RPS19-encoding lentiviral vector and method of using the same

Abstract

A self-inactivating lentiviral vector and method for treating or ameliorating Diamond-Blackfan anemia syndrome. The self-inactivating lentiviral vector includes nucleic acids encoding ribosomal protein S19 operably linked to an EF1α promoter or a MND promoter, wherein the nucleic acids encoding RPS19 are codon optimized for expression in human cells and are devoid of aberrant splice acceptor sites, thereby improving the safety and efficacy of the lentiviral vector.

Description

RPS19 -ENCODING LENTIVIRAL VECTOR ANDMETHOD OF USING THE SAMEREFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority from U.S. Provisional Application Serial Number 63 / 715,767, filed November 4, 2024, the contents of which are incorporated herein by reference in their entireties.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (name: SJ0119WO_ST26.xml; size: 22,784 bytes; and date of creation: October 25, 2025) is herein incorporated by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0003] This invention was made with government support under grant numbers HL053749 and DK134844 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0004] Diamond-Blackfan anemia syndrome (DBAS) is a rare inherited bone marrow failure disorder that typically presents in infancy with macrocytic anemia, reticulocytopenia and bone marrow erythroid hypoplasia. Approximately 50% of affected individuals also have developmental anomalies. Later in life, some DBAS patients develop multilineage cytopenias and reduced bone marrow cellularity, suggesting impaired hematopoietic stem cell (HSC) maintenance. Most cases of DBAS are caused by heterozygous loss-of-function mutations in one of 24 different ribosomal protein genes. The most commonly mutated gene isribosomal protein S19 (RPS19), which accounts for approximately 25% of DBAS patients. How ribosomal protein haploinsufficiency causes selective hematopoietic defects is not fully understood.

[0005] Medical therapy for DBAS includes corticosteroids and chronic red blood cell (RBC) transfusions. Approximately 80% of patients exhibit improved erythropoiesis with corticosteroids but more than half of responders eventually become RBC transfusion-dependent. Both of these therapies are associated with serious long-term toxicities and suboptimal quality of life. Moreover, erythroid hypoplasia renders DBAS patients exquisitely sensitive to transfusion-associated iron overload, which causes multiorgan damage. DBAS patients are also at increased risk for cancer, particularly colon carcinoma, acute myeloid leukemia and osteosarcoma. Moreover, DBAS patients can experience prolonged cytopenias following chemotherapy, which can predispose to infection and promote tumor progression by delaying treatments. Based on registry data, the estimated median overall survival for Americans with DBAS is 56 years, in contrast to approximately 75 years for the general population. Allogeneic hematopoietic stem cell transplantation can provide a hematopoietic cure for DBAS; recent studies report over 80% survival with restored bone marrow function. Problems associated with hematopoietic stem cell transplantation for DBAS include lack of suitable donors for many patients, graft-versus-host disease (GVHD) and the associated need for prolonged immunosuppression. Patients over the age of 10 years are at increased risk for chronic GVHD and transplant-related mortality, most likely due to preexisting organ damage from iron overload, anti-donor antibodies caused by chronic transfusions and the effects of chronic inflammation associated with DBAS.

[0006] Many limitations of allogeneic hematopoietic stem cell transplantation for monogenic blood disorders can be overcome by transplantation of autologous hematopoietic stem cells that have been genetically modified with lentiviral vectors (LV) to restore expression of the defective gene. The development of third-generation self-inactivating (SIN) LVs has improved the safety of this approach by reducing the risk of leukemogenesis caused by insertional activation of oncogenes. Studies have demonstrated safety and efficacy of LV gene therapy for several forms of severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome, and β-hemoglobinopathies. However, safe and effective LV-based gene therapies for DBAS are needed.SUMMARY OF THE INVENTION

[0007] Provided herein is a self-inactivating lentiviral vector for treating Diamond-Blackfan anemia syndrome comprising nucleic acids encoding ribosomal protein S19 (RPS19) operably linked to an EFla promoter or a MND promoter, wherein the nucleic acids encoding RPS19 are codon optimized for expression in human cells and are devoid of aberrant splice acceptor sites.

[0008] A method of treating or ameliorating Diamond-Blackfan anemia syndrome is also provided, the method comprising administering to a subject in need thereof an effective amount of the self-inactivating lentiviral vector herein, thereby treating or ameliorating the subject' s Diamond-Blackfan anemia syndrome.BRIEF DESCRIPTION OF DRAWINGS

[0009] FIG 1. Schematics of LV vectors encoding RPS19 ± P2A peptide fused to green fluorescent protein (GFP) driven by the EFla short (EFlaS) promoter.

[0010] FIG. 2. Efficient transduction of CD34 + hematopoietic stem and progenitor cells (HSPCs) with RPS19 lentiviral (LV) vector. Normal donor, peripheral blood mobilized CD34 + HSPCs were incubated with LV vectors for 16-20 hours and analyzed 3 days later. Shown are the effects of transduction enhancers prostaglandin E2 (PGE2, 10 mM) and LentiBoost® (LB, 1 mg / mL) on GFP expression after transduction with SJEFS-S19GFP at multiplicity of infection (MOI) 20. Transduction enhancers were used in all subsequent studies.

[0011] FIG. 3. BFU-E colonies per 1000 CD34+HSPCs. Mean + standard deviation (SD) is shown, with each symbol representing data from a different CD34+cell donor.

[0012] FIG. 4. Hematopoietic progenitor cells (HPCs) were generated by in vitro differentiation of induced pluripotent stem cells (iPSCs) from a patient with RPS19-mutated Diamond Blackfan anemia (c.191T>C; p.Leu64Pro) or an isogenic control line in which the mutation was corrected by Cas9-mediated homology directed repair. The HPCs were transduced with SJEFS- S19 LV, then incubated in medium containing erythroid cytokines (StemSpan erythroid expansion supplement, StemCell Technologies). Live cells were quantified over 15 days. Each datapoint represents mean ± SD of 3 technical replicate studies.DETAILED DESCRIPTION OF THE INVENTION

[0013] Preclinical development of lentiviral vector gene therapy for DBAS has been impeded by the limited availability of DBAS patient HSCs. It has been shown that CRISPR / Cas9 disruption of one RPS19 gene in healthy donor CD34+hematopoietic stem and progenitor cells (HSPCs) recapitulates several DBAS phenotypes, including defective erythroid maturation, impaired HSC maintenance, aberrant TP53 activation and a specific block in pre-ribosomal (r) RNA processing (Bhoopalan et al. (2013) JCI Insight 8(1):e161810). A lentiviral vector, referred to as SJEFS-S19, for RPS19-mutated DBAS has now been developed. Transduction of RPS19+ / -HSPCs with SJEFS-S19 at a multiplicity of infection (MOI) of 20 attained a vector copy number (VCN) of approximately three and alleviated DBAS-like phenotypes in vitro and in vivo. Sixteen weeks after xenotransplantation into immunodeficient mice, SJEFS-S19 lentiviral vector-transduced HSPCs exhibited a polyclonal integration profile with no clonal dominance and no detectable aberrant splicing between integrated lentiviral vector and nearby genes. These studies establish a foundation for safe and effective lentiviral vector gene therapy to treat RPS19-mutated DBAS.

[0014] Accordingly, provided herein is a self -inactivating lentiviral vector comprising nucleic acids encoding RPS19 operably linked to an EFla or MND promoter, wherein the nucleic acids encoding RPS19 are codon optimized for expression in human cells and are devoid of aberrant splice sites. Also provided is a method of using the lentiviral vector for treating Diamond-Blackfan anemia syndrome optionally with no clonal dominance and / or no detectable aberrant splicing between integrated lentiviral vector and nearby genes.

[0015] Throughout this specification, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. By "consisting of" is meant including, andlimited 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. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or mandatory, but that no 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.

[0016] The term "lentivirus" refers to a group (or genus) of retroviruses that give rise to slowly developing disease. Viruses included within this group include HIV (human immunodeficiency virus; including HIV type 1, and HIV type 2), the etiologic agent of the human acquired immunodeficiency syndrome (AIDS); visna-maedi, which causes encephalitis (visna) or pneumonia (maedi) in sheep, the caprine arthritisencephalitis virus, which causes immune deficiency, arthritis, and encephalopathy in goats; equine infectious anemia virus, which causes autoimmune hemolytic anemia, and encephalopathy in horses; feline immunodeficiency virus (FIV), which causes immune deficiency in cats; bovine immune deficiency virus (BIV), which causes lymphadenopathy, lymphocytosis, and possibly central nervous system infection in cattle; and simian immunodeficiency virus (SIV), which cause immune deficiency and encephalopathy in sub-human primates. Diseases caused by these viruses are characterized by a long incubation period and protracted course. Usually, the viruses latently infect monocytes and macrophages, from which they spread toother cells. HIV, FIV, and SIV also readily infect T lymphocytes (i. e., T-cells).

