Splice-correcting oligonucleotides restore BTK function in X-linked agammaglobulinemia model

X-linked agammaglobulinemia (XLA) is an inherited immunodeficiency that results from mutations within the gene encoding Bruton’s tyrosine kinase (BTK). Many XLA-associated mutations affect splicing of BTK pre-mRNA and severely impair B cell development. Here, we assessed the potential of antisense, splice-correcting oligonucleotides (SCOs) targeting mutated BTK transcripts for treating XLA. Both the SCO structural design and chemical properties were optimized using 2′-O-methyl, locked nucleic acid, or phosphorodiamidate morpholino backbones. In order to have access to an animal model of XLA, we engineered a transgenic mouse that harbors a BAC with an authentic, mutated, splice-defective human BTK gene. BTK transgenic mice were bred onto a Btk knockout background to avoid interference of the orthologous mouse protein. Using this model, we determined that BTK-specific SCOs are able to correct aberrantly spliced BTK in B lymphocytes, including pro–B cells. Correction of BTK mRNA restored expression of functional protein, as shown both by enhanced lymphocyte survival and reestablished BTK activation upon B cell receptor stimulation. Furthermore, SCO treatment corrected splicing and restored BTK expression in primary cells from patients with XLA. Together, our data demonstrate that SCOs can restore BTK function and that BTK-targeting SCOs have potential as personalized medicine in patients with XLA.

X-linked agammaglobulinemia (XLA) is the most common form of inherited agammaglobulinemia, accounting for 85% of all cases (1, 2). In this disease, a B cell lineage developmental block, manifested at the transition between the pro–B and pre–B cell stages (3), leads to the essential lack of mature B and plasma cells, with a concomitant pronounced reduction of immunoglobulin levels. XLA is caused by defects in the gene encoding Bruton’s tyrosine kinase (BTK) (4–6). BTK is a cytoplasmic, nonreceptor tyrosine kinase belonging to the TEC family of tyrosine kinases (7) and is expressed in all stages of B cell development, with the exception of long-lived, antibody-producing plasma cells (8, 9). Besides B lymphocytes, many other hematopoietic cells also express BTK (5, 8–10), but clinically relevant functional impairment seems to be confined to this lineage. Thus, the severe clinical outcome of BTK mutations demonstrates the essential role of BTK already occurring in pre–B cell receptor signaling, which is a determinant for proliferation, differentiation, and survival of the early B cell stages (3, 11, 12).

Patients with XLA are prone to recurrent infections by pyogenic bacteria, such as pneumococci and streptococci. Affected subjects are also unduly susceptible to enteroviruses, which cause dermatomyositis and fatal chronic encephalomyelitis (13–16). The current treatment for XLA consists of prophylactic, regular intravenous, or subcutaneous gammaglobulin replacement therapy and generous administration of antibiotics. This considerably improves quality of life, but patients still suffer from chronic infections (17, 18). Furthermore, life expectancy is reduced despite appropriate care (16, 19–21).

Splicing defects have been identified as an important cause of genetic disease. Many mutations affecting splicing disrupt the regular 5′ splice site (5′ss) or 3′ss at exon-intron junctions. Further, mutations can create new splice sites, which may result in the inclusion of intronic sequences or loss of part of the exons in the transcript. When such a new splice site occurs in an intron in the vicinity of a suitable pseudo splice site, the intervening intronic region can be included in the mRNA as a cryptic exon. This mechanism has been observed in multiple diseases, including cystic fibrosis (22) and ataxia telangiectasia (23, 24) among others. In XLA, we have previously identified and described 2 such families (25, 26).

The XLA defect studied in this work arises from one of these families, which has an A-to-T transition in intron 4 of the BTK gene generating a novel 5′ss. This, together with a preexisting cryptic 3′ss upstream in the same intron, results in the inclusion of a cryptic exon (exon 4a) of 109 nucleotides between exons 4 and 5 in the mRNA (25). This changes the reading frame and completely abolishes BTK protein expression. The erroneous inclusion of exon 4a prompted us to investigate the possibility of using splice-correcting antisense oligonucleotides (SCOs), which bind to and restore the splicing of the pre-mRNA, a concept also exploited in other diseases, as reviewed recently (27, 28).

Apart from the splice sites themselves, splicing is also controlled by short regulatory sequence motifs in both exons and introns. These motifs are designated exonic or intronic splicing enhancers (ESEs or ISEs, respectively) or exonic or intronic splicing silencers (29). These are also of interest since SCOs targeting exonic splicing silencers have been shown to induce the inclusion of exon 7 in the transcript of the SMN2 gene in spinal muscular atrophy (30). Similarly, exonic splicing silencer regions have been targeted in the case of Duchenne muscular dystrophy (DMD), which is caused by mutations in the DMD gene. In this case, restoration of a disrupted reading frame by exon-skipping SCOs has been used successfully to generate a truncated but partially functional protein (31, 32).

Here, we describe the feasibility of splice correction by preventing cryptic exon inclusion as a personalized therapy for XLA. In order to achieve this, we designed SCOs targeting various sites in the pseudoexon region of BTK pre-mRNA. Different oligonucleotide (ON) chemistries have been developed over recent years in order to improve resistance to degradation, enhance target affinity, and promote cellular uptake. In this study, we investigated SCOs with 2′-O-methyl phosphorothioate (2′OMePS) or phosphorodiamidate morpholino (PMO) backbone chemistries. For simplicity, the splice-correcting PMO-based oligomers are also referred to as SCOs.

