Genomics, Gene Therapy and Proteomics

Correction of B-cell development in Btk-deficient mice using lentiviral vectors with codon-optimized human BTK


X-linked agammaglobulinemia (XLA) is the most common primary immunodeficiency (PID) in man and caused by mutations in the Bruton’s tyrosine kinase (BTK) gene. XLA is characterized by a B-cell differentiation arrest in bone marrow, absence of mature B cells and immunoglobulins (Igs), and recurrent bacterial infections. We used self-inactivating lentiviral vectors expressing codon-optimized human BTK under the control of three different ubiquitous or B cell-specific promoters. Btk−/− mice engrafted with transduced cells showed correction of both precursor B-cell and peripheral B-cell development. Lentiviral vectors containing the wildtype BTK sequence did not correct the phenotype. All treated mice with codon-optimized BTK exhibited the recovery of B1 cells in the peritoneal cavity, and of serum IgM and IgG3 levels. Calcium mobilization responses upon B-cell receptor stimulation as well as in vivo responses to T cell-independent antigens were restored. Viral promoters overexpressing BTK >100-fold above normal resulted in erythro-myeloid proliferations independent of insertional mutagenesis. However, transplantation into secondary Btk−/− recipients using cellular promoters resulted in functional restoration of peripheral B cells and IgM levels, without any adverse effects. In conclusion, transduction of human BTK corrects B-cell development and antigen-specific antibody responses in Btk−/− mice, thus indicating the feasibility of lentiviral gene therapy for XLA, provided that BTK expression does not vastly exceed normal levels.


Primary immunodeficiencies (PIDs) form a group of rare but severe diseases due to genetic defects that affect the development and function of immune cells.1 PID can be treated with transplantation of normal hematopoietic stem cells (HSCs) from healthy donors. Stem cell transplantation (SCT) generally has a good outcome, particularly when human leukocyte antigen-matched sibling donors are used. However, such optimal donors are frequently not available. Gene therapy, in which a patient’s autologous HSCs are genetically corrected, represents an alternative treatment for patients with PID, which can avoid the immunological risks of allogeneic SCT, overcomes the lack of suitable donors and confers similar benefits.2, 3 Recent clinical trials using gene therapy have led to immune restoration in patients with X-linked severe combined immune deficiency, adenosine deaminase-deficient SCID and chronic granulomatous disease. However, severe complications arose in several of the patients in whom the integrated retroviral vectors led to the development of leukemia as an adverse consequence of the therapy (see below).

X-linked agammaglobulinemia (XLA) is the most common PID disease in man. The disease is characterized by an arrest of B-cell development, at the pre-B-cell stage in bone marrow (BM) and absence of immunoglobulins (Igs) due to the lack of peripheral B cells and plasma cells. Patients often develop recurrent and severe bacterial infections caused by pyogenic bacteria, such as pneumococci and streptococci. Current treatment includes regular intravenous Ig infusion and prompt administration of antibiotics in case of infections. So far, SCT or gene therapy has not been considered as a regular treatment for XLA patients.

XLA is caused by mutations in the Bruton's tyrosine kinase (BTK) gene.4, 5 The Btk protein is a cytoplasmic non-receptor tyrosine kinase and belongs to the Tec-family of tyrosine kinases which include Btk, Tec, Itk and Bmx.6 Btk is expressed not only in B cells6 but also in erythroid cells and myeloid (monocytes/granulocytes) cells.7, 8, 9, 10, 11, 12 In erythroid cells, Btk is associated with the Epo-receptor8, 13 whereas in (pre) B cells, Btk is coupled to the (pre) B-cell receptor (BCR).10, 11, 12, 14, 15 In XLA patients, BTK mutations can be found throughout the gene and these frequently result in the absence of the Btk protein,16, 17 but amino acid replacements also occur.18 XLA has a heterogeneous phenotype and this heterogeneity might be related to the nature of the mutation in combination with other genetic factors.19

The major blockade caused by BTK mutations in B-cell differentiation is at the transition of pre-B-I cells into large pre-B-II cells.9 This results in the accumulation of pro-B cells and pre-B-I cells in the BM. In this transitional stage, pre-B-cell receptor signaling regulates clonal expansion. As Btk is associated with the pre-BCR signaling pathway, absence of BTK due to mutation leads to disruption of the pathway. In mice, absence of Btk will lead to X-linked immunodeficiency (Xid) phenotype. The Xid mice have a missense mutation at a conserved arginine residue within the plekstrin domain of Btk, which results in conformational changes of the protein and subsequently lack of ability to bind to the cell membrane. In comparison with human XLA, the Xid phenotype is less severe because of the redundancy of other Tec kinases, which apparently can compensate for the lack of BTK in murine B cells.20, 21, 22, 23

Current intravenous Ig treatment for XLA is fairly effective and the quality of life of most patients has significantly improved over the last two decades, but it is not possible to prevent all infections. Despite prompt administration of antibiotics when needed, recurrent infections can cause progressive and irreversible organ damage, particularly of the lung and consequently represent a life-threatening risk.24 Most patients have a reduced life expectancy and many patients suffer from major infections after puberty. Moreover, current treatment is non-curative and is extremely costly: € 2.5 million to 4.0 million per patient lifetime for intravenous Ig treatment only. Furthermore, current conditioning for allogeneic transplantation has risks that could be avoided with gene therapy.