[0017] Lentiviruses are RNA viruses that use reverse transcriptase during their replication cycle. The lentiviral genomic RNA is converted into double-stranded DNA by reverse transcriptase. This double-stranded DNA form of the virus is capable of being integrated into the chromosome of the infected cell; once integrated, it is referred to as a "provirus." The provirus serves as a template for RNA polymerase II and directs the expression of RNA molecules which encode the structural proteins and enzymes needed to produce new viral particles.

[0018] At each end of the provirus are structures called "long terminal repeats" or "LTRs." The term "long terminal repeat (LTR) " refers to domains of base pairs located at the ends of retroviral DNAs which, in their natural sequence context, are direct repeats and contain U3, R, and U5 regions. LTRs generally provide functions fundamental to the expression of retroviral genes (e. g., promotion, initiation and polyadenylation of gene transcripts) and to viral replication. The LTR contains numerous regulatory signals including transcriptional control elements, polyadenylation signals and sequences needed for replication and integration of the viral genome. The viral LTR is divided into three regions called U3, R, and U5. The U3 region contains the enhancer and promoter elements. The U5 region is the sequence between the primer binding site and the R region and contains the polyadenylation sequence. The R (repeat) region is flanked by the U3 and U5 regions. The LTR composed of U3, R and U5 regions, appears at both the both the 5 ' and 3 ' ends of the viral genome. In one embodiment, the promoter within the LTR, including the 5 ' LTR, is replaced with a heterologous promoter to produce a hybridvector. Examples of heterologous promoters that may be used include, for example, the cytomegalovirus (CMV) promoter.

[0019] The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. The term "expression vector" includes any vector containing a gene construct in a form suitable for expression by a cell (e. g., linked to a promoter). The term "retroviral vector" refers to a vector containing structural and functional genetic elements that are primarily derived from a retrovirus. The term "lentiviral vector" refers to a vector containing structural and functional genetic elements outside the LTRs that are primarily derived from a lentivirus. Particular lentiviral vectors of use herein are described by Pawliuk et al. (2001) Science 294: 2368 and Imren et al. (2002) PNAS 99: 14380. These vectors can be constructed and engineered using art-recognized techniques to increase their safety for use in therapy and to include suitable expression elements and a therapeutic gene encoding RPS19 for treating conditions including, but not limited to, Diamond-Blackfan anemia syndrome.

[0020] In consideration of the potential toxicity of lentiviruses, the vector herein may be designed in different ways to increase its safety in gene therapy applications. For example, the vector may be made safer by separating the necessary lentiviral genes (e. g., gag and pol) onto separate vectors as described, for example, in U. S. 6, 365, 150, the contents of which are incorporated by reference herein. Thus, recombinant lentivirus can be constructed in which part of the lentiviral coding sequence (gag, pol, env) is replaced by a gene of interest rendering the lentivirus replication defective. The replication defective lentivirus is then packaged into virions through the use of a helper virus or apackaging cell line by standard techniques. Protocols for producing recombinant lentiviruses and for infecting cells in vitro or in vivo with such viruses can be found in Current Protocols in Molecular Biology, Ausubel, F. M. et al. (eds. ) Greene Publishing Associates, (1989), Sections 9.10-9.14 and other standard laboratory manuals.

[0021] In addition, the development of packaging cell lines, which produce only replication-defective lentiviruses, has increased the utility of lentiviruses for gene therapy, and defective lentiviruses are well-characterized for use in gene transfer for gene therapy purposes. Accordingly, in one embodiment, packaging cell lines are used to propagate vectors (e. g., lentiviral vectors) herein to increase the titer of the vector virus. The use of packaging cell lines is also considered a safe way to propagate the virus, as use of the system reduces the likelihood that recombination will occur to generate wild-type virus. In addition, to reduce toxicity to cells that is caused by expression of packaging proteins, packaging systems may be used in which the plasmids encoding the packaging functions of the virus are only transiently transfected by, for example, chemical means.

[0022] In another embodiment, the vector may be made safer by replacing certain lentiviral sequences with non-lentiviral sequences to produce a hybrid vector. Thus, lentiviral vectors herein may contain partial (e. g., split) gene lentiviral sequences and / or non-lentiviral sequences (e.g., sequences from other retroviruses) as long as its function (e.g., viral titer, infectivity, integration and ability to confer high levels and duration of therapeutic gene expression) are not substantially reduced. Elements that may be cloned into the viral vector include, but are not limited to, a left (5 ' ) retroviral long terminal repeat (LTR); a packaging signal(psi); a lentiviral reverse response element (RRE); a promoter, or active portion thereof, and a locus control region (LCR), or active portion thereof, operably linked to a gene of interest (e. g., RPS19); and a right (3 ' ) retroviral LTR. In some embodiments, a 5' LTR of use in the vector and method herein has the following nucleotide sequence; gggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaaccca ctgctta gcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttg tgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagc a (SEQ ID NO: 14).

[0023] A lentiviral vector herein has preferably been modified to improve its safety by modifying a LTR region. In some embodiments, the 3 ' LTR is modified to generate a self-inactivating vector or SIN vector. The term "self-inactivating vector" or "SIN vector" refers to a vector herein in which the right (3 ' ) LTR enhancer-promoter region, known as the U3 region, has been modified (e. g., by deletion or substitution) to prevent viral transcription beyond the first round of viral replication. Consequently, the vector is capable of infecting and then integrating into the host genome only once, and cannot be passed further. This is because the right (3') LTR U3 region is used as a template for the left (5 ' ) LTR U3 region during viral replication and, thus, the viral transcript cannot be made without the U3 enhancer-promoter. If the viral transcript is not made, it cannot be processed or packaged into virions, hence the life cycle of the virus ends. Accordingly, SIN vectors greatly reduce risk of creating unwanted replication-competent virus since the right (3 ' ) LTR U3 region has been modified to prevent viral transcription beyond the first round of replication, hence eliminating the ability of the virus to be passed. In some embodiments, a 3' LTR of use in the vector and method herein has the following nucleotide sequence:tggaagggctaattcactcccaaagaagacaagatctgctttttgcctgtactgggtctc tctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgctta agcctcaataaagcttgccttg (SEQ ID NO: 15).

[0024] Lentiviral vectors may further contain a central polypurine tract (PPT) or DNA flap. The PPT / DNA flap is used to increase viral titers and transduction efficiency. In a particular embodiment, the PPT / DNA flap is from HIV-1. In some embodiments, a PPT sequence of use in the vector and method herein has the following nucleotide sequence: aattccctacaatccccaaagtcaaggagtagtagaatctatgaataaagaattaaagaa aattataggacaggtaagagatcaggctgaacatcttaagacagcagtacaaatggcagt attcatccacaattttaaaagaaaaggggggattggggggtacagtgcaggggaaagaat agtagacataatagcaacagacatacaaactaaagaattacaaaaacaaattacaaaaat tcaaaattttcgggttt ttacagggacagcagaaatccactttggaaaggaccagcaaa gctcctctggaaaggtgaaggggcagtagtaatacaagataatagtgacataaaagtagt gccaagaagaaaagcaaagatcattagggattatggaaaacagatggcaggtgatgattg tgtggcaagtagacaggatgaggattagaacatggaaaagtttagtaaaacaccata (SEQ ID NO: 16).

[0025] When used in gene therapy applications for treating or ameliorating Diamond-Blackfan anemia syndrome (DBAS), the lentiviral vector further includes nucleic acids encoding "ribosomal protein S19" or "RPS19, " which is a ribosomal structural protein of the 40S subunit. Mutations in this gene cause DBAS. The amino acid sequence of wild-type human RPS19 (i. e., a RSP19 that does not include a mutation known to result DBAS) is as follows:MPGVTVKDVN QQEFVRALAA FLKKSGKLKV PEWVDTVKLA KHKELAPYDE NWFYTRAAST ARHLYLRGGA GVGSMTKIYG GRQRNGVMPS HFSRGSKSVA RRVLQALEGL KMVEKDQDGG RKLTPQGQRD LDRIAGQVAA ANKKH (SEQ ID NO: 17).

[0026] In embodiments of the vector and method herein, the nucleic acids encoding RPS19 are (i) codon optimized forexpression in human cells and (ii) devoid of aberrant splice sites. As used herein the term "codon optimized" refers to a nucleic acid that has been adapted for expression in the cells of a given organism by replacing at least one, or more than one, or a significant number, of codons with one or more codons that are more frequently used in the genes of that organism.