The PMO was also tested in a cell-penetrating peptide–conjugated (CPP-conjugated) version. CPPs are a class of peptides with the ability to translocate rapidly into cells and to transport various macromolecular cargos. PMO-based SCOs conjugated with CPPs have been shown to be very efficient as therapeutics against splice abnormalities, especially in the mdx mouse model of DMD (33). Furthermore, we investigated nucleic acid modifications, such as locked nucleic acid (LNA), since they have been used successfully in many different settings, such as antisense gapmers, siRNAs, anti-microRNAs (antagomirs), and anti-gene approaches (34, 35). An LNA-containing ON with its 3′-endo conformation is considered to be an RNA mimic, making it effective toward structured RNA regions, which can be beneficial when targeting pre-mRNAs. This is due to the fact that position-dependent substitution with LNA bases changes the thermodynamic property of the duplexes (34, 36). Additionally, we also investigated LNA in combination with other derivatives, such as unlocked nucleic acid (UNA) or amino-glycyl-LNA (34). Moreover, since the nonbase modifier N,N-diethyl-4-(4-nitronaphthalen-1-ylazo)-phenylamine (ZEN) has been shown recently to have potentiating effect for antagomirs, it was also tested (37).

We targeted primary patient cells as well as lymphocytes from a genetically altered mouse created for this purpose. This transgenic mouse has several unique features; it carries the entire human mutant BTK locus and is devoid of endogenously expressed BTK protein. Notably, in contrast to the treatment concept for DMD, in which a truncated protein with reduced function is formed, the strategy in XLA permits the restoration of the authentic reading frame generating a fully functional protein. Importantly, because of the developmentally regulated expression of BTK, it may become feasible to achieve long-term correction of the disease phenotype. Moreover, besides proving the viability of splice-modifying therapy for a novel target disease both ex vivo and in vivo, we demonstrate for the first time that both primary B cells and primary monocytes are amenable to splice correction strategies.

Design of SCOs using bioinformatics tools. Different SCOs directed against the BTK pseudoexon region were designed, taking into consideration not only the 3′ss and 5′ss of exon 4a, but also putative ESE sites. For this purpose, we used 3 different algorithms from the web-based servers ESE Finder, PESX, and RESCUE-ESE. The sequence hits were aligned to exon 4a, as depicted in Figure 1, which also shows the studied mutation and the expected outcomes with or without splice correction. This analysis prompted us to generate a set of SCOs, as schematically indicated in Figure 1. They were designed to cover the 3′ss and 5′ss and the ESEs sites that demonstrated the most concordance between the algorithms used. The first set of SCOs was composed of the commonly used 2′OMePS modification, taking into consideration the GC content, self-dimerization, and nucleic acid melting temperature (Tm) values (38). Additional sets were then designed with various modifications and lengths. A schematic representation of the SCOs is shown in more detail in Supplemental Figure 1 (supplemental material available online with this article; doi:10.1172/JCI76175DS1), and the modifications are indicated in Table 1.

Figure 1

Design of SCOs using bioinformatic tools to search for ESE sites in the disease-causing pseudoexon. (A) Three different algorithms from the corresponding web-based servers, ESE-Finder, RESCUE-ESE, and PESX, were used, and the hits were aligned to the pseudoexon sequence to find locations with the highest correlation between algorithms. SCOs targeting the probable ESE regions were subsequently designed. SCOs are schematically presented with their binding-positions along the pseudoexon. (B) Predicted outcome of splicing, with or without SCOs, indicated schematically.

Table 1

SCOs used in the study

SCOs restore luciferase mRNA in a reporter cell line model. We first designed a reporter construct containing the expression cassette for a fusion of EGFP and firefly luciferase but interrupted by the mutated patient-derived BTK intron 4. When uncorrected, this minigene produces a corrupt EGFP-luciferase mRNA. However, introduction of an SCO capable of rescuing the splicing of the mutated intron would promote restoration of EGFP-luciferase fusion protein expression. The model was constructed as a first functional readout for SCO activity and used to generate a stable U2OS reporter cell line expressing the above-mentioned corrupt transcript. Subsequently, the fully 2′OMePS-modified SCOs (sequences depicted in Table 1) were transfected, and luciferase enzymatic activity was determined (Figure 2A). Several of the tested SCOs showed promising results when compared with the unrelated, control SCO 705 directed against a mutated β-globin intron (39). SCO 187, covering the 5′ss, performed the best in these experiments, whereas SCO 165, covering the 3′ss, demonstrated very low efficacy. SCOs 186, 187, and 190 were chosen for further experiments. SCOs 186 and 190 were selected because of their activity and the fact that they were not targeted to splice sites but instead to the predicted ESEs.

Figure 2

Splice correction–induced upregulation of reporter minigene activity. The U2OS cell line stably carrying a minigene interrupted by the mutated BTK intron 4 was transfected with different SCOs, and efficacy was measured as luciferase activity or mRNA restoration. (A) The relative luciferase activity in comparison to nontreated cells (mock) 24 hours after SCO transfection. “705” indicates an unrelated control SCO, targeting position 705 of a mutated β-globin intron, not complementary to the BTK pre-mRNA. All tested SCOs are 2′OMePS based (Table 1). Data represent mean + SD of 3 independent experiments, each with 2 replicates. **P ≤ 0. 01, ***P ≤ 0.001, versus 705. (B) Increase in luciferase activity by 2′OMePS-modified SCOs versus LNA SCOs is presented as fold increase over nontreated cells (mock). Graph represents mean + SD of 3 independent experiments, each with 2 replicates. **P ≤ 0. 01, ****P ≤ 0.0001, 186.18 versus 186.15-5LNA. (C) Total RNA RT-PCR showing the 389-bp and 271-bp bands corresponding to aberrant and corrected (with BTK intron 4 excised) mRNA bands, respectively, with a lower band of ribosomal RNA (18S) serving as an RNA quality control. The lane on the right represents RNA input from nontreated (NT) cells. “Scr” represents the control LNA/2′OMePS SCO not complementary to the reporter gene sequence (Table 1). A representative gel from 2 independent experiments is shown. Statistical significance was determined using 1-way ANOVA, followed by Bonferroni’s multiple-comparison test.