Theoretically, SCT could be a curative treatment option for XLA patients but the current conditioning for SCT and the non-identical allogeneic SCT have many risks. Autologous stem cell-based gene therapy represents a promising alternative for current therapies, because it is curative and prevents the risks of SCT. In general, requirements for clinical application of HSC-based gene therapy are: the availability of a mouse model resembling human disease (such as the Btk−/−, Xid or Btk−/−Tec−/− mice), the disease should, in principle, be curable by SCT, and suitable vectors that can also be used in patients should be in place, that is, vectors encoding the normal human therapeutic protein without tags or heterologous sequences, such as auto fluorescent proteins (GFP).

Earlier studies have shown that this approach is feasible using a Btk− or Btk/Tec-deficient mouse model. Yu and his coworkers25, 26 showed a full restoration of the B-cell defect in the Btk/tec −/− mice using a gamma-retroviral vector expressing the human BTK gene. Although this study showed promising progress in development of gene therapy for XLA, the use of gamma-retroviral long terminal repeat (LTR)-driven vectors carries a significant risk of insertional mutagenesis, as is now well documented. Therefore, other vector systems have been developed.27, 28

Gene therapy using the murine Moloney leukemia virus gamma-retroviral vectors has been successful in the treatment of other PIDs such as X-linked severe combined immune deficiency and adenosine deaminase-deficient SCID.29, 30, 31 Although these vectors have proven to be useful for gene transfer into HSCs, the strong enhancer activity of the LTR driving the transgene expression and the recently highlighted integration bias of these vectors towards gene regulatory regions near the transcriptional start site are the major risks for insertional mutagenesis in gene therapy trials.3, 32, 33 As an alternative, HIV-1 derived lentiviral self-inactivating (SIN) vectors could be useful to overcome these obstacles as they have several advantages. First, lentiviral vectors offer more flexibility for inclusion of heterologous sequences that allow strict control of transgene expression throughout hematopoietic differentiation, for instance, the use of lineage specific promoters. Second, in both lentiviral and gamma-retroviral vectors, endogenous viral sequences can be removed to prevent the enhancer activity of the viral promoter in so-called SIN vectors, which should improve the safety of these vectors. Third, lentiviral vectors have been shown to be superior to gamma-retroviral vectors for transducing quiescent cells, such as HSCs. Fourth, the risk of insertional oncogenic events may also be reduced as these vectors do not preferentially target transcriptional start sites to the same extent as gamma-retroviral vectors do.34, 35

Several mouse models have been reported to have predictive value of the potential risks of insertional mutagenesis. Shou et al.36 showed in Arf−/− IL2rg−/− double-deficient mice, high incidence of integration-dependent T-cell lymphomas. Montini et al.34 determined the viral integration sites in HSCs after SIN lentiviral or gamma-retroviral transduction. They observed a high frequency of retroviral insertion near oncogenes and cell-cycle related genes in the early onset tumors, while lentiviral vectors showed more favorable integration profiles despite the higher integration levels and gene-expression levels. A caveat in this study is the fact that a retroviral LTR was compared with a lentiviral SIN LTR, which per se creates different expression levels. In vitro cell culture systems have also been developed to address the detection and frequency of insertional mutagenesis.37 Modlich et al.38 reported that SIN gamma-retroviral vectors with strong internal promotor/enhancer may still be capable of insertional mutagenesis, although with a lower frequency than retroviral LTR-driven vectors. These results were further confirmed in vivo with serial transplantation.

To investigate whether stem cell-based gene therapy using the new generation of vectors could provide a novel treatment option for XLA, we tested SIN lentiviral vectors encoding the human BTK gene to correct B-cell defects in Btk−/− mice. Our results show the correction of B-cell development and B-cell function after lentiviral gene transfer. Moreover, using cellular promoters (rather than strong viral promoters) no severe adverse mutagenesis events were observed during 10 months follow up, including secondary transplantation, strongly supporting the higher degree of safety using these lentiviral vectors.

Materials and methods


C57BL/6 and Btk−/− mice were generated as previously described39 and were bred and maintained at the Experimental Animal Center (EDC) of the Erasmus MC, Rotterdam, The Netherlands under specified pathogen-free condition. All procedures were approved by the Animal Ethics committee (DEC) of the Erasmus MC.

Lentiviral vectors and virus production

Human BTK complementary DNA10 was cloned into a third generation SIN lentiviral vector40 with three different promoters, including a 990 bp fragment of the human CD19 promoter (CD19),41 the elongation factor 1a, short form (EFS), and the spleen focus-forming virus (SFFV).42 In addition, we used a safety-improved woodchuck hepatitis posttranscriptional regulatory element (WPRE) devoid of X protein sequences and 4 ATG mutations (see Figure 1).43 Codon optimization of the human BTK gene was performed by Geneart (Regensburg, Germany). Helper constructs pRSV.Rev, pMDLg/p.RRE (expressing gag/pol) and pMD.G (for vesicular stomatitis virus glycoprotein) were described previously.44 Large scale helper plasmids were produced by Plasmid Factory (Bielefeld, Germany). High-titer lentiviral vectors were produced using a four-plasmid system in 293T cells. Briefly, 293T cells were transfected transiently with transfer and helper plasmids using Fugene6 (Roche Nederland, Woerden, The Netherlands). Supernatants were harvested 24, 36 and 48 h after transfection. Virus supernatant was filtered, pooled and stored at −80 °C before concentration. Pooled viral supernatants were concentrated by ultracentrifugation for 16 h at 18 000 g at 4 °C. Viral pellets were pooled and stored at −80 °C until use.