[0027] In general, highly expressed genes in an organism are biased toward codons that are recognized by the most abundant tRNA species in that organism. One measure of this bias is the "codon adaptation index" or "CAI," which measures the extent to which the codons used to encode each amino acid in a particular gene are those which occur most frequently in a reference set of highly expressed genes from an organism. The Codon Adaptation Index is described in more detail in Sharp & Li (1987) Nucleic Acids Research 15: 281-1295.

[0028] A codon optimized sequence may be further modified for expression in a particular organism, depending on that organism' s biological constraints. For example, large runs of "As" or "Ts" (e. g., runs greater than 3, 4, 5, 6, 7, 8, 9, or 10 consecutive bases) may affect transcription negatively. Therefore, it may be useful to remove a run by, for example, replacing at least one nucleotide in the run with another nucleotide. Furthermore, specific restriction enzyme sites may be removed for molecular cloning purposes by replacing at least one nucleotide in the restriction site with another nucleotide. Additionally, the DNA sequence can be checked for direct repeats, inverted repeats and mirror repeats with lengths of about 5, 6, 7, 8, 9 or 10 bases or longer. Runs of "As" or "Ts", restriction sites and / or repeats can be modified by replacing at least one codon within the sequence with the "second best" codons, i. e., the codon that occurs at the secondhighest frequency for a particular amino acid within the particular organism for which the sequence is being optimized.

[0029] In some embodiments, a codon optimized sequence may be further modified to remove aberrant splice sites. A "splice acceptor site" refers to the junction between an exon and an intron in a pre-mRNA (unspliced RNA) molecule (also known as a "splice junction"). A "cryptic splice acceptor site" or "aberrant acceptor splice site" is a splice site that is not typically used but may be used when the usual splice site is blocked or unavailable or when a mutation causes a normally dormant site to become an active splice site. In embodiments of the vector and method herein, the codon optimized RPS19 nucleic acids and / or lentiviral vector as a whole are devoid or substantially devoid of aberrant splice acceptor sites, e. g., the presence of fusion transcripts between host genes and the lentiviral sequence are not detectable as determined by, e.g., RNASeq analysis.

[0030] Many organisms display a bias for use of particular codons to code for insertion of a particular amino acid in a growing peptide chain. Codon preference or codon bias, differences in codon usage between organisms, is afforded by degeneracy of the genetic code, and is well documented among many organisms. Codon bias often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, inter alia, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization.

[0031] In some embodiments, a codon optimized nucleic acid sequence encoding RPS19 comprises, consists of, or consists essentially of any one of SEQ ID NOs: 8-10. In some embodiments, a codon optimized nucleic acid sequence encoding RPS19 that is devoid of aberrant splice acceptor sites comprises, consists of, or consists essentially of any one of SEQ ID NOs: 8-10.

[0032] To effect expression of RPS19, the nucleic acids encoding RPS19 are operably linked to promoter. The term "promoter" as used herein refers to a recognition site of a DNA strand to which the RNA polymerase binds. The promoter forms an initiation complex with RNA polymerase to initiate and drive transcriptional activity. In some embodiments, the promoter may be "endogenous" or "exogenous" or "heterologous." An "endogenous" promoter is one which is naturally linked with a given gene in the genome. An "exogenous" or "heterologous" promoter is one which is placed in juxtaposition to a gene by means of genetic manipulation (i. e., molecular biological techniques) such that transcription of that gene is directed by the linked promoter.

[0033] In some embodiments, the promoter is an EFla promoter. An "EFla promoter" or "elongation factor 1 alpha promoter" refers to the human eukaryotic translation elongation factor 1 alpha promoter which regulates transcription in a broad range of cell types. In some embodiments, the EFla promoter of the vector and / or method herein has the nucleotide sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 11 or SEQ ID NO: 12.

[0034] In some embodiments, the promoter is an MND promoter. An "MND promoter" or "myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primerbinding site substituted promoter" refers to a syntheticpromoter that contains the U3 region of a modified Moloney murine leukemia virus with long terminal repeats and enhancer from myeloproliferative sarcoma virus. In some embodiments, the MND promoter of the vector and / or method herein has the nucleotide sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 13.

[0035] Efficient expression of recombinant nucleic acid sequences in eukaryotic cells requires expression of signals directing the efficient termination and polyadenylation of the resulting transcript. Transcription termination signals are generally found downstream of the polyadenylation signal. The term "poly A site" or "poly A sequence" as used herein denotes a nucleic acid sequence which directs both the termination and polyadenylation of the nascent RNA transcript. Efficient polyadenylation of the recombinant transcript is desirable as transcripts lacking a poly A tail are unstable and are rapidly degraded. Accordingly, in some embodiments, the nucleic acid sequence encoding RPS19 includes a poly A signal. The poly A signal used in a vector and method herein may be "heterologous" or "endogenous." An endogenous poly A signal is one that is found naturally at the 3 ' end of the coding region of a given gene in the genome. A heterologous poly A signal is one which is one which is isolated from one gene and placed 3 ' of another gene.

[0036] The term "operably linked" refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. In one embodiment, the term refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter) and a second nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.

[0037] In some embodiments, the lentiviral vector herein further includes a 5' long terminal repeat (LTR), a packaging signal (psi), a rev response element (RRE), and / or a 3 ’ LTR comprising at least one deletion compared to a wild-type 3 ' LTR.

[0038] As used herein, the term "packaging" refers to the process of sequestering (or packaging) a viral genome inside a protein capsid, whereby a virion particle is formed. This process is also known as encapsidation. As used herein, the term "packaging signal" or "packaging sequence" refers to sequences located within the lentiviral genome which are required for insertion of the viral RNA into the viral capsid or particle. Several lentiviral vectors use the minimal packaging signal (also referred to as the psi [i| / ] sequence) needed for encapsidation of the viral genome. Thus, as used herein, the terms "packaging sequence," "packaging signal," "psi" and the symbolare used in reference to the noncoding sequence required for encapsidation of retroviral RNA strands during viral particle formation. In some embodiments, a psi sequence of use in the vector and method herein has the following nucleotide sequence: ctcggcttgctgaagcgcgcacggcaagaggcgaggggcggcgactggtgagtacgccaa aaattttgactagcggaggctagaaggagagagatgggtgcgagagcgtcagtattaagc gggggagaattagatcgcgatgggaaaaaattcggttaaggccagggggaaagaaaaaat ataaattaaaacatatagtatgggcaagcagggagctagaacgattcgcagttaatactg gcctgttagaaacatcagaaggctgtagacaaatactgggacagctacaaccatcccttc agacaggatcagaagaacttagatcattatataatacagtagcaaccctctattgtgtgc atcaaaggatagagataaaagacaccaaggaagctttagacaagatagaggaagagcaaa acaaaagtaagaaaaaagcacagcaagcagcag (SEQ ID NO: 18).

[0039] The term "export element" refers to a cis-acting post- transcriptional regulatory element which regulates the transport of an RNA transcript from the nucleus to thecytoplasm of a cell. Examples of RNA export elements include, but are not limited to, the human immunodeficiency virus (HIV) rev response element (RRE) (see, e. g., Cullen et al. (1991) J. Virol. 65: 1053; and Cullen et al. (1991) Cell 58: 423), and the hepatitis B virus post-transcriptional regulatory element (PRE) (see, e. g., Huang et al. (1995) Molec. Cell. Biol.15 (7): 3864; Huang et al. (1994) J. Virol. 68 (5): 3193; Huang et al. (1993) Molec. Cell. Biol. 13 (12): 7476), and U. S.5, 744, 326). Generally, the RNA export element is placed within the 3 ’ UTR of a gene, and can be inserted as one or multiple copies. RNA export elements can be inserted into any or all of the separate vectors generating the packaging cell lines herein. In some embodiments, a rev response element sequence of use in the vector and method herein has the following nucleotide sequence: aattggagaagtgaattatataaatataaagtagtaaaaattgaaccattaggagtagca cccaccaaggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaataggagct ttgttccttgggttcttgggagcagcaggaagcactatgggcgcagcgtcaatgacgctg acggtacaggccagacaattattgtctggtatagtgcagcagcagaacaatttgctgagg gctattgaggcgcaacagcatctgttgcaactcacagtctggggcatcaagcagctccag gcaagaatcctggctgtggaaagatacctaaaggatcaacagctcctggggatttggggt tgctctggaaaactcatttgcaccactgctgtgccttggaatgctagttggagtaataaa tctctggaacagatttggaatcacacgacctggatggagtgggacagagaaattaacaat tacacaagcttaatacactccttaattgaagaatcgcaaaaccagcaagaaaagaatgaa caagaattattggaattagataaatgggcaagtttgtggaattggtttaacataacaaat tggctgtggtatataaaattattcataatgatagtaggaggcttggtaggtttaagaata gtttttgctgtactttctatagtgaatagagttaggcagggatattcaccattatcgttt cagacccacctcccaaccccgaggggaccg (SEQ ID NO: 19).