SCO chemistry affects splice correction activity in reporter cell line. We modified the selected SCOs with LNA bases, which are known to significantly enhance Tm and increase the efficiency of ONs both in vitro and in vivo (40–44). More specifically, we chose a 2′OMe RNA backbone with LNA modifications, as this combination has previously demonstrated high potency (45–48). Since size-reduced SCOs are generally considered to be more efficient (34, 42, 49), with lowered risk of off-target effects and improved uptake, we designed LNA-modified 15-mer versions of the 186, 187, and 190 series (Supplemental Figure 1 and Table 1). Figure 2 shows the activity of LNA-modified SCOs in the U2OS reporter cell line, as measured by luciferase expression and as also confirmed by the correction of mRNA determined by RT-PCR. It is clearly shown that the LNA-modified SCOs mediate enhanced luciferase activity at low concentrations. The same tendency was confirmed at the RNA level, at which we observed a reduction of the aberrant mRNA band and a concomitant appearance of the corrected form at low SCO concentrations. The 190 series of SCOs was not further assessed, since at higher concentrations there were signs of toxicity in the form of cell death 24 hours after transfection (data not shown).

Based on these results we further modified the 186 and 187 series, including a reduction of the ON length further to 10 to 12 nucleotides. We also synthesized 3 additional SCOs, referred to as the GAA series. These were designed to target GAA-triplets, known to be present in certain splicing-enhancers (26, 50), as shown in Supplemental Figure 1 and Table 1. Supplemental Figure 2 depicts the effect of SCOs derived from the 186, 187, and GAA series, as analyzed by RT-PCR. The doses were lowered, as compared with previous experiments (Figure 2), in order to identify the most potent SCOs. As can be seen in Supplemental Figure 2, LNA-modified SCOs displayed higher potency as compared with their corresponding 2′OMePS versions. 186.15-5LNA and 186.15-3LNA (with 5 and 3 LNA substitutions, respectively) showed the best efficiency in the reporter cell assay. The 12-mer versions of both the 186 and 187 series did not exhibit any enhanced activity under these in vitro conditions, and the 10-mer of SCO 187 was inactive (Supplemental Figures 2 and 3). Moreover, the newly designed GAA series did not show better efficiency compared with 186.15-5LNA or 186.15-3LNA and was therefore not studied further.

In addition, we investigated 3 more chemical modifications (Table 1): amino-glycyl-LNA, UNA, and the nonbase modifier ZEN. The amino-glycyl modification adds a glycyl residue to the N2′ position of 2′-amino-LNA, thereby providing a positive charge (51). UNA is known to decrease the Tm and destabilize the duplex, which potentially could increase target specificity if appropriately designed (52). ZEN is a naphthyl-azo modifier; when placed at or near both ends of a 2′OMe ON, it increases Tm and enhances the stability against endo- and exonucleases (37). These new modifications were also tested in the reporter cell line model. As can be seen in Supplemental Figure 3, the glycyl-modified 12-mer versions of 186 and 187 series showed reduced activity with increasing numbers of amino-glycyls correlating to further reduction in activity. This was somewhat unexpected, since the reduced net negative charge potentially could have enhanced their splice-correcting capacity by allowing better hybridization to the cognate pre-mRNA. On the other hand, decreased net negative charge might also have reduced the complexation with the transfection reagent Lipofectamine 2000, which potentially could have altered both the complex conformation and the amount transfected into cells. Conversely, UNA incorporation into the 186 series of SCOs caused reduced activity at low LNA content, whereas with higher LNA content the activity was somewhat restored. Moreover, ZEN-modified SCO (186.15-5LNA-Zen), as compared with 186.15-5LNA (Supplemental Figure 2), did not show much difference in activity. The slightly longer 186.18-ZEN had almost the same effect as 186.15-5LNA and 186.15-3LNA. This might suggest that ZEN modification is more useful when used in the context of a pure 2′OMe backbone and not when combined with shorter LNA ONs. Taken together, these additional modifications did not show enhanced activity and were not studied further.

LNA-based SCOs and PMOs conjugated to a CPP are efficiently taken up (gymnosis) in the reporter cell line. We next investigated the naked uptake efficiency in the reporter cell line under “gymnosis” conditions. The term gymnosis was coined by Stein et al. to describe uptake of LNA gapmers by cells in the absence of any transfection reagent (53). The authors suggested that this mode of in vitro delivery better correlated to in vivo uptake, making this method of general interest. Compared with lipofection, considerably higher concentrations of ONs are needed for gymnosis. We selected the most efficient SCOs based on the results from the lipofection experiments and assessed their gymnotic activity. Although 186.15-5LNA exhibited good activity, we continued with the similarly efficient 186.15-3LNA, owing to the fact that reduced LNA content potentially could reduce off-target effects (45). Although ZEN-modified SCOs had similar activity compared with the LNA-containing 186.15 versions, they could not be studied, since their availability was limited.

In addition, we also investigated the PMO backbone (54, 55), with the 186 sequence in the gymnosis setting. Owing to the rules of PMO design, this SCO was further extended to be 25 nucleotides (Supplemental Figure 1 and Table 1). The rationale for lengthening PMOs is the lower target affinity (56). We further conjugated the PMO with a CPP moiety named B-peptide (B-PMO), since it is known to be very active for exon skipping in the mdx mouse model (57). Thus, the PMOs tested were either unmodified or covalently conjugated to B-peptide.

As can be seen from Figure 3, in gymnosis setting, 186.15-3LNA and B-peptide–conjugated PMO 186.25 (B-PMO) showed high potency in the reporter cells when analyzed by RT-PCR. We also tested higher (up to 10 μM) concentrations of PMO 186.25, which were devoid of any CPP; however, the exon-skipping effect was very low.

