, about 25°, about 26°, about 27°, about 28°, about 29°, about 30°, about 31°, about 32°, about 33°, about 34°, about 35°, about 36°, about 37°, about 38°, about 39°, about 40°, about 41°, about 42°, about 43°, about 44°, about 45°, about 46°, about 47°, about 48°, about 49°, about 50°, about 51°, about 52°, about 53°, about 54°, about 55°, about 56°, about 57°, about 58°, about 59°, about 60°, about 61°, about 62°, about 63°, about 64°, about 65°, about 66°, about 67°, about 68°, about 69°, about 70°, about 71°, about 72°, about 73°, about 74°, about 75°, about 76°, about 77°, about 78°, about 79°, about 80°, about 81°, about 82°, about 83°, about 84°, about 85°, about 86°, about 87°, about 88°, aboutPCT / US25 / 50889 14 October 2025 (14.10.2025)89°, about 90°, about 91°, about 92°, about 93°, about 94°, about 95°, about 96°, about 97°, about 98°, about 99°, or about 100°. In some aspects, this increase is measured about 3 months, or about 6 months, or about 12 months, or about 15 months, or about 18 months, or about 21 months, or about 24 months after the administration of the first population of modified B cells to the subject. In some aspects, these increases are measured about 6 months after the administration of the first population of modified B cells to the subject. In some aspects, these increases are measured about 12 months after the administration of the first population of modified B cells to the subject.

[0143] In some aspects, the administration of the populations of the modified B cells of the present disclosure in the amounts described above results in a decrease in the concentration of glycosaminoglycans (GAGs) in the urine of the subject. In some aspects, the decrease is at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%. In some aspects, the decrease is about 5%, or about 10%, or about 15%, or about 20%, or about 25%, or about 30%, or about 35%, or about 40%, or about 45%, or about 50%, or about 55%, or about 60%, or about 65%, or about 70%, or about 75%. In some aspects, this decrease is measured about 3 months, or about 6 months, or about 12 months, or about 15 months, or about 18 months, or about 21 months, or about 24 months after the administration of the first population of modified B cells to the subject. In some aspects, these decreases are measured about 6 months after the administration of the first population of modified B cells to the subject. In some aspects, these decreases are measured about 12 months after the administration of the first population of modified B cells to the subject. In some aspects, the concentration of GAGs in urine is measured as mg / mmol creatinine.

[0144] In some aspects, the administration of the populations of the modified B cells of the present disclosure in the amounts described above results in a decrease in the concentration of heparan sulfate in the cerebrospinal fluid (CSF) of the subject. In some aspects, the decrease is at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%. In some aspects, the decrease is about 5%, or about 10%, or about 15%, or about 20%, or about 25%, or about 30%, or about 35%, or about 40%, or about 45%, or about 50%, or about 55%, or about 60%, or about 65%, or about 70%, orPCT / US25 / 50889 14 October 2025 (14.10.2025) about 75%. In some aspects, these decreases are measured about 6 months after the administration of the first population of modified B cells to the subject. In some aspects, these decreases are measured about 12 months after the administration of the first population of modified B cells to the subject. In some aspects, these increases are measured about 6 months after the administration of the first population of modified B cells to the subject. In some aspects, the concentration of heparin sulfate in the CSF is measured as ng / ml.

[0145] In some aspects, the administration of the populations of the modified B cells of the present disclosure in the amounts described above results in a decrease in the concentration of non-reducing end (NRE) I0S0 in the CSF of the subject. In some aspects, the decrease is at least about 1%, or at least about 2%, or at least about 3%, or at least about 4%, or at least about 5%, or at least about 6%, or at least about 7%, or at least about 8%, or at least about 9%, or at least about 10%, or at least about 11%, or at least about 12%, or at least about 13%, or at least about 14%, or at least about 15%, or at least about 16%, or at least about 17%, or at least about 18%, or at least about 19%, or at least about 20%, or at least about 21%, or at least about 22%, or at least about 23%, or at least about 24%, or at least about 25%, or at least about 26%, or at least about 27%, or at least about 28%, or at least about 29%, or at least about 30%, or at least about 31%, or at least about 32%, or at least about 33%, or at least about 34%, or at least about 35%. In some aspects, the decrease is about 1%, or about 2%, or about 3%, or about 4%, or about 5%, or about 6%, or about 7%, or about 8%, or about 9%, or about 10%, or about 11%, or about 12%, or about 13%, or about 14%, or about 15%, or about 16%, or about 17%, or about 18%, or about 19%, or about 20%, or about 21%, or about 22%, or about 23%, or about 24%, or about 25%, or about 26%, or about 27%, or about 28%, or about 29%, or about 30%, or about 31%, or about 32%, or about 33%, or about 34%, or about 35%. In some aspects, these decreases are measured about 6 months after the administration of the first population of modified B cells to the subject. In some aspects, these decreases are measured about 12 months after the administration of the first population of modified B cells to the subject. In some aspects, the concentration of NRE I0S0 in CSF is measured as ng / ml.