[0040] Lentiviral vectors may also comprise elements that control selection of the transduced cell. In one embodiment, the lentiviral vector comprises a nucleic acid cassette which allows for in vivo selection of the transduced cell. Forexample, the nucleic acid cassette could contain the cDNAs for methylguanine methyltransferase {MGMT) or the human glutathione-S-transf erase pi (GST pi) which have both been successfully used as in vivo selection markers for transduced cells. These transgenes provide chemoprotection to the combination of 06-benzylguanine (BG) and 1, 3-bis (2-chloroethyl) -1-nitrosourea (BOND) or to transduced cells against post-transplant treatment with cyclophosphamide, respectively. In another embodiment, the lentiviral vector contains a suicide gene operably linked to a promoter. Examples of suicide genes include, but are not limited to, herpes simplex virus (HSV) thymidine kinase (HSV-Tk).

[0041] Standard techniques for the construction of expression vectors suitable for use herein are well-known to those of ordinary skill in the art and may be found in such publications as Sambrook et al. ( 1989) Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor, N. Y. A variety of strategies are available for ligating fragments of DNA, the choice of which depends on the nature of the termini of the DNA fragments and which choices may be readily made by the skilled artisan.

[0042] Production of infectious viral particles in cells may be carried out using conventional techniques, such as standard cell culture growth techniques. If desired, lentiviral stock solutions may be prepared using the vectors and methods herein. Methods of preparing viral stock solutions are known in the art and are illustrated by, e. g., Soneoka et al. (1995) Nucl. Acids Res. 23: 628-633 and Landau et al. (1992) < J. Virol.66: 5110-5113. In a method of producing a stock solution, lentiviral-permissive cells (referred to herein as producer cells) are transfected with the vector herein. The cells are then grown under suitable cell culture conditions, and thelentiviral particles collected from either the cells themselves or from the cell media. Suitable producer cell lines include, but are not limited to, the human embryonic kidney cell line 293, the equine dermis cell line NBL-6, and the canine fetal thymus cell line Cf2TH.

[0043] The step of collecting the infectious virus particles also may be carried out using conventional techniques. For example, the infectious particles may be collected by cell lysis, or collection of the supernatant of the cell culture, as is known in the art. Optionally, the collected virus particles may be purified if desired. Suitable purification techniques are well-known to those skilled in the art.

[0044] Other methods relating to the use of viral vectors in gene therapy may be found in, e. g., Kay (1997) Chest 111 ( 6 Supp. ): 138S-142S; Ferry & Heard (1998 ) Hum. Gene Then. 9: 1975-81; Shiratory et al. (1999) Liver 19: 265-74; Oka et al. (2000) Curr. Opin. Lipidol. 11: 179-86; Thule & Liu (2000) Gene Ther.7: 1744-52; Yang (1992) Grit. Rev. Biotechnol. 12: 335-56; Alt (1995) J. Hepatol. 23: 746-58; Brody & Crystal (1994 ) Ann. N. Y. Acad. Sci. 716: 90-101; Strayer (1999) Expert Opin. Investig. Drugs 8: 2159-2172; Smith-Arica & Bartlett (2001) Curr. Cardiol. Rep. 3: 43-49; and Lee et al. (2000) Nature 408: 483-8.

[0045] Retroviral vectors, including lentiviral vectors, as described above may be administered in vivo to subjects by any suitable route, as is well-known in the art. The term "administration" refers to the route of introduction of a formulated vector into the body. For example, administration may be intravenous, intramuscular, topical, oral, or by gene gun or hypospray instrumentation. Thus, administration may be direct to a target tissue or through systemic delivery. Administration directly to the target tissue may involveneedle injection, hypospray, electroporation, or the gene gun. See, e.g., WO 93 / 18759.

[0046] Alternatively, the lentiviral vector herein may be administered ex vivo or in vitro to cells or tissues using standard transfection techniques well-known in the art.

[0047] The lentiviral vector herein may also be transduced into host cells, including embryonic stem cells, somatic stem cells, or progenitor cells. Examples of progenitor host cells that may be transduced by the lentiviral vector herein include precursors of erythrocytes and hematopoietic stem cells, including CD34+hematopoietic stem and progenitor cells. In another embodiment, the host cell is an erythrocyte. Transduced host cells may be used as a method of achieving erythroid-specific expression of RPS19 in the treatment of Diamond-Blackfan anemia syndrome.

[0048] Another embodiment pertains to pharmaceutical compositions including the lentiviral vector herein. In one embodiment, the pharmaceutical composition includes a lentiviral vector in a therapeutically effective amount sufficient to treat or prevent (e. g. ameliorate the symptoms of Diamond-Blackfan anemia syndrome), and a pharmaceutically acceptable carrier. A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, such as treatment or prevention of Diamond-Blackfan anemia syndrome. A therapeutically effective amount of lentiviral vector may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the lentiviral vector to elicit a desired response in the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. A therapeutically effective amount is also one in which any toxic or detrimental effectsof the lentiviral vector are outweighed by the therapeutically beneficial effects. The potential toxicity of the lentiviral vector herein may be assayed using cell-based assays or art-recognized animal models and a therapeutically effective modulator may be selected which does not exhibit significant toxicity. In one embodiment, a therapeutically effective amount of a lentiviral vector is sufficient to treat Diamond-Blackfan anemia syndrome.

[0049] As used herein "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. In one embodiment, the carrier is suitable for parenteral administration. Preferably, the carrier is suitable for administration directly into a target tissue. The carrier may be suitable for intravenous, intraperitoneal or intramuscular administration. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is well-known in the art. Except insofar as any conventional media or agent is incompatible with the lentiviral vector, use thereof in the pharmaceutical compositions herein is contemplated. Supplementary active compounds may also be incorporated into the compositions.

[0050] Sterile injectable solutions may be prepared by incorporating lentiviral vector in the required amount in an appropriate solvent with one or a combination of ingredients enumerated herein, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the vector into a sterile vehicle that containsa basic dispersion medium and the required other ingredients from those enumerated here. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0051] A method of treating or ameliorating Diamond-Blackfan anemia syndrome is also provided by administering to a subject in need thereof, an effective amount of a self-inactivating lentiviral vector comprising nucleic acids encoding RPS19 operably linked to an EFla or MND promoter, wherein the nucleic acids encoding RPS19 are codon optimized for expression in human cells and are devoid of aberrant splice sites. "Treating" or "treatment" as used herein covers the treatment of Diamond-Blackfan anemia syndrome in a subject, preferably a human, and includes arresting the development of the syndrome; relieving the syndrome, i. e., causing regression of the syndrome; slowing progression of the syndrome; and / or inhibiting, relieving, or slowing progression of one or more symptoms of the syndrome. In some embodiments, treatment results in a measurable (i. e., at a statistically significant level) decrease or reduction in one or more signs or symptoms of Diamond-Blackfan anemia syndrome, e. g. one or more of defects in ribosomal RNA processing, erythropoiesis, macrocytic anemia, reticulocytopenia, bone marrow erythroid hypoplasia, developmental anomalies, multilineage cytopenias, and bone marrow cellularity.

[0052] "Subject" or "subject in need thereof" refers to a warm-blooded animal such as a mammal, preferably a human, or a human child, which is afflicted with, or has the potential to be afflicted with Diamond-Blackfan anemia syndrome.Subjects benefiting from any one of the above referenced treatments include, but are not limited to, subjects at risk of having, having or predisposed to have Diamond-Blackfan anemia syndrome.

[0053] In some embodiments, the self-inactivating lentiviral vector herein is administered to a subject by introducing (e. g., transducing) the lentiviral vector into donor host cells. In some embodiments, the donor host cells are autologous / autogeneic ("self") or non-autologous ("non-self," e.g., allogeneic, syngeneic or xenogeneic). "Autologous, " as used herein, refers to cells from the same subject. "Allogeneic," as used herein, refers to cells of the same species that differ genetically to the cell in comparison. "Syngeneic," as used herein, refers to cells of a different subject that are genetically identical to the cell in comparison. "Xenogeneic, " as used herein, refers to cells of a different species to the cell in comparison. In some embodiments, the cells used in the method herein are allogeneic. In some embodiments, the donor host cells are embryonic stem cells, somatic stem cells, or progenitor cells, in particular CD34 + HSPCs.