Figure 3

Splice correction–induced upregulation of reporter minigene activity following naked uptake of SCOs. SCOs from the 186 series, including PMO 186.25, with and without the CPP moiety (B-PMO 186.25), were tested, and activity was measured as restoration of mRNA. A concentration of 6 μM was used for the scrambled control SCO (Scr) (Table 1). A representative gel from 2 independent experiments is shown.

Generation of a transgenic mouse carrying the mutated human BTK locus. In order to have access to an unlimited supply of mutated B-lineage cells as well as a relevant in vivo model, we generated a mouse carrying the corresponding human BTK locus. A suitable BAC clone named RP11-125I5 was chosen for the development of BAC transgenic mice. This clone encompasses 175 kb and contains an extended human genomic BTK locus. The BAC was subsequently subjected to site-specific mutagenesis, generating a single-point mutation at position 602 of BTK intron 4, the very same A-to-T change as seen in the XLA-family described previously. We confirmed that the BAC transgenic mice carry the mutated human BTK gene by sequencing of chromosomal DNA (data not shown). The founder animals were then backcrossed onto Btk knockout (KO) mice in order to generate transgenic animals with a disrupted, nonfunctional endogenous Btk locus, which were therefore only capable of expressing the mutated human BTK gene (Supplemental Figure 4). Throughout this report we refer to these mice as BAC transgenic mice. We also verified that the insertion of the transgene did not influence hematopoietic development; Supplemental Figure 5 shows that the common lymphoid progenitors and B cell compartment are similar in Btk KO and BAC transgenic mice.

SCOs correct mRNA and restore BTK protein in primary B cells ex vivo. To test the activity of the SCOs in the context of a full-length pre-mRNA transcribed from the mutant human BTK gene, we isolated splenic B cells from mice expressing the human mutant BTK transcript. As control, we used B cells from WT and Btk KO mice. For transfections of SCOs, we optimized a protocol for electroporation of mouse primary B cells. At 48 hours after electroporation, cells were collected, and human BTK RNA was analyzed by semiquantitative RT-PCR, using primers detecting only human BTK. Another primer set was used to specifically amplify mouse Btk RNA in order to verify that this transcript is unaffected by the SCOs (Figure 4A). The results clearly show that, by using SCO 186.15-3LNA, splicing of the full-length human mutant BTK pre-mRNA was corrected, as demonstrated by the appearance of a lower molecular weight amplicon corresponding to the size of unmutated, native BTK mRNA. The correction was also verified by sequencing the cDNA (data not shown).

Figure 4

Full-length human mutated BTK pre-mRNA corrected by SCOs yielding normal-sized transcripts and detectable BTK protein. (A) B cells from human BAC transgenic mice were treated with 1.6 μM or 3.2 μM 186.15-3LNA SCO and analyzed by RT-PCR. To identify human RNA in cells from BAC transgenic and KO mice, a primer pair only detecting human BTK was used. To measure endogenous mouse Btk mRNA levels in splenocytes derived from WT mice, a different primer set selective for mouse Btk was used. A representative gel from 3 independent experiments is shown. Ribosomal RNA (18S) was used as an RNA quality control. (B) FACS analysis of cells 48 hours after treatment with 186.15-3LNA. B cells were stained with antibodies directed against CD19 or BTK protein (recognizing both mouse and human protein). Histograms for BTK expression in each sample from a representative experiment as well as the percentage of corrected cells from 3 independent experiments are shown (mean + SD). A concentration of 3.2 μM was used for the scrambled control SCO (Table 1). Mean fluorescence intensity, WT NT: 2,314; BAC SCO treated: 1,210. Percentage of WT BTK RNA was calculated as WT RNA fraction × 100/(misspliced + WT RNA fractions).

The RT-PCR results were also confirmed by flow cytometry analysis on cells collected 48 hours after transfection (Figure 4B). As can be seen from Figure 4B, treatment with 186.15-3LNA generated a mean of over 40% of cells expressing substantial levels of BTK protein. These experiments demonstrate that not only the reporter minigene-derived transcript carrying the mutated BTK intron, but also the full-length human BTK pre-mRNA is amenable to SCO therapy.

SCOs restore functional BTK in primary B cells from the BAC transgenic mouse. Since we could conclusively show the presence of BTK protein by flow cytometry analysis, we investigated whether the synthesized protein is also functionally active. For this purpose, 2 functional assays were carried out, cell viability and phosphorylation of the BTK regulatory loop tyrosine (Y551).

The rationale for the cell viability test is that impaired maturation and survival of B cells are hallmarks of XLA (1, 2, 13). Moreover, it is known that BTK-deficient mouse B cells do not proliferate in response to anti-IgM stimulation, while WT cells do (58, 59). Therefore, we stimulated BAC transgenic mouse B cells 48 hours after SCO treatment with anti-IgM and incubated them for another 24 or 48 hours in the absence of the survival-promoting CpG stimulus (see Methods), conditions rather harsh for primary B cells. Figure 5A shows the viability of primary B cells after 24 or 48 hours of incubation with anti-IgM. As seen, under these experimental conditions SCO-treated BAC transgenic B cells showed enhanced survival compared with nontreated or scrambled, SCO-treated cells. As a sign of the harsh environment, WT mouse B cells were also reduced in numbers. As a second proof of BTK functionality, we analyzed the BTK phosphorylation after B cell receptor stimulation. In normal B cells, upon anti-IgM treatment, B cell receptor–induced signaling leads to tyrosine Y551 phosphorylation of BTK. The corresponding tyrosine is conserved in all cytoplasmic protein-tyrosine kinases and upon phosphorylation significantly enhances their catalytic activity owing to a conformational change (3, 13). SCO-treated B cells from BAC transgenic mice were compared with B cells from WT mice. 48 hours after electroporation, cells were serum starved for 6 hours and subsequently incubated with 20 μg/ml anti-IgM before harvest. BAC transgenic B cells treated with 186.15-3LNA SCO showed pronounced phosphorylation when activated with anti-IgM (Figure 5B). The level was almost as high as in WT cells, suggesting functional restoration of BTK.