Figure 1

Schematic representation of the lentiviral vectors used in this study and resultant lentivirally-regulated BTK protein expression. (a) Native BTK (b) codon-optimzed BTK. Packaging signal (Ψ), rev-responsive-element (RRE), central polypurine tract sequence (cPPT) and 3′ regulatory sequences with viral enhancer and promoter sequences deleted (ΔU3). The latter feature generates the so-called self-inactivating vector (SIN). (c) Detection of BTK protein after transduction with lentiviral vectors containing codon-optimized BTK. B220+ cells were stained for intracellular fluorescence-activated cell sorting analysis using a BTK-specific antibody.

Lentiviral gene transfer and in vivo transplantation

C57BL/6 wild type (Wt) and Btk−/− BM cells were collected by flushing both femurs and tibiae with RPMI supplemented with 2.5% heat-inactivated fetal calf serum, and washed twice with the medium. Lineage depletion of BM cells was performed using Lineage Cell Depletion Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's protocol. The Wt and Btk−/− BM Lin− cells were cultured overnight with StemSpan serum free medium (Stemcell Technologies, Vancouver, BC, Canada) supplemented with 100 ng/ml recombinant mouse FLT3-L, 10 ng/ml recombinant thrombopoietin and 100 ng/ml recombinant mouse stem-cell factor. Btk−/− cells were transduced with CD19.coBTK, EFS.coBTK (7500 particles/cell) or SFFV.coBTK (2000 particles/cell) by spin occulation at 800 g for 60 min at 32 °C. At 2 days after transduction, 500 000 transduced cells were injected intravenously via tail vein along with 300 000 Btk−/− splenic cells into lethally irradiated Btk−/− recipient mice. As controls, lethally irradiated Btk−/− mice were also transplanted with cultured C57BL/6 Wt or Btk−/− BM Lin− cells. The mice were given ciprofloxin-containing water ad lib and monitored daily.

For secondary transplantation, 3 × 106 total BM cells from primary recipients were mixed with 300 000 Btk−/− spleen cells and were transplanted into lethally irradiated Btk−/− secondary recipients. Reconstitution in peripheral blood was monitored every 4 weeks after transplantation. At 20 weeks after transplantation, mice were killed and analyzed for reconstitution in tissues as described previously.45

Flow cytometric analysis

At 17 weeks after primary transplantation or 20 weeks after secondary transplantation, mice were killed. Peripheral blood samples were collected via heart puncture in heparin-containing microtubes. Peritoneal cavity fluid was collected in ice cold phosphate-buffered saline. Erythrocytes were depleted by treatment with lysis buffer, containing of NH4Cl (8.3 g/l) and KHCO3 (1 g/l) on ice, for 5 min and washed twice immediately with RPMI medium supplemented with 10% fetal calf serum. Single-cell suspension from spleens were made using 70 μm cell strainer (BD Biosciences, San Jose, CA, USA). BM cells were collected by flushing both tibiae and femurs with RPMI medium and washed twice with 1% fetal calf serum/phosphate-buffered saline. The phenotype of the cells was determined by staining with monoclonal antibodies against the following antigens: B220 (Ra3-6B2), CD3 (145-2C11), CD4 (L3T4), CD8 (53-6.7), CD11b/Mac1 (M1/70), CD19 (ID3), CD23 (B3B4), CD71 (C2), c-kit/CD117 (2B8), Gr1 (RB6-8C5), IgD (11-26c), IgM (R6-60.2), NK1.1 (PK136), Sca1 (E13-161.7), TCRβ (H57.597) and Ter119, all from Pharmingen/Becton Dickinson, San Jose, CA, USA; B220-PE-Cy7, CD5 (53-7.3)-APC, CD34-Alexa Fluor 647 and CD93/AA4.1-PE from eBiosciences (eBioscience, San Diego, CA, USA). For detection of biotinylated antibodies, PE-Cy7- or APC-Cy7-conjugated streptavidin was used. As isotype controls, FITC-, PE-, PerCP-, APC-, PE-Cy7- and APC-Cy7-conjugated mouse IgG1 and IgG2a/2b were used. Stained cells were measured with a FACSCanto II (Becton Dickinson) and analyzed with FlowJo software (TreeStar Inc., Ashland, OR, USA).

Gene expression and detection of viral copy number

To assess the viral titers, gene expression and viral copy number, real-time quantitative PCR was developed to detect WPRE, coBTK and ABL gene expression. For viral titers and gene expression, total RNA from viral supernatant and transduced cells were extracted using the RNeasy Total RNA extraction kit (Qiagen Benelux B.V. Venlo, The Netherlands). To assess the viral copy number, genomic DNA was extracted using the GeneElute mammalian Genomic DNA miniprep kit (Sigma-Aldrich, Zwijndrecht, The Netherlands), and the WPRE sequence was quantified by use of quantitative PCR analysis using WPRE forward primer, 5′-IndexTermCCGTTGTCCGTCAACGTG-3′; reverse primer, 5′-IndexTermAGTTGACAGGTGGTGGCAAT-3′; probe, 5′-IndexTermFAM-TGCTGACGCAACC CCCACTGGC-TAMRA-3′. For the quantification of viral integrations and copy number, standard curves were made by 10-fold serial dilution of lentiviral vector of known concentration. The expression of coBTK was detected using the following primers: forward primer, 5′-IndexTermAGACCGCCAAGAACGCTATG-3′; reverse primer, 5′-IndexTermCTTGGTCTTTCTGTGGCTGCT-3′; probe, 5′-IndexTermFAM-CTGCCAGATTCTGGA AAACCGGAACA-TAMRA-3′. All gene-expression levels were normalized to the ABL gene as household gene. The ABL expression was detected using the following primers; forward primer, 5′-IndexTermTGGAGATAACACTCTAAGCATAACTAAAGGT-3′; reverse primer, 5′-IndexTermGATGT AGTTGCTTGGGACCCA-3′; probe, 5′-IndexTermFAM-CCATTTTTGGTTTGGGCTTCACACCATT-TAMRA-3′. ABL Fusionquant standards (Ipsogen Inc., Marseille, France) were used for the quantification of ABL expression.