[0054] In some embodiments, host cells to be used in the treatment of DBAS (e. g., HSPCs) are transduced with the selfinactivating lentiviral vector described herein at a multiplicity of infection (MOI) of between about 10 and about 200, e. g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200, or any value or range therebetween. In some embodiments, host cells are transduced with the self-inactivating lentiviral vector described herein at a MOI of at least 20 and not more than 100.

[0055] It is to be noted that dosage values may vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens may be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.

[0056] Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of vector calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[0057] As demonstrated herein, computational approaches were used to identify potential splice acceptor sites in the lentiviral vector encoding RPS19 thereby improving the safety of the same. In particular, aberrant splicing was identified by transducing human hematopoietic stem cells with the lentiviral vector, conducting RNASeq analysis of the transduced cells, identifying hybrid reads that map to the human genome and to the lentiviral vector sequence, and normalizing the same to determine the degree of aberrant splicing as compared to a control, e. g., a lentiviral vectorknown to possess cryptic splice acceptor sites. Upon identification of aberrant splicing, the gene of interest is modified by changing the coding sequence to eliminate aberrant splice sites, while maintaining the same amino acid sequence of the protein encoded by the gene of interest. Accordingly, also provided herein is a method for improving the safety and / or efficacy of a self-inactivating lentiviral vector, in particular a lentiviral vector including nucleic acids encoding RPS19, by transducing a suitable host cell (e. g., HSPC) with the self-inactivating lentiviral vector, conducting RNASeq analysis of the transduced cells, identifying hybrid human-lentiviral vector transcripts indicative of aberrant splice sites in the self-inactivating lentiviral vector, and removing the aberrant splice sites in the self-inactivating lentiviral vector thereby improving the safety and / or efficacy of a self-inactivating lentiviral vector. As used herein, "RNASeq" refers to the use of second-generation high throughput sequencing technology to perform cDNA sequencing, to obtain all transcripts of a particular organ or tissue of a species in a certain state. See, e. g., De Ravin et al. (2022) Nature Commun. 13: 3710. In some embodiments, the method is used to improve a self-inactivating lentiviral vector encoding RPS19.

[0058] The following non-limiting examples are provided to further illustrate the present invention.EXAMPLE 1: Materials and Methods

[0059] CD34+ Hematopoietic Stem and Progenitor Cells (HSPCs) Isolation, Culture and Differentiation. Human mononuclear cells were collected from healthy adult volunteer donors who underwent apheresis following mobilization with granulocyte colony stimulating factor (G-CSF, CGT Global). CD34+ HSPCswere isolated using an AutoMACS instrument (Miltenyi Biotec) following manufacturer' s instructions and cells were cryopreserved until needed. CD34+cells were cultured in HSPC maintenance medium: X-VIVO”*-10 (Lonza) medium supplemented with 100 ng / µL human stem cell factor (hSCF; R& D systems), 100 ng / µL human thyroid peroxidase (hTPO; R& D systems) and 100 ng / µL human Fms-like tyrosine kinase 3 (hFlt-3) ligand (R& D systems) at a concentration between 0.2-1.0×106cells / mL. HSPCs were differentiated in vitro by seeding 500-1000 CD34 + HSPCs in 1 mL of methylcellulose media (StemCell Technologies) in meniscus-free culture plates (StemCell Technologies). Colonies were assessed and counted 14 days after seeding.

[0060] Genome Editing. HSPCs were edited using CRISPR / Cas9 and sgRNAs as previously described (Bhoopalan et al. (2023) JCI Insight 8 (1): e161810). Briefly, ribonucleoprotein (RNP) complex was prepared by incubating single guide RNA (sgRNA, Synthego) and Cas9-3XNLS protein (St. Jude Protein Production Facility) at a molar ratio of 1:3 at room temperature for 15 minutes. Next, 0.2-1.0×106CD34 + HSPCs were washed and suspended in P3 buffer (Lonza) before RNP was added to make a total volume of 20 pL and a final Cas9 concentration of 0.08 mg / mL. Cells were electroporated using a Lonza 4D-Nucleofector®, using program DS-130, following manufacturer' s instructions, and suspended in HSPC maintenance medium. Editing efficiency was determined through next generation sequencing (NGS) and analyzing for percentage of insertiondeletion (indel) mutations as previously described (Bhoopalan et al. (2013) JCI Insight 8(1):e161810).

[0061] Production and Titration of Lentiviral Vector (LV). Production and titration of LV were performed as described previously (Bauler et al. (2024) Mol. Ther. Methods Clin. Dev.32 (2): 101270; Bauler et al. (2020) Mol. Ther. Methods Clin.Dev. 17: 58-68). Briefly, SJ293TS-DPB cells were transfected with the transfer vector and the helper plasmids pCAG-kGPl-1R-AF, pCAG-VSVG-AF and pCMV-Rev-AF using PEIpro® (Polyplus Transfection, Strasbourg, France). Six hours posttransfection, transfected cells were diluted with an equal volume of media containing Benzonase® (Millipore-Sigma, Burlington, MA) to achieve a final concentration of 6.25 U / mL. Vector supernatants were collected 48 hours post-transfection, clarified by centrifugation at 330×g for 5 minutes and 0.22 µM filtered. LV containing supernatants were adjusted to 300 mM NaCl, 50 mM Tris pH 8. 0 and loaded onto an Acrodisc® Mustang Q membrane (Pall Life Sciences, NY) according to the manufacturer' s instructions using an Akta Avant chromatography system (GE Healthcare Bio-Sciences, Pittsburgh, PA). After washing the column with 10 column volumes of 300 mM NaCl and 50 mM Tris pH 8.0, viral particles were eluted from the column using 2 M NaCl and Tris pH 8.0. Viral particles were formulated in X-VIVO™-10 serum-free hematopoietic media (Lonza, Walkersville, MD) using a PD10 desalting column (GE Healthcare) according to the manufacturer' s instructions to achieve an approximate 50-fold concentration from the starting material, 0.22 µm sterile filtered, aliquoted and stored at -80°C.

[0062] Titration of LV was performed by transducing HOS-6A5 cells with serially diluted vector preparations in the presence of polybrene (5-8 µg / mL; Millipore Sigma, Burlington, MA). Four days post-transduction, genomic DNA was isolated from transduced cells and, after digestion with the restriction enzyme MspI, was used as a template in PCR using a QX200 digital droplet PCR system (Bio-Rad, Carlsbad, CA). The following primer-probe sets were used to amplify the HIV psi sequence and the endogenous control gene, RPP30, 5' -ACTTGAAAGCGAAAGGGAAAC-3' (SEQ ID NO: 1), 5' -CACCCATCTCTCTCCTTCTAGCC-3' (SEQ ID NO: 2) and probe 5'-56-FAM-AGCTCTCTC linked to GACGCAGGACTCGGC-3'IABkFQ-3' (SEQ ID NO: 3) via a ZEN™ quencher (FAM, fluorescein; lABkFQ, Iowa Black™ FQ quencher) and 5' -GCGGCTGTCTCCACAAGT-3' (SEQ ID NO: 4), 5' -GATTTGGACCTGCGAGCG-3' (SEQ ID NO: 5) and probe 5' -5HEX-CTGACCTGA linked to AGGCTCT-3'IABkFQ-3' via a ZEN™ quencher (HEX, Hexachloro-fluorescein), respectively. Vector titers and copy number were determined by calculating the number of copies of HIV psi to every two copies of RPP30, multiplied by the number of cells transduced, and if necessary, multiplied by the dilution factor.

[0063] LV Transduction of HSPCs. CD34+cells were thawed and cultured in X-VIVO™ medium containing 1000× dilution of hSCF, Fit 3, and hTPO, overnight. The transduction assays were conducted with a concentration of 2×106cells / mL with the specified multiplicity of infection (MOI). The transduction medium was prepared with culture medium containing 1 mg / mL of LentiBOOST® transduction enhancer (Sirion Biotech) and 10 µM of prostaglandin E2 (Cayman Chemical). After 16-20 hours, the transduction medium was replaced with culture medium, and the cells were maintained for an additional 3 days before being collected.