Figure 5

Enhanced survival and anti-IgM–induced BTK tyrosine phosphorylation in SCO-treated BAC transgenic B cells. (A) Spleen B cells from BAC transgenic mice were treated with 3.2 μM SCO (186.15-3LNA or scrambled control) and stimulated with 20 μg/ml anti-IgM 48 hours after electroporation (time 0). Cells were counted, and viability was determined by trypan blue exclusion 24 and 48 hours after stimulation. The initial number of cells from both WT and BAC transgenic mice was 2 × 10⁶. Data represent 2 independent experiments with duplicates. Note that nontreated and scrambled ON–treated cell numbers overlap. Statistical significance was analyzed by using 1-way ANOVA followed by Bonferroni’s multiple-comparison test. ****P ≤ 0.0001 for BAC SCO treated versus BAC Scr and NT at 24 hours. (B) Analysis of BTK tyrosine phosphorylation after 3.2 μM SCO (186.15-3LNA) treatment and anti-IgM stimulation (20 μg/ml). Blots show pY551 phosphorylation of BTK and total BTK. Whole cell lysate (WCL) analysis shows BTK protein and actin. Each duplicate (–/+) corresponds to starved or anti-IgM–activated conditions. A representative blot is shown from 2 independent experiments. Note that the higher molecular weight band, located immediately above the pY551-BTK band in activated samples (IP pY551 gel), is of unknown origin and unrelated to BTK, since it also exists in B cells obtained from Btk KO mice following stimulation. The bar graph shows the quantitative analysis of Y551 phosphorylation as a percentage of relative intensity signal from the blots according to ImageJ Software.

B-PMOs efficiently promote splice correction in primary B cells. We next evaluated the effects of naked delivery into primary cells. The non-PMO SCOs, initially evaluated in the reporter gene assay (Supplemental Figure 2), were again selected based on potency and assessed in primary B cells. In contrast to the gymnotic effect seen in the reporter cell line, we were unable to detect any significant reproducible correction of the BTK mRNA in primary B cells under these conditions (data not shown). We subsequently evaluated the B-PMO 186.25 as well as the corresponding control, PMO 186.25. Notably, treatment with 3 μM B-PMO 186.25 produced corrected BTK mRNA (Figure 6A) as well as restored BTK protein expression (Figure 6B) in B cells from the BAC transgenic mice.

Figure 6

B-PMO–mediated restoration of BTK mRNA and protein in primary spleen B cells from human BAC transgenic mice. Cells were treated with PMO 186.25 or B-PMO 186.25 in different concentrations. “Scr” indicates a B-PMO SCO for an unrelated target (DMD). (A) RT-PCR products from SCO-treated B cells at 1 and 3 μM concentrations. Ribosomal RNA (18S) serves as an RNA quality control. Of note, for the splenocytes derived from WT mice, a different primer set selective for mouse Btk was used. The percentage of WT BTK RNA was calculated as WT RNA fraction × 100/(misspliced + WT RNA fraction). (B) Western blot from the same treatment. Experiments were repeated 3 times, and representative gels are depicted. Of note, for this experiment, anti-BTK from BD (see Methods) was used, yielding different unspecific bands. The bottom band originates from BTK, while the top band is unspecific. The lanes separated by a black line were run on the same gel, but since some lanes were considered uninformative, they were omitted as indicated by the black line. The bar graph shows the quantitative analysis of BTK protein as a percentage of relative intensity signal according to ImageJ Software. Scr, treated with scrambled ON.

SCOs restore BTK protein expression in pro–B cells from the BAC transgenic mouse. Patients with XLA are devoid of mature B cells but harbor differentiation-arrested pro–B cells in their bone marrow (13, 60). Because of this fact, it was important to establish whether these cells are also amenable to SCO treatment as a proof of concept. For this reason, we first isolated and differentiated c-KIT⁺ progenitor cells from the bone marrow of BAC transgenic mice. With support of OP9 stromal cells, Flk-2/Flt3 ligand, KIT ligand, and IL-7, the expansion of pro–B cells was induced in vitro (see Methods). After 9 days, an essentially pure pro–B cell population was obtained (Supplemental Figure 6). We subsequently transfected these cells with the 186.15-3LNA SCOs and assessed BTK protein expression at 48 hours. Encouragingly, as many as 33.8% of the pro–B cells were BTK positive (Figure 7).

Figure 7

Restoration of BTK protein expression after splice correction in pro–B cells from BAC transgenic mice. Cells were electroporated with the 186.15-3LNA SCO. After 48 hours, cells were stained with anti-CD19 and anti-BTK (recognizing both mouse and human protein) for FACS analysis. Numbers in the 3 right upper quadrants indicate the percentage of CD19-positive and BTK-positive cells. The experiment was repeated twice with a similar outcome, and a representative result is presented.

In vivo administration of SCOs restores BTK expression in BAC transgenic mice. In order to investigate whether the SCOs also had the capacity to correct the defect in vivo, 4 animals were treated with a multiple-dose regimen using B-PMO 186.25. They received B-PMO 186.25 (30 mg/kg) 4 times: 3 doses intravenously every second day during 1 week, followed by a single subcutaneous dose; animals were assayed after another 3 or 4 days, respectively. As is depicted in Figure 8, A and B, and Supplemental Figure 7, a robust therapeutic effect was observed both in the bone marrow and in the spleen. In contrast to control mice, all treated mice expressed corrected BTK mRNA as well as BTK protein. Moreover, we isolated B cells from 2 treated and 2 untreated animals in order to be able to analyze BTK restoration specifically in this cell type. As shown in Figure 8C, we found that B-PMO was taken up by B cells both in bone marrow and spleen, as evidenced by the restored BTK expression.