Ligation-mediated (LM)-PCR and analysis of viral integration sites

Primer extension starts with the ligation of a biotinylated primer 5′-IndexTermGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAG-3′ followed by ligation of the following linker 5′-IndexTermCCTAACTGCTGTGCCACTGAATTCAGATCTCCCGGGTC-3′. PCR amplification was performed with the following LTR-specific primers 5′-IndexTermGAACCCACTGCTTAAGCCTCA-3′—LvLTRI




OCII (5′-IndexTermAGTGGCACAGCAGTTAGG-3′). The resulting amplicon of 225 bp serves as internal control.

Serum Ig detection and immunization in vitro

Serum levels of IgM and IgG3 were determined by isotype-specific enzyme-linked immunosorbent assay. To determine the T-cell independent type II immune response, mice were immunized intraperitoneally with 50 μg TNP–Ficoll in phosphate-buffered saline (Bioresearch Tec Inc., Novato, CA, USA). After 7 days, serum was collected, and TNP-specific IgM and IgG3 were analyzed. Serum from pre-immunization was also collected and it served as control. For TNP-specific enzyme-linked immunosorbent assay, plates were coated with TNP–KLH and serum dilutions used incubated for 2 h. Subsequent steps were biotinylated anti-IgM of anti-IgG3, streptavidin-coupled peroxidase and 2′-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid as substrate.

Calcium mobilization response

Calcium flux analysis was performed as described previously.46 Briefly, total spleen cells were washed in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid containing Hanks’ balanced salt solution supplemented with 5% heat-inactivated fetal calf serum (loading buffer) at a concentration of 10 × 106 cells/ml. Spleen cells were incubated with indo-1 acetoxymethyl ester (Molecular Probes, Breda, The Netherlands; 6 mg/ml) for 45 min at 37 °C and were washed with CaCl2 (1 mM) containing lithium borate buffer (LB Flux buffer). Cells were incubated with flux buffer at a concentration of 10 × 106 cells/ml for 60 min at 37 °C and were aliquoted to 1 × 106 cells/ml (1:10 diluted). Measurements were performed 1 min w/o stimulus, followed by 10 min with 20 mg/ml anti-IgM F(ab’)2 fragments (Jackson ImmunoResearch Laboratories, Suffolk, UK). Finally ionomycin (2 mg/ml) was added to determine the maximum response.

Statistical analysis

The statistical significance of assessments of B-cell populations was determined using a two-tailed t-test of independent sample means. The statistical analyses were performed by comparing the results obtained from mice that received lentiviral transduced Btk−/− BM cells or WT BM cells versus mice receiving mock transduced Btk−/− BM cells. Statistical results are indicated in the figures by bars representing the s.d.


To evaluate the feasibility of lentiviral gene transfer into HSCs for correction of XLA, we initially generated lentiviral vectors containing the human BTK cDNA. Human BTK was cloned into SIN lentiviral vectors under the control of two promoters, a 990 bp fragment of the human CD19 promoter (CD19.BTK)41 or the elongation factor 1a, short form (EFS.BTK) (Figure 1a). Lineage-depleted Btk−/− BM cells were transduced with these lentiviral vectors. Transduced cells were transplanted into lethally irradiated Btk−/− recipient mice. Btk−/− mice received untransduced Btk−/− Lin− BM cells or C57Bl/6 Lin− BM cells as negative and positive controls, respectively. We found on average, 5.7 viral copy numbers and a BTK expression of 0.25 relative to the housekeeping gene ABL per Lin− BM cells that were transduced with CD19.BTK lentiviral vector. For cells transduced with EFS.BTK lentiviral vector, we detected 1.5 viral copies per cell and a BTK expression of 0.03 relative to ABL. This low level of BTK gene expression in both CD19.BTK- and EFS.BTK-transduced cells was not sufficient to significantly restore any of the major features of Btk−/− mice, such as low percentages of IgM−/IgD+ B cells in the periphery and low serum IgM levels (data not shown). Only rarely, we observed low levels of reconstitution in individual mice. In conclusion, because of the low BTK expression achieved, lentiviral gene transfer with the wt, native human BTK cDNA under the control of CD19 or EFS promoters, was not sufficient to correct the phenotype of Btk-deficient mice in vivo.

Codon optimization of BTK

To increase the level of BTK expression in transduced cells, we performed the so-called ‘codon optimization’ of the human BTK cDNA. The genomic sequence of BTK was altered to optimal codon usage for translation into protein, furthermore cryptic splice sites, polyA sites, extensive mRNA secondary structures and other destabilizing sequences were removed. We cloned the codon-optimized BTK (coBTK) in the same CD19 (CD19.coBTK) and EFS (EFS.coBTK) lentiviral vectors described earlier. In addition, we also generated a third lentiviral vector, in which the expression of coBTK was placed under the control of SFFV promoter (SFFV.coBTK),42 as this is a very strong promoter and should lead to high BTK expression (Figure 1b). Recombinant lentiviruses were produced and tested for viral titer and transgene expression. We observed a 9-fold (CD19.coBTK) to 25-fold (EFS.coBTK) increase in lentiviral titer compared with lentiviral vectors containing the wt and native BTK cDNA. More importantly, the expression per integration increased 14-fold and 48-fold for CD19.coBTK and EFS.coBTK, respectively (Table 1). For SFFV, we observed very high expression but we did not have a native version to compare the effects of codon optimization. This increased BTK expression was also seen at the protein level in B cells that developed in vivo after transplantation of Lin− stem/progenitor cells that were transduced with coBTK under the control of various promoters (Figure 1c). This indicates that high expression of the therapeutic transgene can be obtained with a lower titer and a lower number of integrations per cell. The latter feature is important to reduce the risk of genotoxicity.

