Development Of Lymphoid Leukemia Research Articles

The relevance of the CDK4/6–p16INK4A axis for the development of lymphoid leukemia

The relevance of the CDK4/6–p16INK4A axis for the development of lymphoid leukemia

Globin Lentiviral Vector Insertions Can Perturb the Expression of Endogenous Genes in β-thalassemic Hematopoietic Cells

Although hematopoietic cell gene therapy using retroviral vectors has recently achieved success in clinical trials, safety issues regarding vector insertional mutagenesis have emerged. Vector insertion, resulting in transcriptional activation of proto-oncogenes, played a role in the development of lymphoid leukemia in an X-linked severe combined immunodeficiency trial, and caused myeloid clonal dominance in a trial for chronic granulomatous disease. These events have raised the question of whether gene therapy for other disorders such as beta-thalassemia and sickle cell disease may hold a similar risk. In this study, we prospectively evaluated whether gamma-globin lentiviral vectors containing enhancer elements from the beta-globin locus control region could alter the expression of genes near the vector insertion. We studied this question in primary, clonal murine beta-thalassemic erythroid cells, where globin regulatory elements are highly active. We found an overall incidence of perturbed expression in 28% of the transduced clones, with 11% of all genes contained within a 600-kilobase region surrounding the vector-insertion site demonstrating altered expression. This rate was higher than that observed for a lentiviral vector containing a viral long-terminal repeat (LTR). This is the first direct evidence that lentiviral vectors can cause insertional dysregulation of cellular genes at a frequent rate.

Assessment of Changes in Gene Expression Caused by Insertions of a Globin Lentiviral Vector Containing Globin Regulatory Elements or a Lentiviral Vector Containing Retroviral LTR Elements.

The development of lymphoid leukemia in two children with X-SCID who underwent gene therapy was partially due to activation of the LMO-2 proto-oncogene by the retroviral LTR of the vector which inserted nearby (Hacein-Bey-Abina et al., Science 2003), highlighting the importance of vector design on the potential to activate genes near vector integration sites. As gene therapy vectors for other blood disorders are evaluated, it seems prudent to assess the safety issues regarding insertion for each particular vector in appropriate pre-clinical models. We have focused on developing γ-globin lentiviral vectors for gene therapy of the hemoglobin disorders and have documented correction of a murine model of β-thalassemia in the absence of observable adverse events (Persons et al., Blood 2003; Hanawa et al., Blood 2004). To more thoroughly evaluate the potential for vector-induced genotoxicity, we have examined whether self-inactivating (SIN) γ-globin lentiviral vectors containing erythroid-specific, β-globin locus enhancer elements can alter the expression of genes nearby the vector insertion site, as the retroviral LTR did in the X-SCID trial. To ascertain whether an integrated globin vector could influence endogenous transcriptional activity in erythroid precursors, 15 clonal spleen colony erythroblast populations (≥ 95% erythroid) containing lentiviral globin vector insertions and 15 untransduced control clones were derived from bone marrow cells of β-thalassemic mice. The transcriptional profile of each clone was determined using the Affymetrix Mouse 430A microarray (representing ~15,000 genes). Expression of 4500–6000 genes was observed in all samples. Ligation-mediated PCR was used to obtain the vector-genomic DNA junction sequences, allowing identification of vector insertion locations in 13 of the clones using the NCBI database. Of these, 6 globin vector clones had 16 genes, including N-ras, which were located within 100kb of the vector insertion site and were represented on the array. Only one gene, D3Jfr1, encoding a “cold shock” DNA binding protein and which was disrupted by an intronic vector insertion, had a change in signal value relative to the mean signal value of the controls. Real time RT-PCR confirmed a 4-fold reduction in expression of this gene. Both microarray and real time RT-PCR demonstrated that expression of N-ras was unchanged. For comparison, 15 clones with insertions of a lentiviral vector containing the MSCV retroviral LTR, were also derived, along with 10 additional mock control clones. We are currently analyzing the expression of some 116 genes that lie within 300kb of the vector insertions, relative to the mean expression level in the 25 mock transduced clones. Additionally, we have expanded analysis of the globin vector clones to evaluate changes in expression of 107 genes located within 300kb of the vector insertions. These data should prove useful to assess whether integrated SIN globin lentiviral vectors containing erythroid-specific regulatory elements have a propensity to alter transcriptional activity in the progeny of genetically modified hematopoietic stem cells, relative to vectors containing viral LTR elements.