[0064] iPSC Culture, Differentiation and Analysis. Induced pluripotent stem cells (iPSCs) were maintained in mTeSR® Plus medium (StemCell Technologies) and passaged every 3 to 4 days using ReLeSR® dissociation medium (StemCell Technologies). For the differentiation of hematopoietic progenitor cells, the protocol outlined in the STEMdiff® Hematopoietic Kit (StemCell Technologies) was followed. Briefly, iPSC aggregates were seeded onto Geltrex®-coated plates. After 24 hours, the medium was changed to hematopoietic differentiation medium A. On day3, the medium was replaced with hematopoietic differentiation medium B, and subsequently refreshed every two days until day 10. On day 10, suspension cells were collected and isolated using CD43 magnetic beads (Miltenyi Biotec). Following isolation, 50,000 CD43+cells per well were seeded into ultra-low-attachment 24-well plates with SFEM II medium (StemCell Technologies) supplemented with StemSpan® Erythroid Expansion Supplement (StemCell Technologies). The medium was refreshed on day 4, day 7, and day 10 until day 14. Erythroid cells were harvested on day 14 for subsequent flow cytometry analysis. Cells were washed once with phosphate-buff ered saline (PBS) and then collected by centrifugation at 300g for 5 minutes. The cell pellet was resuspended in 100 µL of flow staining buffer (PBS containing 2% fetal bovine serum (FBS) ) and incubated with the following antibodies: CD45-FITC (BD Biosciences), CD71-BV711 (BD Biosciences), and CD235a-PE-Cy7 (BD Biosciences). Incubation was carried out for 30 minutes at room temperature in the dark. Following antibody labeling, cells were washed with 1 mL of flow staining buffer and resuspended in 200 µL of flow staining buffer. Viability staining was performed using DAPI (Invitrogen™). Data acquisition was conducted on a BD LSRFortessa™ Cell Analyzer. Flow cytometry data were analyzed using Flow Jo v. 10. 10.0.

[0065] Xenotransplantation Studies. 5- to 8-week-old female NSGW mice bred in-house were used for all experiments. 3-5×105HSPCs were washed and suspended in PBS containing 2% FBS and administered via tail vein injection, 2 days after mice being conditioned with low-dose busulfan (10 mg / kg) (Everette et al. (2023) Nat. Biomed. Eng. 7(5):616-28). Mice were euthanized and examined 16 weeks post-xenotransplantation. Recipient bone marrow cells were treated with lineage-specific antibodies for both mouse and human and sorted using a BD FACSAria® II cellsorter (BD Biosciences). Indel frequencies were assessed through NGS analysis of mouse bone marrow or purified human hematopoietic lineages.

[0066] Northern Blot. RNA was isolated from HSPCs using a RNeasy® kit (Qiagen) according to the manufacturer' s protocol. After gel fractionation on a 1.5% formaldehyde-agarose gel, the RNA was transferred onto zeta-probe nylon membranes (BioRad). Subsequently,32P-labeled ITS1 probes were hybridized with the membrane overnight at 37 °C in hybridization buffer (Ambion) prior to phosphorimaging.

[0067] Quantitative Shearing Linear Amplification Polymerase Chain Reaction (qsLAM-PCR). Lentiviral insertion sites were determined as described (Yan et al. (2023) Sci. Adv.9 (40): eadg9959; Zhou et al. (2015) Hum. Gene Ther. Methods 26(1):1-12). Briefly, 1 µg of genomic DNA was sheared to generate fragments between 200 and 800 bp using a Bioruptor® Pico sonication device (Diagenode). DNA underwent end-repair, dA-tailing, and ligation of adaptors with New England BioLabs (NEB) Modules (NEBNext® End Repair Module, NEBNext® dA-Tailing Module, and NEBNext® Quick Ligation Module, respectively). Adaptors were compatible with the NEBNext® Multiplex Oligos for Illumina®. Following ligation, linear amplification was performed for 50 cycles with a 5' biotinylated primer (5' -AGTAGTGTGTGCCCGTCTGT-3'; SEQ ID NO: 6) specific to the vector' s long terminal repeat (LTR) using KAPA HiFi HotStart ReadyMix (Roche (KAPA BIOSYSTEMS) ). Amplified products were selected and purified using Dynabeads™ M-270 Streptavidin beads (ThermoFisher Scientific). Indexing PCR was performed using an LTR-specific primer (5' - AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCT CTTCCGATCTNNNagttaGATCCCTCAGACCCTTTTAGTC-3'; SEQ ID NO: 7 ) and NEBNext® Multiplex Oligos for Illumina® (Index Primers Set 1and 2, NEB). All reactions were combined, and size-selection was performed on E-Gel™ EX Agarose Gels (ThermoFisher Scientific) for amplicons between 350 and 800 bp. Targeted sequencing of the libraries was performed using Illumina MiSeq® with a 150 bp paired-end read length.

[0068] Vector Integration Site Analysis. The bioinformatics pipeline for calling vector integration sites from sequencing data of the qsLAM PCR assay focused on chimeric reads where the LV are found at the 5' end (Yan et al. (2023) Sci. Adv.9 (40): eadg9959). PCR adaptor and 3' LTR of the vector were trimmed from the chimeric reads. The human sequences were mapped to hgl9 and the starting genomic coordinates were used to establish vector integration sites. To compare vector integration sites across samples, sites that were within 8 bp were regarded to be the same. List of 119 cancer related genes were obtained from previously published report (Ma et al. (2018 ) Nature 555 (7696): 371-6). The pipeline for processing sequencing data from the qsLAM PCR assay and the subsequent integration site analysis can be found in the github repository.

[0069] Splice Analysis. CD34 + HSPCs from three different donors were transduced with specified LVs at an MOI of 100, and resuspended in X-VIVO™-10 media with cytokines as described above. Four days later, RNA was extracted using RNeasy® Plus kit (Qiagen). Total stranded paired-end RNA sequencing was performed using NovaSeq® 6000 platform (Illumina, Inc. ). RNA-Seq reads were mapped to a reference genome composed of hg38 human genome sequence and the corresponding proviral sequence for the sample using STAR (2. 7. 9a) (Dobin et al. (2013) Bioinformatics 29(1):15-21). The *. Chimeric. out. junction output files were parsed to retain only chimeric reads that spanned both the proviral sequenceand human genome. If the predicted human-proviral splice junction in the chimeric reads was > 5 bases away from a known splice donor site in the human genome, then the read pairs were excluded. Predicted splice acceptor sites within five bases of the 5 ' start position in the proviral sequence were excluded from subsequent analysis as they are likely to arise from exonic integration. Coordinates of vector splice sites in 3 ’ LTR were adjusted to the corresponding coordinates in 5 ' LTR. Splice acceptor locations were tallied for each sample and normalized to total read pairs mapped to chromosomal genes. Normalized counts were averaged from replicates. For in silico prediction of potential splice acceptor sites, the webservers for Netgene2 and BDGP were used (Brunak et al. (1991) J. Mol. Biol. 220 (1): 49-65; Reese et al. (1997) J. Comput. Biol. 4 (3): 311-23).

[0070] Codon Optimized RPS19. Three versions of codon optimized RPS19 were generated (Table 1).TABLE 1*Underlined residues show splice acceptor sites.

[0071] Protein Extraction and Western Blot Assays. Cells (0.5-1x106) were collected and washed with PBS, the pellets were resuspended in cell lysis buffer (PBS:2xLaemmli sample buffer=1:1, Sigma-Aldrich). Protein samples were loaded onto 4%-12% Bis-Tris Plus gels (Life Technologies), subjected to electrophoresis (100 Volts, 100 minutes). The gels were transferred to a nitrocellulose membrane (295 mA, 100 minutes). Membranes were blocked with Intercept® blocking buffer (LI-COR) at room temperature for 1 hour, and incubated with Ubiquityl-Histone H2A (Lys119) (D27C4 ) primary antibody (Cell Signaling Technology) using a 1:2000 dilution, 4 °C, overnight. The membranes were washed with IX TBST (Trisbuffered saline + 0.1% TWEEN® 20) and incubated with secondary antibody at room temperature for 1 hour (IRDye 800 CW anti-Rabbit, 1:10,000 dilution, LI-COR). (3-actin was used as an internal control, which was detected with anti-actin primary (Sigma-Aldrich Anti-p-actin antibody, 1: 8000 dilution) and secondary (IRDye 800 CW anti-Mouse, 1:10,000 dilution, LI-COR) antibody at the same condition as the samples. Blots were acquired and analyzed with both fluorescence channels byOdyssey Clx imaging system (LI-COR). Quantitative analysis was performed with Image Studio software by normalizing the sample band to the corresponding actin loading control.