Table 1 Effects of codon optimization

Lentiviral transduction with coBTK leads to restoration of B-cell development

To evaluate the effects of codon optimization on B-cell reconstitution in vivo, lineage depleted (Lin−) Btk−/− BM cells were enriched and transduced with recombinant lentiviruses containing coBTK. Reconstitution of the hematopoietic system was monitored by tail bleeding of the mice every 4 weeks.

After 17 weeks of transplantation, all mice were killed and cells from BM, spleen, peripheral blood and peritoneal cavity were collected. Phenotype analysis using lineage-specific markers was performed by flow cytometry Compared with the BTK−/− group, no significant differences were observed in the absolute numbers of CD3+ T cells and CD11b+ myeloid cells in the lentiviral-treated groups (Figure 2a), whereas the overall levels in B lymphocytes in the EFS and CD19 groups were restored to over half of the normal (spleen) or equal to the normal numbers (blood) (see Figure 2b).

Figure 2

Reconstitution of B-cell development after lentiviral gene transfer with codon-optimized (co) BTK. Lineage depleted Btk−/− BM cells were transduced with lentiviral vectors containing codon-optimized BTK. At 17 weeks after transplantation, mice were killed. Cells from BM, spleen, peripheral blood and peritoneal cavity were harvested, evaluated by flow cytometry and analyzed for the presence of B, T and myeloid reconstitution. (a) Absolute number of B cells (B220+CD19+), T cells (CD3+) and myeloid cells (CD11b+) were calculated based on the total cell counts and the percentage of positive cells detected by flow cytometry. (b) Flow cytometric analysis of surface IgM/IgD expression, gated on B220+/CD19+ B cells in BM, spleen and peripheral blood. (c) Representative analysis of cells collected from peritoneal cavity in Btk−/− recipient mice. First, B220+/CD19+ cells were gated on total cells and within this population CD19+/CD5+ and CD19+/CD5− cells were shown. Data shown are representative of six mice examined within each group. (d) Restoration of pre-B cell block in BM by lentiviral gene therapy. BM cells gated on B220+IgM− cells were stained for CD2, surrogate light chain (SLC) and cytoplasmic IgM. The development of CD2+cIgM− cells is largely restored, as well as that of CD2+SLC− cells.

Erythro-myeloid proliferation without clonal integration sites

In the SFFV group, four out of five mice developed an erythro-myeloproliferation 4–6 weeks after transplantation, apparently because of exceedingly high BTK levels (>100 fold above normal levels, Table 2). The BTK-expression levels per cell basis were high in BM and spleen, but reached extremely high levels in the tumors. PCR-based analysis of the viral integration sites using LTR primers revealed a polyclonal integration pattern (Figure 3), indicating that solely the high BTK level, but not additional effects of insertion near proto-oncogenes, was responsible for development of these proliferations. To support the polyclonality of the eythro-myeloproliferations, we cloned 49 integration sites and sequenced the areas upstream and downstream of the insertion sites (Supplementary Table 1). No common integration sites and obvious oncogenes were identified, although the SP8 and TCF7 (effector of Wnt signaling)47 were of interest. Therefore, given the speed of development after transplantation and the polyclonal nature of the integration sites, the myeloproliferations are unlikely to be caused by insertional mutagenesis. The kinetics of myeloproliferation development (within a few weeks after transplantation) also makes insertional mutagenesis with subsequent leukemogenesis an unlikely event, as such events tend to take much longer.48 High tyrosine kinase, that can no longer be regulated in its activity, is the most likely explanation for the myeloproliferations. Whatever the explanation for myeloproliferations in the SFFV promoter group, we further focussed on mice receiving BTK under the control of CD19 and EFS promoters.

Table 2 Viral copy number and relative BTK expression of SFFV.coBTK-transduced mice
Figure 3

Lack of insertional mutagenesis in SFFV–BTK induced leukemias. On top, a schematic representation of the PCR analysis of insertion sites is shown. A 225 bp internal control band is generated and bands of other lengths indicate insertion sites. The gel shows BM and spleen samples from four mice that developed leukemia. The similarity of BM and spleen samples indicates the same tumor, the polyclonal patterns indicate that there is no major insertion site, which was confirmed by full sequencing of the integration sites leading to the identification of a number of different genes (Supplementary Table 1).

To characterize the B-cell compartment in detail, the B220+CD19+ B cells were investigated based on their expression of surface membrane IgM and IgD. We observed significant changes between the untreated Btk−/− animals and the gene therapy-treated animals. Data in Figure 2b illustrate the phenotypic analysis of IgM/IgD CD19+ cells in BM, spleen and peripheral blood from representative animals of all groups. We observed that the relative frequency and the absolute number of the most mature IgM−/IgD+ cells was significantly increased in all tissues of the gene therapy-treated animals compared with the BTK−/− group. The low frequency of these cells is the single, most obvious phenotypic abnormality that can be observed in the peripheral blood of Btk−/− mice (Figure 2b). These results show that lentiviral gene transfer of coBTK gene into repopulating hematopoietic cells is sufficient to overcome the late B-cell differentiation block associated with the murine BTK deficiency.