[0146] In some aspects, the administration of the populations of the modified B cells of the present disclosure in the amounts described above results in a decrease in the concentration of NRE I0S6 in the CSF of the subject. In some aspects, the decrease is at least about 1%, or at least about 2%, or at least about 3%, or at least about 4%, or at least about 5%, or at least about 6%, or at least about 7%, or at least about 8%, or at least about 9%, or at least about 10%, or at least about 11%, or at least about 12%, or at least about 13%, or at least about 14%, or at least about 15%, or at least about 16%, or at least about 17%, or at least about 18%, or at least aboutPCT / US25 / 50889 14 October 2025 (14.10.2025)19%, or at least about 20%, or at least about 21%, or at least about 22%, or at least about 23%, or at least about 24%, or at least about 25%, or at least about 26%, or at least about 27%, or at least about 28%, or at least about 29%, or at least about 30%, or at least about 31%, or at least about 32%, or at least about 33%, or at least about 34%, or at least about 35%. In some aspects, the decrease is about 1%, or about 2%, or about 3%, or about 4%, or about 5%, or about 6%, or about 7%, or about 8%, or about 9%, or about 10%, or about 11%, or about 12%, or about 13%, or about 14%, or about 15%, or about 16%, or about 17%, or about 18%, or about 19%, or about 20%, or about 21%, or about 22%, or about 23%, or about 24%, or about 25%, or about 26%, or about 27%, or about 28%, or about 29%, or about 30%, or about 31%, or about 32%, or about 33%, or about 34%, or about 35%. In some aspects, these decreases are measured about 6 months after the administration of the first population of modified B cells to the subject. In some aspects, these decreases are measured about 12 months after the administration of the first population of modified B cells to the subject. In some aspects, the concentration of NRE I0S6 in CSF is measured as ng / ml.

[0147] Incorporation by Reference

[0148] All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entireties for all purposes. However, mention of any reference, article, publication, patent, patent publication, and patent application herein is not, and should not be, taken as acknowledgment or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.

[0149] Experimental Examples

[0150] EXAMPLE 1: Production ofIDUA Expressing B Cells

[0151] Sleeping Beauty transposon and transposase constructs for transposition and expression of human IDUA were generated. Transposons assembled to achieve IDUA gene integration and expression in B cells are shown in FIG 1. The EEK promoter, consisting of promoter and enhancer elements from the human immunoglobulin gene as well as other regulatory elements previously described in WO2018201071, were used to achieve high level expression in B cells.

[0152] EXAMPLE 2: Production of ISP-001

[0153] ISP-001 consists of ex vivo expanded autologous B cells that have been genetically engineered using the SB transposon system to express and secrete IDUA. PBMCs are harvested from a patient’s blood by leukapheresis. The B cells are then immuno-magnetically enriched by positive selection for CD 19+ cells using Miltenyi-based separation techniques. The isolatedPCT / US25 / 50889 14 October 2025 (14.10.2025)CD 19+ B cells are then activated for 2 days by incubation in the presence of a proprietary combination of cytokines. Following activation, the B cells are modified to express the IDUA gene by means of the SB transposon system. SB is a non-viral vector system for gene therapy, which combines the advantages of an integrating vector with the simplicity of uncomplexed DNA. The SB transposon system consists of two components: a transposon containing a geneexpression cassette flanked by inverted repeat / direct repeats (IR / DRs) and a source of transposase enzyme. For the treatment of MPS IH / S and MPS IS, the SB transposon system consists of the IDUA transposon construct in combination with messenger RNA encoding SB100X, which serves as the source of SB transposase. The SB transposon system is further explained in Example 1. In the case of ISP- 100, the transposon used was pKT2 / EEK-IDUA, described above and in FIG. 1.