[0072] Genomic DNA Extraction and Measurement of Vector Copy Number (VCN). Genomic DNA was extracted from transduced cells using DNeasy® Blood and Tissue Kit (Qiagen) following manufacturer' s instructions. VCN was determined by qRT-PCR using Taqman® probe, the endogenous reference control used was RNaseP. The Jurkat VCN was set as the reference VCN, and the copy number of the samples was analyzed by the comparative CT (2-DDCT) method, where DDCT = (CTtarget - CTreference)Sample X- (CTtarget - CTreference)Sample 1. Sample 1 was the reference Jurkat without any treatment, and Sample X represents the treated samples.

[0073] Statistical Analysis. All the experiments were replicated at least three times using CD34+HSPCs from at least two different donors. The data were analyzed statistically using Prism vlO. O (GraphPad, Boston, MA). Unless otherwise specified in the figure legend, data from all experiments are shown as mean ± standard deviation (SD). The data analyses were performed by unpaired t-test, the asterisk indicates a significant difference (*P<0.05, **P<0.01, ***P<0.001).EXAMPLE 2: Efficient Transduction of CD34+ HSPCs with RPS19-Encoding LVs

[0074] A third-generation, SIN LV encoding RPS19 and green fluorescent protein (GFP) has been described and was separated by ribosome-skipping P2A sequence driven by EFla short (EFlaS) promoter (Bhoopalan et al. (2023) JCI Insight 8 (1): el61810). This LV, called SJEFS-S19GFP (FIG. 1), used a backbone similar to the one used in a previous clinical trial with one change involving removal of chicken hypersensitivity site 4 (cHS4 )insulator element (Mamcarz et al. (2019) N. Engl. J. Med.380 (16): 1525-34). SJEFS-S19GFP was able to rescue erythroid defect and in vivo bone marrow repopulation defect caused by CRISPR / Cas9-mediated heterozygous knockout of RPS19 in CD34+HSPCs from healthy donors (Bhoopalan et al. (2013) JCI Insight 8(1):e161810). With the goal of performing Investigational New Drug ( IND) -enabling studies toward a future DBAS gene therapy trial, the P2A. GFP segment was removed to create SJEFS-S19 (FIG. 1). GFP expression in S JEFS-S19GFP-transduced cells was used to optimize transduction of CD34+HSPCs using flow cytometry to measure percentage of GFP+cells 3 days after LV treatment. Transduction was significantly increased by the use of transduction enhancers, prostaglandin E2 (PGE2) and poloxamer synperonic F108 (LentiBOOST® or "LB"), either alone or in combination (PC0.001) (FIG. 2) (Jang et al. (2020) Gene Therapy. 27(12):545-56). All subsequent transduction experiments were performed with the PGE2 and LB combination. To determine the optimal MOI for further studies, healthy CD34 + HSPCs were transduced with SJEFS-S19GFP at different MOIs ranging from 5 to 100, and percentage GFP+cells were measured by flow cytometry and VCN per diploid genome by quantitative PCR (qPCR). A linear increase in transduction efficiency and a corresponding increase in VCN was noticed with increasing MOI. SJEFS-S19 LV showed a similar linear increase in VCN ranging from 2.5 to 6.3 / diploid genome (dg) with increasing MOI. Both LVs resulted in a VCN of 2-4 / dg at an MOI of 20 which is a relatively safer VCN and was used for subsequent experiments. The VCN did not plateau even at high MOIs of 50 and 100, which highlights the relative safety of higher doses of RPS19 LVs and the quality of the LV preparation. In support of this, SJEFS-S19 LV caused a negligible change in cell viability at increasing MOIs.EXAMPLE 3: RPS19-Encoding LVs Rescue Defects in RPS19+ / - HSPCs

[0075] To test the efficacy of RPS19-encoding LVs, a previously published approach was used to model RPS19 haploinsufficiency in healthy donor CD34 + HSPCs by using CRISPR / Cas9 (Bhoopalan et al. (2013) JCI Insight 8(1):e161810). CD34 + HSPCs from healthy donors were electroporated with ribonucleoprotein (RNP) complex composed of 3x-NLS-SpCas9 (Protein Production Core, St. Jude) and sgRNAs targeting either RPS19 or AAVS1 locus as a control. RNP-treated HSPCs were transduced with or without LV followed by differentiation in methylcellulose-based medium to generate erythroid colonies. To determine the optimal promoter for future clinical use, three SIN LVs encoding RPS19. P2A. GFP sequence driven by different clinically relevant promoters of varying strengths were generated: EFla long (EFlaL), EFla short (EFlaS) and MND (Table 2).TABLE 2

[0076] Compared to control HSPCs, RPS19 RNP-treated HSPCs generated ~75% fewer burst-forming unit— erythroid (BFU-E) colonies (P < 0.001) (FIG. 3). Treatment with any one of the three LVs led to a comparable rescue of this in vitro erythroid defect (P < 0.001) (FIG. 3). EFlaS promoter was chosen for further evaluation due to its relatively smaller size, potentially favorable safety profile due to lack of enhancer activity, and extensive clinical experience for HSC gene therapy with this promoter. To further test the efficacy of SJEFS-S19 LV, iPSCs generated from a DBAS patient with RPS19 c. l91T> C (p. Leu64Pro) were used (Osuna et al. (2024 ) Stem Cell Res. 79: 103479). Isogenic control lines were developed by Cas9-mediated homology-directed repair using a single-stranded oligodeoxynucleotide donor template (Osuna et al. (2024) Stem Cell Res. 79: 103479). Using a monolayer differentiation technique, iPSCs were differentiated to generate hematopoietic progenitor cells (HPCs), which were then transferred to erythroid differentiation medium following transduction with SJEFS-S19 LV (Ruiz et al. (2019) Stem Cell Res. 41: 101600). DBAS iPSC-derived HPCs showed minimal expansion in erythroid culture, compared to isogenic control cells (FIG. 4 ). In contrast, SJEFS-S19 LV-transduced DBAS HPCs rescued this defect resulting in nearly 4-fold expansion at day 14 of erythroid differentiation (P<0.01) (FIG. 4). Flow cytometric analysis at day 14 showed that SJEFS-S19 LV treatment led to significantly increased expression of the erythroid maturation markers, CD71 and CD235a, in DBAS HPCs. Impaired processing of 21S to 18SE pre-rRNA species is characteristic of RPS19 deficiency, as observed in RPS19 RNP-treated HSPCs by northern blot analysis (Flygare et al. (2007) Blood 109 (3): 980-6). Transduction with SJEFS-S19 LV rescued this impairment. Together, these data show that SJEFS-S19 LV is efficacious in rescuing hematopoietic and pre-rRNA processing defects in various in vitro models of DBAS.EXAMPLE 4: SJEFS-S19 LV Rescues Bone Marrow Repopulation by RPS19+ / - HSPCs

[0077] It has been shown that RPS19+ / -HSPCs have a competitive bone marrow repopulation defect following xenotransplant that was partly TP53-dependent (Bhoopalan et al. (2013) JCI Insight 8(1):e161810). To test if this HSC function defect could be rescued by SJEFS-S19 LV, RNP-treated HSPCs were treated with SJEFS-S19 LV at an MOI of 20 and the cells were transplanted into NSGW mice, which were euthanized 16-18 weeks later and analyzed by flow cytometry and NGS. Human cell chimerism wasdetermined by percentage of human CD45+cells in recipient mice, which was similar between control cells and RPS19 RNP-treated HSPCs + SJEFS-S19 LV. Indel mutations created by RNP treatment were determined by NGS and used to track the proportion of gene-edited alleles. In control cells treated with AAVS1 RNP, the input cells had an indel frequency of 77. 4±8.9%, which dropped to 69.4±11% post-transplant. In comparison, RPS19 RNP-treated HSPCs had an input indel frequency of 77.2% ± 6.3% which dropped to <0.5%, below the limit of detection by NGS. Treatment with SJEFS-S19 LV improved engraftment of RPS19-edited HSPCs, as the indel frequency was 23.01% ± 12.5% post-transplant (P < 0.0001). Similarly, indel analysis in human donor HSPC-derived B-lymphocytes, myeloid cells, HSPCs and erythroid cells isolated from recipient mouse bone marrow showed a similar dropout of RPS19 indels, which were rescued by transduction with SJEFS- S19 LV while the proportion of human cells in each lineage was similar across the different groups. The VCN in input HSPCs was approximately 3 / dg which decreased to about 1. 6 / dg in engrafted cells at 16 weeks post-transplant. To further look at the distribution of VCN at a single-cell resolution, individual BFU-E colonies generated by CD34+ cells pretransplant or from CD34+HSPCs isolated from recipient mouse bone marrow at 16 weeks post-transplant were used. The majority of the erythroid colonies had a VCN <5 / dg in both input and post-transplant HSPCs, with no evidence of preferential of expansion of cells with higher VCNs following xenotransplant. These data indicate that modest amounts of LV-derived RPS19 are adequate to rescue competitive bone marrow repopulation defect seen in RPS19 haploinsuf f icient HSPCs.EXAMPLE 5: Safety of HSPC Transduction with RPS19 LV