Another characteristic phenotype of Btk−/− deficiency is the absence of CD19+CD5+ B1 cells within the peritoneal cavity. These cells constitute a separate lineage of murine B cells with important functional properties different from the conventional B cells, such as the production of natural antibodies. To investigate whether this phenotype can be corrected after lentiviral gene therapy, peritoneal cells were collected and analyzed for the presence of CD19+CD5+ B1 cells. Figure 2c shows the >10-fold reduced percentage of CD19+CD5+ B1 cells in Btk−/− mice (1%) compared with wt mice (11%). In all gene therapy-treated animals, the CD19+CD5+ B1 cells were restored to normal or even above normal levels (Figure 2c). These results indicate that CD19+CD5+ B1 cells can be fully restored by lentiviral gene transfer. Thus, both conventional and B1 lineage B-cell defects can be corrected by lentiviral gene therapy in Btk−/− mice.

In human XLA, almost complete block in B-cell differentiation in the BM is observed.19 In Btk−/− mice this block is much more subtle but can be observed by careful flow cytometric analysis of the pro-B and pre-B-cell compartments. In Btk−/− mice, a clear block can be observed in the development of cytoplasmic IgM+ CD2− to CD2+ pre-B cells, which is largely overcome by both CD19.coBTK and EFS.coBTK transduced cells (Figure 2d). Similarly, in Btk−/− mice, the development into CD2+ SLC (surrogate light chain) –pre-B cells is hampered, which is also largely restored in the gene therapy-treated mice.

Restoration of IgM and IgG3 production in gene therapy-treated mice

BTK deficiency is characterized by major defects in serum Ig levels. Btk−/− mice exhibit reduced levels of serum IgM and IgG3, and they fail to respond to T-cell independent type II (TI–II) antigens, such as TNP–Ficoll. Figure 4 shows the serum IgM and IgG3 levels at 16 weeks after transplantation. High serum levels of IgM were detected in Wt, CD19.coBTK and EFS.coBTK animals, whereas in Btk−/− animals the IgM levels were significantly lower (Figure 4a) High levels of IgG were also observed in Wt animals, however, in the absence of specific immunization in CD19.coBTK and EFS.coBTK animals, IgG3 levels were not elevated compared with the Btk−/− animals. To investigate whether lentiviral gene transfer also restores TI–II immune response, at 16 weeks after transplantation, mice were immunized with TNP–Ficoll. After 7 days, serum was collected and TNP-specific IgM and IgG3 antibodies were assessed by enzyme-linked immunosorbent assay. Serum collected before immunization served as control. Indeed, we observed high levels of TNP-specific IgM in WT, CD19.coBTK and EFS.coBTK, after immunization the level of total IgM was only slightly increased (Figure 4b). Btk−/− animals did not show any antigen-specific response to TNP immunization. High levels of IgG3 were also observed in Wt animals after immunization; moreover compared with pre-immunization, CD19.coBTK and EFS.coBTK animals showed significantly increased IgG3 level after immunization. Btk−/− animals did not have any detectable levels of IgG3. Taken together, lentiviral gene therapy is capable of restoring the major antibody defects associated with BTK deficiency.

Figure 4

Restoration of IgM and IgG3 production in gene therapy-treated mice. At week 16 (pre-immunization) and at week 17 (post-immunization) after transplantation, serum was collected from individual mice. At week 16, mice were immunized with TNP–Ficoll and 1 week later serum was collected to measure the effect of immunization on antibody levels. (a) Total IgM and IgG3 levels collected before immunization. For the quantification of IgM and IgG3 concentrations, standard curves were made by 10-fold serial dilution of mouse IgM and IgG3. Data shown are of six mice examined within each group. (b) Anti-TNP-specific IgM and IgG3 antibodies were accessed by specific ELISA 1 week after immunization. Plates were coated overnight with TNP–KLH, incubated with serial dilution of collected serum and detected by biotinylated anti-IgM or IgG3.

Lentiviral gene transfer restores BCR stimulation in Btk–/– mice

To further investigate whether the membrane-proximal events associated with BCR signaling also are functionally rescued, we tested the splenocytes from gene therapy-treated animals for calcium mobilization in response to BCR stimulation with anti-IgM-F(ab’)2 antibodies. As expected, splenocytes derived from Btk−/− animals did not show a calcium flux upon stimulation (Figure 5); whereas, a clear calcium flux was detected in Wt-derived splenocytes. Moreover, calcium mobilization was also detected in splenocytes derived from CD19.coBTK and EFS.coBTK animals, although the magnitude of the flux observed in both groups was considerably lower than the levels found in the Wt-derived splenocytes (Figure 5). This is explained by the fact that not all B cells are transduced to reach the BTK levels required for full restoration of the calcium flux. These results show that coBTK is functionally active in Btk−/− mice that underwent lentiviral gene transfer, in line with the rescue of the developmental defects.

Figure 5

Lentiviral gene transfer with coBTK restores BCR stimulation in Btk−/− recipient mice. Total spleen cells from primary recipients were tested for calcium mobilization in response to BCR stimulation. After incubation with fluorescent dye indo-1 in the presence of CaCl2-containing buffer, total spleen cells from control mice (Wt or Btk−/−) or gene therapy-treated (CD19.coBTK and EFS.coBTK) mice were stimulated with anti-IgM F(ab)2 fragment and measured for the Ca2+ mobilization. For assessment of the maximum response, ionomycin was added to the cells. Blue indicates the BCR-induced calcium flux and red indicates the maximum response obtained by ionomycin.

BTK expression in lentiviral gene therapy treated Btk−/− mice

We observed sustained expression of BTK in BM, spleen, peripheral blood and peritoneal cavity in gene therapy-treated Btk−/− recipients 17 weeks after transplantation and in secondary transplanted mice, suggesting that the correction of B-cell development in Btk−/− recipients was due to efficient transduction of immature HSCs with long-term repopulation capacity (Table 3 and Figure 1c).