Figure 8

Restoration of BTK expression upon splice correction after in vivo treatment of BAC transgenic mice. Four mice were treated with B-PMOs, as described in Methods, and assayed for BTK restoration. (A) RT-PCR analysis of total spleen and total bone marrow cells from B-PMO–treated transgenic mice. A representative gel from 2 animals, both from the treated and the untreated group, is shown. “WT-2” represents RNA from normal mice, and the slightly smaller amplicon in this lane results from a differential primer set discriminating human and mouse RNA. To ascertain the detection of corrected transcripts for samples from treated and untreated BAC transgenic animals, we decided to use 3 times more cDNA and 5 more amplification cycles. (B) Western blot analysis of BTK restoration in 2 of 4 treated animals; total cells from bone marrow and spleen. (C) Western blot analysis of the isolated B cells both from spleens and bone marrow, showing BTK expression from 2 representative animals. The percentage of WT BTK RNA was calculated as WT RNA fraction × 100/(misspliced + WT RNA fraction). Bar graphs show the quantitative analysis of BTK protein as a percentage of relative intensity according to ImageJ Software. T, treated.

Collectively, this suggests that the splice correction concept could become a viable treatment option for patients with XLA, although standardization of multiple parameters is needed prior to any clinical approach.

SCOs correct splicing and restore BTK protein in monocytes from patients with XLA ex vivo. As a final proof of concept in a human setting, we applied our strategy to primary patient cells. Similar to B cells, monocytes express the BTK gene but, in contrast, do not need BTK protein for normal development or survival (9, 13). Since patients with XLA essentially lack circulating B cells but do have normal numbers of circulating monocytes, we obtained monocytes from a patient with the BTK intron 4 cryptic splice site mutation. Since splicing factors in mice and humans are similar but not identical, it was important to ascertain that splice correction of full-length pre-mRNA also could take place in primary patient cells.

Electroporation conditions for transfection of SCOs into human monocytes were optimized, and their therapeutic effect was assessed. Using 186.15-3LNA, splice correction was observed for BTK both at the RNA (Figure 9A) and protein level (Figure 9B). Moreover, we also evaluated naked uptake of B-PMO 186.25 in patient monocytes, and SCOs readily restored BTK mRNA in these cells (Figure 9C) as well as yielded the corresponding protein dose dependently (Figure 9D). Even at a very high concentration (10 μM), PMO 186.25, devoid of the CPP moiety, did not restore BTK protein synthesis, suggesting the crucial importance of the CPP.

Figure 9

SCO-induced BTK restoration in patient monocytes. (A) Results from RT-PCR 48 hours after electroporation of monocytes from a patient with XLA with the intron 4 mutation. Monocytes were transfected with 1 μM 186.15-3LNA. Human control monocytes were also included. Ribosomal RNA (18S) serves as an RNA quality control. (B) Western blot showing BTK protein 48 hours after electroporation with or without treatment with 1 μM 186.15-3LNA. The right lane with a mouse B cell sample serves as a positive control for BTK (mouse and human BTK protein are both composed of 659 amino acids and recognized by the same antiserum). (C) RT-PCR results from patient monocytes treated with B-PMO 186.25 (3 μM and 6 μM). (D) Western blot showing BTK protein from B-PMO 186.25– and PMO 186.25–treated patient monocytes. Note that the anti-BTK antibody used for the monocyte lysates generates a strong, nonspecific band appearing just below the BTK-specific band, while the specific band is completely absent from the nontreated patient control cells (lane 1). Actin served as a loading control. Representative gels from 3 independent experiments are shown. The lanes separated by a black line were run on a different gel. The percentage of WT BTK RNA was calculated as WT RNA fraction × 100/(misspliced + WT RNA fraction). The bar graphs show the quantitative analysis of BTK protein as a percentage of relative intensity according to ImageJ Software. MΦ, monocytes.

Thus, the results obtained in splice-corrected monocytes derived from a patient with XLA are in concordance with the findings in B lymphocytes from BAC transgenic mice both in vitro and in vivo. Collectively this demonstrates that splicing of full-length mutant human BTK pre-mRNA can be efficiently corrected by the SCOs, thereby suggesting that our strategy is viable in a human setting.

In this report, we describe a novel BAC transgenic mouse model for human XLA and demonstrate its feasibility for the study of splice correction therapy. To the best of our knowledge, there is only a single existing human transgenic animal model for hematopoietic disease caused by aberrant splicing (61). The unique features of our transgenic mouse are that the entire affected locus is of human origin and that the corresponding endogenous gene has been deleted. This is also the first human transgene model for treatment strategies caused by defective splicing in the lymphoid compartment. Furthermore, owing to the fact that the BAC also encompasses the HNRNPH2 splice-factor encoding gene, these mice express an additional component of human origin, making this model even more similar to the situation in XLA. Taken together, this suggests that our human BAC transgenic mouse could become of broad interest.

Mutations interfering with splicing events are estimated to account for a substantial amount of all alterations leading to disease (62), and XLA is no exception, with 14% of all mutations being caused by defects related to splicing (63, 64). In this study, we investigated a case of XLA caused by a single nucleotide replacement (A to T) in intron 4 of the BTK gene, which leads to aberrant splicing resulting in the inclusion of a cryptic exon containing an in-frame termination codon. The aim was to promote the exclusion of the erroneous exon and restore normal splicing in both patient with XLA and mouse model cells. Here, we have tested a number of ON chemistries and demonstrated their different properties and utility as therapeutics in leukocytes.

Design of efficient SCOs requires careful examination of both the Tm and the uniqueness of the target sequence and, when possible, analysis of the secondary structure of the target site. Furthermore, the in vivo activity of any SCO is not only dependent on hybridization properties, but also on resistance to degradation and clearance as well as on uptake and intracellular release in the target cell. We initially tested a battery of 2′OMePS RNA ONs in a model cell line expressing an EGFP-luciferase fusion reporter gene interrupted by the BTK intron 4 with the same mutation as in the studied family with XLA. We scanned the whole exon 4a by designing different SCOs primarily targeting ESE sites. Using the reporter cell line readout, we could observe robust splice correction, as evidenced by restored mRNA, thus confirming the potential of this approach, and the best-performing SCO candidates were selected for the next round of experiments. Of note here, SCOs complementary to the 3′ss of the cryptic exon showed lower efficiency compared with the other targeted sites. This might be due to the presence of secondary structures in the 3′ss, making the SCOs less efficient.