[0078] As observed by clonal studies, LV transduction, even at modest doses, can lead to some cells with high VCNs which are not eliminated post-transplant. Therefore, to assess the safety of HSPCs with high RPS19 VCNs, cells were transduced with MOIs up to 100. Western blot analysis of RNP-treated HSPCs transduced with SJEFS-S19GFP LV showed that endogenous RPS19 can be detected along with LV-derived RPS19-P2A and GFP proteins. Quantification of relative intensities of RPS19-P2A and GFP, normalized to actin, showed that GFP protein levels increased with increases in MOI but RPS19-P2A levels did not. Similarly, RPS19-P2A levels were stable with increasing MOI in unedited HSPCs. This is consistent with observations that ribosomal protein levels are tightly regulated by the proteasomal system (Sung et al. (2016) Mol. Biol. Cell.27 (17): 2642-52; Lam et al. (2007) Curr. Biol. 17 (9): 749-60). To determine if high doses of SJEFS-S19 LV impact in vitro differentiation potential of healthy, unedited HSPCs, transduced cells were differentiated in methylcellulose-based medium. HSPCs, treated with or without different doses of SJEFS-S19 LV, generated similar proportions of hematopoietic colonies. Assessment of pooled erythroid and myeloid colonies showed similar VCNs even at higher MOIs (e.g., up to 100 MOI). Together, this indicates that high doses of SJEFS-S19 LV do not affect the hematopoietic lineage differentiation potential of CD34+ HSPCs, highlighting the relative safety of RPS19 LV.EXAMPLE 6: No Prominent Aberrant Splicing in SJEFS-S19 LV-Transduced HSPCs

[0079] Gene trapping due to cryptic splice acceptors (SAs) within LVs has been reported in multiple clinical trials where HMGA2 transcript truncation led to oligoclonal expansion ofhematopoietic cells (Cavazzana-Calvo et al. (2010) Nature 467 (7313): 318-22; De Ravin et al. (2022) Nat. Commun.13(1):3710; Cesana et al. (2012) J. Clin. Invest. 122 (5): 1667-76). The splicing in those cases was mediated by a dominant splice acceptor within the cHS insulator element, which is not present in SJEFS-S19 LV backbone (De Ravin et al. (2022) Nat. Commun. 13(1):3710). In silico analysis was performed using previously published tools to identify five potential splice acceptors within the codon-optimized RPS19 sequence (Table 1). These splice acceptors were modified to generate SJEFS-S19v2 LV and an insulator element was inserted therein to generate S JEFS-S19v2-i4r LV as a positive control (Brunak et al. (1991) J. Mol. Biol. 220 (1): 49-65; Reese et al. (1997) J. Comput. Biol. 4(3):311-23). RNASeq data from CD34+ HSPCs transduced with different LVs at an MOI of 100 was analyzed to detect fusion transcripts between host genes and LV sequence as described in the methods section. As expected, a high level of splicing was observed from multiple different genes into the previously identified splice acceptor within cHS insulator (De Ravin et al. (2022) Nat. Commun. 13(1):3710) while minimal splicing was observed in other regions of the proviral sequence, including the five predicted splice acceptors within RPS19 transgene. Further analysis of chimeric reads did not show any dominant splice donor genes suggestive of a polyclonal LV integration profile. Interestingly, HMGA2 was not detected in HSPCs transduced with any of the different LVs. The two versions of RPS19 LV retained similar levels of RPS19 codon optimization as measured by codon adaptability index (Sharp & Li (1987 ) Nucleic Acids Res. 15 (3): 1281-95). These data indicate that SJEFS-S19 LV does not contain any strong cryptic splice acceptor sites and does not cause any prominent aberrant splicing in transduced HSPCs.EXAMPLE 7: Integration Site Analysis of SJEFS-S19 LV in Human CD34+HSPCs

[0080] LVs integrate in a semi -random fashion into transcriptionally active genomic regions with an open chromatin architecture (Yan et al. (2023) Sci. Adv.9 (40): eadg9959). The genome-wide vector integration site (VIS) profile of SJEFS-S19 LV in CD34+ HSPCs was assessed using quantitative shearing linear amplification (qsLAM) PCR as previously described (Zhou et al. (2015) Hum. Gene Ther. Methods 26 (1): 4-12 ). To analyze the VIS profile, RPS19 RNP-treated HSPCs from three different donors were transduced with SJEFS-S19 LV and transplanted into NSGW mice (4-5 mice per donor). DNA was isolated from a fraction of the HSPCs prior to transplant and from mice bone marrow post-transplant, which was used to assess VIS profile. 5920-10908 unique VISs per donor pre-transplant and 118-496 unique VISs per recipient mouse post-transplant were identified. The top 20 most frequent VISs in the pre-transplant HSPCs constituted <5% of total integration sites highlighting the polyclonal diversity of SJEFS-S19 LV-transduced HSPCs. There was minimal overlap in VIS between input HSPCs and donor-derived cells posttransplant, and between individual recipient mice. This could be due to sampling bias as each mouse received only 3-4xl05HSPCs but also underscores the diversity of VIS repertoire and the polyclonality of transduced HSPCs. SJEFS-S19 LV primarily integrated into introns and had a very similar VIS profile to CL20-i4-EFla-hgc-OPT LV used to treat X-linked SCID, consistent with other third-generation, SIN LVs in clinical use (Mamcarz et al. (2019) N. Engl. J. Med. 380 ( 16): 1525-34; Yan et al. (2023) Sci. Adv. 9 (40): eadg9959; Williams & Thrasher (2014) Stem Cells Transl. Med. 3 (5): 636-42 ).Comparing the VIS signature across different donors, genes were identified that are frequently integrated into by SJEFS-S19 LV. The genes, referred to as recurrent integration genes (RIGs), were highly consistent across all three CD34 donors. Additionally, 3% of SJEFS-S19 LV VIS were found to be close to 119 cancer-related genes with most of the insertions near tumor suppressor genes and less frequently near oncogenes. Further, most of these insertions were in the introns or 10-100 kb upstream of transcription start site (TSS). Very rare integration events were detected in LMO2 and MECOM, but the insertions were intronic, consistent with other SIN LVs (Williams & Thrasher (2014) Stem Cells Transl. Med. 3 (5): 636-42). These data show that SJEFS-S19 LV has an integrome signature consistent with other SIN LVs which have demonstrated relative safety in multiple independent clinical trials.

Claims

WHAT IS CLAIMED IS:

1. A self-inactivating lentiviral vector for treating Diamond-Blackfan anemia syndrome comprising nucleic acids encoding ribosomal protein S19 (RPS19) operably linked to an EFla promoter or a MND promoter, wherein the nucleic acids encoding RPS19 are codon optimized for expression in human cells and are devoid of aberrant splice acceptor sites.

2. The self-inactivating lentiviral vector of claim 1, further comprising a 5' long terminal repeat (LTR), packaging signal (psi), rev response element (RRE), and 3 ' LTR comprising at least one deletion compared to a wild-type 3 ' LTR.

3. The self-inactivating lentiviral vector of claim 1, wherein the EFla promoter comprises the nucleotide sequence of SEQ ID NO: 11 or SEQ ID NO: 12.

4. The self-inactivating lentiviral vector of claim 1, wherein the MND promoter comprises the nucleotide sequence of SEQ ID NO: 13.

5. The self-inactivating lentiviral vector of claim 1, wherein the nucleic acids encoding RPS19 comprise the nucleotide sequence of any one of SEQ ID NOs: 8-10.

6. A host cell transduced with the self-inactivating lentiviral vector of claim 1.

7. The host cell of claim 6, wherein the host cell is a CD34+hematopoietic stem cell.

8. A method of treating or ameliorating Diamond-Blackfan anemia syndrome comprising administering to a subject in need thereof an effective amount of the self-inactivating lentiviral vector of claim 1, thereby treating or ameliorating the subject' s Diamond-Blackfan anemia syndrome.

9. The method of claim 8, wherein the self-inactivating lentiviral vector is administered by introducing the selfinactivating lentiviral vector into a donor host cell and administering the transduced donor host cell to the subject.

10. The method of claim 9, wherein the self-inactivating lentiviral vector is introduced into the donor host cell at a multiplicity of infection of between 20 and 100.

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