Table 3 Assessment of gene expression and viral copy number in secondary transplanted mice

Transplantation into secondary recipients shows long-term expression in HSCs

To show that the immature HSCs were faithfully transduced with codon-optimized lentiviral vectors, BM harvested from primary recipients was transplanted into lethally irradiated Btk−/− secondary recipients (n=28 in total), which were monitored by flow cytometric analysis of peripheral blood for B cells (B220, CD19, IgD and IgM), T cells (CD3) and myeloid cells (CD11b/Mac1); see Figure 6. Restoration of IgM−IgD+ B cells was observed at 8 weeks after transplantation in all mice receiving EFS.coBTK-transduced primary recipient BM cells. The increase was maintained to 20 weeks after transplantation (Figure 6a).

Figure 6

Correction of B-cell defects in Btk−/− secondary recipients. BM harvested from Btk−/− primary recipients was re-transplanted into lethally irradiated Btk−/− secondary recipients. (a) Flow cytometric analysis of B-cell reconstitution in peripheral blood collected from secondary recipients 20 weeks after transplantation. Individual mice numbers are indicated. Cells were stained for B cells (B220, CD19, IgM and IgD), T cells (CD3) and myeloid cells and gated B cells are shown. B220+CD19+ cells were gated and subsequently IgD/IgM cells are shown. (b) Total IgM and IgG3 serum levels of Btk−/− secondary recipient mice 20 weeks after transplantation.

At 16 weeks, Btk−/− secondary recipients that received CD19.coBTK-transduced BM cells (n=10) showed only a slightly increased percentage of IgM−/IgD+ cells compared with the Btk−/− group, but at 20 weeks after transplantation, 8 out of 10 mice showed high levels of IgM−IgD+ cells. Similarly, all EFS.coBTK mice showed a high percentage of these cells, even higher than that found in Wt mice. Analysis of the BM revealed that the mild block at the pre-B-cell stage (lower CD2 expression on B cells, lower cytoplasmic (Cy)IgM expression, higher surrogate light chain), associated with Btk deficiency in mice was restored in CD19.coBTK- and EFS.coBTK-treated mice (not shown).

We also assessed the IgM and IgG3 serum levels at 20 weeks after transplantation. In CD19.coBTK secondary recipient mice, 7 out of 10 mice showed elevated Ig levels compared with the Btk−/− mice. In EFS.coBTK secondary recipients, we found all the recipients with increased serum level of IgM (Figure 6b). Consistent with the flow cytometric analysis, no correction of serum IgM and IgG3 level was observed in SFFV.coBTK secondary recipients.

These results show efficient transduction of true HSCs with self-renewal capacity by lentiviral vectors containing codon-optimized BTK, leading to sustained correction of B-cell defects in Btk−/− mice.

Finally, no myeloproliferations or malignancies were observed in EFS.coBTK- or CD19.coBTK-treated mice. The total follow up is 10 months, 5 months in the primary transplantation and 5 months in the secondary transplantation. These results indicate that SIN lentiviral vectors do not lead to overt insertional mutagenic events, provided an internal promoter is used that does not vastly overexpress BTK. Given that the SFFV promoter also is associated with insertional mutagenesis, this promoter is not a good choice for XLA gene therapy. A possible concern is the high level of viral integrations that we observed with EFS.BTK-treated mice (Table 3). For individual mice, integration levels and BTK expression do not correlate and our assay overestimates viral integration as it also detected unintegrated proviral circular DNA. Nevertheless, careful titration of both EFS- and C19-encoding viruses would be required for future clinical application. Notwithstanding this fact, no myeloproliferations or leukemias were detected in any of the secondary transplanted mice, indicating that within the limits of animal experiments this is not a major concern.


Despite the fact that XLA is the most common immunodeficiency in man, representing approximately 85% of the early B-cell defects, gene therapy for this disorder has not yet reached the clinic. This is caused by the delicate clinical situation, in which the development of gene therapy as an alternative to currently available substitute treatment will require a much improved safety profile of the vectors used, when compared with other diseases for which HSC-based gene therapy has been attempted, including X-linked severe combined immune deficiency. To this end, the achievement of efficient BTK expression in the B-lymphoid lineage with our codon-optimized lentiviral SIN vectors, represents the first step towards this aim.

Using a competitive reconstitution model, several groups have shown that in vivo reconstitution by a limited number of normal BM cells was sufficient to correct the B-cell deficiency in Xid mice. Rohrer et al.49 showed that as few as 2500 cells (0.5% of total transplanted cells) could restore IgM and IgG3 serum levels to normal values. Full recovery of immune responses were achieved when 25 000 normal BM cells (5%) were transplanted into BTK-deficient Xid mice.49 These investigators concluded that even under sub-optimal condition (low number of transduced cells), gene therapy could still be beneficial for patients with XLA as long as the corrected cells maintain sufficiently high expression levels of the BTK gene. Here we show that codon optimization represents an important step to increase and sustain transgene expression from a lentiviral SIN vector. For both BTK and RAG1 gene (for which we develop gene therapy in another project), 10–100 fold increased gene expression was obtained after lentiviral transduction with codon-optimized human sequences. Very recently, a similar observation was made by Moreno-Carranza et al.50 in the gp91 phox gene, which is involved in chronic granulomatous disease. In fact, in our initial efforts using BTK cDNA without codon optimization, no restoration of B-cell development was found 16 weeks after transplantation; neither could we observe the correction of B1 cells in the peritoneal cavity. Moreover, serum IgM and IgG3 levels were not increased compared with the Btk−/− mice receiving untransduced cells. Using real-time quantitative PCR, we could show that the unsuccessful treatment was because of the low transduction efficiency of the Lin− BM cells. This resulted in insufficient BTK expression in B cells to overcome the B-cell defects in Btk−/− recipient mice. In BM, spleen and peripheral blood, the low BTK expression relative to ABL was detected. Other researchers have recently reported similar results in Btk-deficient Xid mice.51 Thus, codon optimization is apparently essential for successful BTK gene therapy with lentiviral vectors.