[0154] To produce ISP-001 cells, CD19+cells are collected from peripheral blood of the subject by leukapheresis, genetically engineered using an integrating vector system, expanded ex vivo and differentiated into plasmablasts prior to re-infusion.

[0155] Non-mobilized peripheral blood mononuclear cells are harvested from the patient by apheresis. The cells are shipped to the processing facility, where they are enriched for B cells, expanded in culture and electroporated for introduction of SB transposon DNA encoding for human a-L-iduronidase. The expanded cellular product is then sampled for quality control testing (cell identity and purity by immunophenotyping, viability, potency by iduronidase enzyme assay and intracellular staining for iduronidase expression, and safety by testing sterility, mycoplasma, and endotoxin, etc.). The cells are cryopreserved in Cryostor 10 according to standard operating procedures. The cells are then shipped to the study site where they are thawed, diluted, assayed for viability, and then infused into the patient. No lymphodepleting chemotherapy or other ablative regimens are used prior to IP infusion.

[0156] If an excess of cells is obtained during manufacturing, the remaining cells not required during the scheduled ISP-001 administration are cryopreserved and reserved for potential future use for the subject.

[0157] EXAMPLE 3: Shoulder Flexion Protocol

[0158] Motion occurs in the sagittal plane around a medial-lateral axis.

[0159] A subject is placed supine with knees flexed to flatten the lumbar spine. The shoulder is positioned at 0 degree abduction, adduction, and rotation. The elbow placed in extension so that tension in the long head of the triceps muscle does not limit the motion. The forearm is positioned at 0 degrees of supination and pronation so that the palm of the hand faces the body.PCT / US25 / 50889 14 October 2025 (14.10.2025)

[0160] Glenohumeral joint is stabilized in the scapula to prevent posterior tilting, upward rotation, and elevation of the scapula.

[0161] The thorax is stabilized to prevent the extension of the spine and movement of the ribs. The weight of the trunk may assist in stabilization.

[0162] In sum: the goniometer alignment should be as follows: (1) the center fulcrum is over the lateral aspect of the greater tubercle; (2) stationary arm is parallel to the midaxillary line of the thorax; and (3) the moving arm is in the lateral midline of the humerus. The zero goniometer alignment is shown in FIG. 4B.

[0163] EXAMPLE 4: Results of Subject #1 Treated with ISP -001

[0164] A subject was treated with ISP-001 cells at a dosage of 2.5 x 107cells / kg and the 15 months (or more) results are shown in FIG. 2 - FIG. 4C. The subject was also being treated with enzyme replacement therapy (laronidase). During month 8 following administration of the ISP-001 cells, the enzyme replacement therapy was discontinued. The enzyme replacement therapy was restarted at 9 months after administration of the ISP-001 cells.

[0165] After 15 months there were no adverse events reported. There was no dysplasia, and no increase in blasts (<1%) - No morphologic or immunophenotypic evidence of a hematolymphoid neoplasms.

[0166] The following surprising results were found:• The subject’s pre-diagnosis GAG level was 39.5 mg / mmol creatinine. As shown in FIG. 2, at the eight month mark following ISP-001 administration, the subject exhibited their lowest urine GAG levels to date and exhibited normalized levels. For comparison, the package insert for laronidase explains that no subject in the group receiving laronidase reached the normal range for urinary GAG levels during a 6-month study.• After six months, the subject showed a 25.3% reduction from baseline in total heparan sulfate levels in the CSF, 16.7% reduction from baseline in IOSO levels, and 6.7% reduction from baseline in IOS6 levels. See FIG. 3.• After six months, the subject was able to walk 446 meters in the 6MWT, which is a 31% improvement from the initial screening. See FIG. 4C. Without wishing to be bound by theory, 30 meters is generally considered to be clinically meaningful, with laronidase showing a 20-meter improvement at the time of its approval by the FDA.

[0167] After 12 months, the subject also was able to increase their active and passive range of motion restriction in the shoulder flexion test. As shown in FIG. 4A, in the right arm the subject was able to increase the active range of motion restriction and passive range of motionPCT / US25 / 50889 14 October 2025 (14.10.2025) beyond the initial screening by 19 and 12 degrees, respectively. In the left arm the subject was able to increase the active range of motion restriction and passive range of motion beyond the initial screening by 26 and 16 degrees, respectively. See FIG. 4A. In comparison, laronidase only exhibit a 10 degree improvement in its pivotal trial used for FDA approval. Additionally, the shoulder flexion improvement was still seen even after the discontinuation of enzyme replacement therapy.