Studies in the cell line model allowed us to screen different LNA-modified versions of the SCOs to achieve further enhanced splice correction. Since LNA ONs are normally more potent, we investigated size-reduced versions. By decreasing the length from the initial 18-mers to 15-mers, the idea was that smaller SCOs could provide an advantage in terms of cellular uptake and release and hybridization specificity (49, 65). Thus, the efficiency of 2′OMePS SCOs was compared with the corresponding shorter versions. Hence, in comparison to an 18-mer 2′OMePS RNA, the various 15-mers composed of LNA/2′OMePS RNA had similar, or even higher, activity. The size factor is of relevance, since shorter ONs have reduced risk of accidentally targeting irrelevant RNA sequences if appropriately designed. The resulting enhanced target specificity can potentially allow lower doses to be administered while activity in vivo is maintained (34, 49, 53). However, we also observed that further shortening of the SCOs, as measured in this in vitro model, reduced the splice-correcting activity of both the 186 and the 187 series. This may be secondary to the fact that the avidity of very short ONs simply is too low or to the fact that short ONs are less likely to interfere with splicing.

Next, the activity of the SCOs was assessed in the context of full-length, mutated human BTK pre-mRNA using either cells derived from the human BAC transgenic mouse or from primary patient monocytes carrying the intron 4 mutation. Although not all SCOs with different chemistries and lengths were compared between these 2 readouts, those tested behaved similarly. Thus, there was no indication of any unique secondary pre-mRNA structures imposing an altered sensitivity to the SCO-induced correction. This suggests that the identified SCOs, at least from this perspective, could be applied in a clinical setting.

To the best of our knowledge, this is the first time that exon-skipping SCOs have been successfully attempted as a treatment in primary B lymphocytes, adding this lineage to the list of potential targets for such a therapeutic intervention (66). Of crucial importance was the fact that we had the chance to obtain primary monocytes from a patient with XLA and successfully treat them with SCOs. This further substantiates the therapeutic potential of our SCO-based approach.

To this end, we also wanted to study naked delivery to both primary B cells and patient monocytes. Recently, PMO-based SCOs have proven to be very efficient in DMD animal models, especially when conjugated to a short arginine-rich peptide (i.e., B-PMO) belonging to the CPP group (67). Moreover, a d–amino acid–containing CPP-PMO was used in a thalassemia transgenic mouse model (68). Thus, in the context of splice switching, several CPPs, including the B-PMO, have been used to enhance PMO delivery, allowing the dose to be reduced by 1 order of magnitude or more in vivo (33). Moreover, antisense PMOs (23) and, even more potently, when conjugated to the B-peptide, such SCOs were successfully used by Gatti and collaborators to treat lymphoblastoid cell lines derived from patients with ataxia telangiectasia (24). In the latter report, it was also demonstrated that fluorescently labeled CPP-PMOs could cross the blood-brain barrier.

While almost full restoration was obtained with B-PMO 186.25 in patient monocytes, the correction under gymnosis conditions was weaker in primary B cells as compared with that under electroporation. It should be noted that the restored mRNA, as expected, was more stable than the aberrantly spliced transcript. Here, it is likely that optimization of primary cell culture conditions enabling long-term survival would allow uptake over several days, which is necessary for optimal gymnosis (53). Besides, the phagocytic nature of monocytes might enable easier translocation of CPPs in a shorter time. Since there is frequently a high capacity in endogenous signal transduction pathways, with only a fraction of proteins being, for example, phosphorylated, it is possible that correction of every defective BTK pre-mRNA is not needed for a therapeutic effect. Our findings with CPP-conjugated SCOs are reminiscent of the results obtained in DMD mice (57, 69, 70), in which B-PMOs were extraordinarily active.

This is also the first time that such modified ONs have been proven to work ex vivo in primary lymphoid cells, suggesting that the B-PMO design could become a versatile combination chemistry not restricted to muscle cell delivery. Of note is the fact that we observed a decrease in cell viability with increasing B-PMO concentrations in B cells, whereas monocytes were more resistant. This may be due to the fragile nature of BTK-defective primary B cells compared with monocytes, which are not functionally affected by lack of this protein. Moreover, the fact that lymphoblastoid cell lines treated with B-PMO SCOs showed restoration of the ataxia telangiectasia protein for a period of 21 days following a single treatment (24) demonstrates that at least certain B-lineage cells are highly suitable targets.

Comparing delivery by electroporation in human monocytes and primary B cells, we observed that lower concentrations of LNA-containing SCOs (1 μM) were sufficient for the correction in monocytes. This could be attributed to the electroporation method itself, which monocytes are able to resist better, whereas B cells get more damaged. It is possible that the electroporation conditions per se reduced the actual number of lymphocytes that are both successfully transfected and surviving, which could result in an underestimation of the potency of the SCO after electroporation in B cells. On the other hand, there may be differences in the internalization of the SCOs and their subsequent intracellular routing. Moreover, the fact that primary human monocytes were readily transfectable also suggests that SCOs could represent a treatment option for monocyte disease.

Demonstrating not only protein restoration, but also the corresponding biological activity, was crucial for the splice-switch concept as a potential treatment modality. The fact that 2 different functional tests, cell viability and Y551 phosphorylation of BTK, yielded confirmatory results strongly suggests that the BTK protein generated is fully active.