The BTK gene is functionally expressed in both the B and myeloid lineage. Although the B-cell defects clearly are the most prominent defects underlying the clinical phenotype of XLA, important myeloid defects have also been described for XLA.52 Thus, it may be important to not only express BTK in B lymphocytes but also in monocytes to fully restore immunity in XLA patients. Hence, besides the B-cell restricted CD19 promoter, we have tested the SFFV and EFS promoters to direct the expression of BTK. Our data show that the SFFV promoter leads to extremely high BTK levels (>100 fold of Wt levels) that could not be tolerated in immature erythroid cells, resulting in a erythro-myeloid proliferative syndrome without any indication of insertional mutagenic events (Figure 3). Previous data show that up to 15-fold overexpression of human BTK in Btk−/− mice actually functions as tumor suppressor gene.53, 54 This level of expression far exceeds what is reached with EFS and CD19 promoters but is lower than that with the viral SFFV promoter. With this promoter, levels of BTK expression were very high and 80% of mice developed erythromyeloid proliferations within a few weeks after transplantation. Despite the polyclonal nature of the insertion sites, the very high levels of BTK induce proliferation and might consequently lead to leukemogenesis. Therefore, our data suggest that a therapeutic window exists for BTK protein expression and that SFFV-directed expression of BTK might not be a safe option for clinical application. On the other hand, moderate and sustained expression of BTK was achieved under EFS and CD19 promoters to comparable levels in normal cells. Transduction with EFS.coBTK, and to a large extent with CD19.coBTK, could rescue B-cell development and restore serum Ig levels to normal values. Importantly, use of both CD19 and EFS promoters did not lead to any adverse effects. Moreover, in secondary transplantations, which significantly increase the likelihood of detecting such events,55 no myeloproliferations or leukemias were found. Given that EFS also allows BTK expression in the myeloid lineage and performs slightly better in secondary transplantations than the CD19-directed BTK expression, we would prefer EFS for use in clinical applications after extensive viral titrations and for demonstration of efficacy in models using cells from XLA patients.

The potential risks of severe adverse events (in particular leukemias) remain a major obstacle in gene therapy trials. Therefore, it is important to predict whether the lentiviral vectors and the therapeutic gene will potentially cause such severe adverse events in recipient mice. Several mouse models have been described that may enhance the sensitivity in the detection of leukemogenic events. These tumor-prone mouse models showed that retroviral transduction lead to high frequency of integration-dependent T-cell tumors and furthermore that a favorable integration profile was observed using lentiviral vectors compared with retroviral vectors.24 Modlich and coworkers27 showed that serial transplantation could remarkably enhance the sensitivity of detection potential toxicity effect of SIN viral vectors. These investigators showed that no tumors were observed in primary recipients but when BM was re-transplanted, vector-related leukemias arose in 26% (12 out of 46) of secondary recipients using retroviral vectors. In the current study, we did not observe any leukemogenic events both in primary (n=25) and in secondary recipients (n=28), suggesting a high safety of the procedure and of the lentiviral CD19 and EFS vectors used in this study. A larger cohort is needed to come up with a frequency of leukemogenic events. Our safety results thus far indicate a superior safety profile of SIN lentiviral vectors compared with gamma-retroviral vectors.

In conclusion, lentiviral-based expression of the BTK gene in transplanted HSCs can restore all immunological defects of BTK-deficiency thus indicating the feasibility of lentiviral gene therapy for XLA, provided BTK expression does not vastly exceed the normal levels. Previous work has shown gamma-retroviral transduction of BTK in mouse models of XLA to be successful.25 However, gamma-retroviral vectors are no longer considered as prime vectors for clinical gene therapy applications because of the oncogenic adverse effects.55, 56, 57 Our study therefore provides proof-of-principle for gene therapy of XLA in mice. When this paper was under submission, Rawlings and coworkers5 published an elegant study, showing lentiviral correction with a GFP-containing vector in a mouse model for XLA, confirming some of our work albeit with a vector not readily suited for treatment of patients.58 As our vectors do not contain any other elements besides BTK (that is, no fluorescent markers, tags or such), they are more likely acceptable to regulatory agencies for clinical use. The next steps will therefore comprise of transduction of human BTK-deficient cells with the same vectors as reported here, which will then be transplanted into immunodeficient mice as the final step before clinical application of this promising technique.


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We would like to thank Tiago Luis, Brigitta Naber, Edwin de Hass and Machteld Tiemessen (Staal laboratory) for their technical assistance and Yvette Caljouw for her assistance at the EDC. This work was supported in part by a Grant from the Translational Gene Therapy Research Program of ZonMw—the Netherlands Organization for Health Research and Development (project no. 43100016).

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Correspondence to F J T Staal.

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Ng, Y., Baert, M., Pike-Overzet, K. et al. Correction of B-cell development in Btk-deficient mice using lentiviral vectors with codon-optimized human BTK. Leukemia 24, 1617–1630 (2010).

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  • XLA
  • BTK
  • stem cells
  • B cells
  • lentivirus
  • gene therapy

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