[0168] EXAMPLE 5: Re-dosing of Subject #1

[0169] Subject #1 (described in Example 4 above) was re-dosed at 18 months following the initial administration of ISP-001 cells. In the re-dosing, the subject was administered 5.0 x 107cells / kg. Following re-dosing, the subject’s urine GAG amounts continue to normalize. No adverse events have been reported as of 20 months following the initial dose of ISP-001 cells.

[0170] EXAMPLE 6: Dosing of Subject #2 with ISP-001 Cells

[0171] A second subject (Subject #2) was administered ISP-001 cells at a dosage of 7.5 x 108cells / kg. Like Subject #1, Subject #2 was also being treated with enzyme replacement therapy (laronidase) at the time of dosing. Similar to Subject #1, Subject #2 has shown clinical relevant improvements following the administration of the ISP-001 cells, including a normalization in the urine GAG amounts, as well as improvements in functional endpoints such as shoulder flexion.

Claims

What is claimed is:

1. A method of treating MPS I in a subject, the method comprising administering to the subject a population of modified B cells in an amount of:• 2.5 x 107cells / kg body weight of the subject;• 5.0 x 107cells / kg body weight of the subject; or• 7.5 x 107cells / kg body weight of the subject, wherein the population of modified B cells has been modified such that an expression cassette comprising an EEK promoter and a nucleic acid sequence encoding iduronidase (IDUA) has been inserted into the genome of the modified B cells via a transposon system.

2. A population of modified B cells for use in a method of treating MPS I in a subject, wherein the method comprises administering to the subject the population of modified B cells in an amount of:• 2.5 x 107cells / kg body weight of the subject;• 5.0 x 107cells / kg body weight of the subject; or• 7.5 x 107cells / kg body weight of the subject, wherein the population of modified B cells has been modified such that an expression cassette comprising an EEK promoter and a nucleic acid sequence encoding IDUA has been inserted into the genome of the modified B cells via a transposon system.

3. The method or use of claim 1 or claim 2, wherein the method further comprises administering to the subject at least one additional population of modified B cells in an amount of:• 2.5 x 107cells / kg body weight of the subject;• 5.0 x 107cells / kg body weight of the subject; or• 7.5 x 107cells / kg body weight of the subject.

4. The method or use of any one of the preceding claims, wherein the MPS I is MPS H4.

5. The method or use of any one of the preceding claims, wherein the MPS I is MPS IS.

6. The method or use of any one of the preceding claims, wherein the MPS I is MPS HUS.

7. The method or use of any one of the preceding claims, wherein the transposon system is the Sleeping Beauty transposon system.

8. The method or use of any one of the preceding claims, wherein the EEK promoter comprises the nucleic acid sequence of SEQ ID NO: 4.

9. The method or use of any one of the preceding claims, wherein the nucleic acid sequence encoding IDUA comprises the nucleic acid sequence of SEQ ID NO: 5.

10. The method or use of any one of the preceding claims, wherein the method further comprises administering to the subject an enzyme replacement therapy.

11. The method or use of any one of the preceding claims, wherein the subject has been previously treated with an enzyme replacement therapy.

12. The method or use of claim 10 or claim 11, wherein the enzyme replacement therapy is laronidase.

13. The method or use of any one of the preceding claims, wherein six months after administration of the population of modified B cells, the concentration of heparan sulfate (HS) in the cerebral spinal fluid (CSF) of the subject is at or below the upper limit of a normal concentration of HS in CSF in a subject not having MPS I.

14. The method or use of any one of the preceding claims, wherein six months after administration of the population of modified B cells, the subject exhibits improved flexibility in shoulder flexion as compared to before the administration of the population of modified B cells, preferably wherein the subject exhibits an at least 25% increase in flexibility in shoulder flexion.

15. The method or use of any one of the preceding claims, wherein after six months after administration of the population of modified B cells, the subject can walk a longer distance in the six-minute walk test (6MWT) as compared to before the administration of the population of modified B cells, preferably wherein the subject can walk a 25% longer distance.

16. The method or use of any one of the preceding claims, wherein the subject exhibits at least one of the following: i) abnormal liver size; ii) decreased respiratory function; iii) decreased mobility; iv) moderate to several corneal clouding; v) a history of hydrocephalus or increased ventricular size; vi) thickening of the mitral valve; vii) evidence of dysostosis multiplex; viii) lower extremity skeletal disease; and ix) history of impaired growth.

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