Moreover, we also demonstrate that B-PMO 186.25 could be systemically administered in vivo, correcting the defect in mice lacking the endogenous protein but expressing the mutated human BTK gene. We could show that treated animals had substantial levels of BTK expression both in the bone marrow and spleen, organs considered challenging for SCO therapy. Moreover, we could also demonstrate that isolated B cells, both from the bone marrow and spleen, expressed BTK, further proving that B-PMO reached the target cells and induced a therapeutic effect. At this stage, we have not made measurements allowing the detailed comparison between transcript and protein levels, for example, but rather, the analysis should be regarded as semiquantitative. The observation that upon treatment the relative amount of BTK in bone marrow cells seems to be lower is not unexpected, since it is likely that the splenocyte exposure to SCOs will be greater, due to the fact that more of the cardiac output reaches the spleen. One can also speculate that a fraction of BTK-expressing B-lineage cells in the bone marrow might constitute progenitor cells, adding a further therapeutic value. In this study, only a single multidose regimen was investigated, so it remains to titrate the dose and frequency of treatments to identify an optimal dosage. Importantly, treatment for just 1 week was sufficient for obtaining significant BTK restoration. Thus, it is quite possible that treatment periods longer than a week will further increase the number of corrected cells. The optimal mode of administration also remains to be tested. Since the treated mice were subject to combined intravenous and subcutaneous delivery, their respective contribution to the final treatment result cannot be estimated but deserves further studies.

Splenic B cells are easily accessible and constitute the preferred experimental source of mouse B cells. However, for obvious reasons, splenocytes are not the candidate target cells for SCO therapy in XLA and, instead, bone marrow–resident pro–B cells are. Therefore, we differentiated primary bone marrow progenitor cells into pro–B cells and subsequently transfected them with SCOs. Already after 48 hours, substantial amounts of BTK protein were detected in a high proportion of these cells, approximately to the same extent as that observed in mature B lymphocytes. This is an important piece of information, which suggests that the SCO treatment concept could become applicable in a patient setting. Furthermore, it is known that pro–B cells from human bone marrow can be expanded ex vivo as described before (71). It was gratifying to observe that in vivo administration of B-PMOs yielded BTK restoration not only in the spleen, but also in the bone marrow. This bodes well for the concept of correcting human pro–B cells.

We also investigated the possibility of using a dual approach, namely to complement the SCO therapy with treatment with antagomirs targeting miR-185, which has been reported to negatively regulate BTK expression in primary mouse cells (72). However, in preliminary experiments, the antagomir strategy was not sufficiently robust (unpublished observations). A single antagomir design was used in these experiments, and it may well be that further optimization could improve the outcome. Thus, combining different ON strategies remains an attractive option with recent success in experimental DMD (73).

A key feature of XLA is that BTK is expressed in all B-lineage stages, except for the mature plasma cell (8, 9). This opens the possibility that even a transient correction of B-lineage cells could result in a long-term treatment effect. Thus, if BTK expression could be transiently restored at the pro–B cell stage, yielding a polyclonal native B cell repertoire, such cells are expected to respond to immunization. Since infectious agents represent a major threat to patients with XLA, vaccination against them could potentially yield an adequate humoral immune response by SCO-corrected B cells. The primary B lymphocytes responding to the vaccine would subsequently maturate into BTK-independent long-lived plasma cells exerting long-term protection. Due to the fact that the B cell receptor is not expressed in plasma cells, this strategy is valid for all primary immunodeficiencies caused by splice site mutations affecting this receptor complex (1).

From a therapeutic point of view, it might be feasible to obtain pro–B cells, carry out the treatment ex vivo, and return the splice-corrected cells to the patient or, alternatively, to administer the SCOs systemically in vivo. Ex vivo treatment has the advantage that any potential side effect resulting from the uptake of SCOs in non–B-lineage cells could be avoided. On the other hand, such a treatment would be more cumbersome. Thus, in vivo administration might seem more straightforward, and even if the dosage has not been studied in detail, it is likely that rather large doses will be required. If such a medicine would become available, there would be additional aspects to take into consideration, e.g., the cost of synthesis, which is likely to be higher for in vivo delivery. Whether splice correction will ever become a treatment option for B cell disorders, or other hematopoietic diseases, remains to be seen. Here, we have developed a unique animal model allowing both ex vivo and in vivo research along this line to be conducted in a preclinical setting.

View Supplemental data

The project was supported by Swedish Research Council, Swedish County Council (ALF project), Swedish Cancer Society, and the Ragnar Söderberg Foundation. We would like to thank the people working in the animal facility at Karolinska Institutet; our special thanks to Maria Ottosson for the excellent help with the mouse breeding. We would like to thank A. Charlotta Asplund for performing mutation analysis of the BTK gene and Janne Turunen for critical reading of the manuscript. D.K. Mohammad holds a PhD fellowship from the Ministry of Higher Education and Scientific Research in the Kurdistan Regional Government (Erbil, Iraq). M.O. Gustafsson holds a PhD scholarship from Södertörn University College. The work in the laboratory of M.J. Gait was supported by the Medical Research Council (MRC programme no. U105178803).

Address correspondence to: C.I. Edvard Smith, Clinical Research Center, Department of Laboratory Medicine, Karolinska Institutet, Novum, Hälsovägen 7 SE-141 57 Huddinge, Sweden. Phone: 46.8.5858.3651; E-mail: edvard.smith@ki.se.

Conflict of interest: Patent applications have been filed relating to the technologies described herein, which are assigned to IDT. Mark A. Behlke is employed by IDT but does not personally own any shares or equity in IDT. IDT is not a publicly traded company.

Reference information: J Clin Invest. 2014;124(9):4067–4081. doi:10.1172/JCI76175.

Amer F. Saleh’s present address is: AstraZeneca R&D, Alderley Park, Safety Assessment UK, Cheshire, United Kingdom.

Tolga Sutlu’s present address is: Nanotechnology Research and Application Center, Sabanci University, Istanbul, Turkey.

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