915. Insertional Analyses in Rhesus Monkey Blood Cells after Non-Myeloablative Hematopoietic Stem Cell Marking with a Therapeutic Onco-Retroviral Vector for X-Linked Chronic Granulomatous Disease

Wild-type murine onco-retroviruses cause cancer in part by insertional mutagenesis, but until recently, cancers had not been observed in humans or animals subjected to marking or treatment with the highly modified murine retrovirus vectors in current use. Recently, mutagenesis activation of the LMO-2 gene was observed to be associated with the development of lymphoid leukemia several years after two infants with X-SCID had their immune systems restored by gene therapy with a therapeutic onco-retrovirus vector. Insertional mutagenesis activation of other oncogenes associated with development of cancers has been reported in vector-marked mice. These observations highlight the importance of encouraging many investigators to conduct large animal marking studies which follow the insertional pattern of vectors planned for human treatments. We have recently reported that a very high titer RD114 pseudotyped MFGS-gp91phox vector achieves unprecedented levels of correction of the oxidase defect in CD34+ cells from patients with X-linked chronic granulomatous disease in the NOD/SCID mouse model. In anticipation of considering clinical use of this vector, we transduced mobilized peripheral blood CD34+ cells (PBSC CD34+) of two healthy rhesus macaques with the RD114-MFGS-gp91phox. Transduced PBSC CD34+ with an average two vector copies per cell were transplanted on culture day 4 into two non-myeloablative (2× 300 Rads) irradiated animals. The moderately high levels of in vivo marking (>6%) seen in the first months following gene marking decreased and stabilized at about 8 months post transplant. Marking for animal J976 and D406 at 15 and 19 months, respectively is 1.3% and 0.3% in B lymphocytes, 0.3% and 0.3% in neutrophils and 0.7% and 0.4% in T lymphocytes at which time the animals remained healthy. Vector insertion analysis was performed by LAM-PCR, using gel electrophoresis and GeneScan analysis to demonstrate that despite the relative low marking at 10 months and later animal D406 had more than 25 and animal J976 had more than 40 separate insertional events. 34% of integration sites are located in coding regions. No predominant clone was seen to emerge over time and no patterns of insertion site preferences were detected up to this point.

5. Evaluation of the Genotoxic Potential of a Self-Inactivating γ-Globin Lentiviral Vector

The development of lymphoid leukemia in two children with X-SCID who underwent gene therapy appears to be partially due to activation of the LMO-2 proto-oncogene by the retroviral vector LTR (Hacein-Bey-Abina et al., Science 302:415, 2003), underscoring the importance of vector design on the potential to activate genes near integration sites. In order to determine whether integrated, self-inactivating (SIN) globin lentiviral vectors which lack a viral LTR also have the potential to cause transcriptional activation, we initially compared the mobilization rate of an integrated SIN globin lentiviral vector containing β-globin locus enhancer elements with that of a SIN lentiviral vector containing the MSCV U3 LTR region. 293T cells were transduced to a high vector copy number with the SIN globin vector or a SIN lentiviral vector containing the LTR element, followed by transfection of viral packaging genes to rescue aberrantly transcribed vector genomes. Although both vectors had a comparable transducing titer (globin 2 × 107 ± 4 × 106 TU/ml, n=4 vs. MSCV 4 × 107 ± 2 × 106 TU/ml, n=9), the globin vector had a “mobilized” titer 60-fold less than that of the MSCV LTR vector (p<0.0001). We estimate that 1 in 300,000 globin vector integration events were mobilized, compared to a frequency of 1 in 12,000 with the MSCV vector, consistent with the possibility of less transcriptional activation attributable to the globin vector. To ascertain whether an integrated globin vector could influence endogenous transcriptional activity in red cell presursors, clonal spleen colony erythroblasts containing a single globin vector insertion were derived from transduced bone marrow cells of β-thalassemic mice. The transcriptional profile of 21 single-copy globin vector clones and 15 untransduced, control clones was determined using the Affymetrix Mouse 430A microarray (representing ~15,000 genes). Expression of 4500–6000 genes was observed in all samples. Using a 2-fold difference in the level of expression as a cutoff, only 0.02% of all the genes among the 21 clones had potentially different expression compared to the control clones, well below the expected 2% rate of random differences (Genomics 83:321, 2004). Ligation-mediated PCR was then used to clone the sequences of the vector-genomic DNA junctions, allowing identification of vector insertion locations using the NCBI database. Six clones had 16 genes, including N-ras, that were located within 100kb of the vector insertion site and were represented on the array. Of those genes that were expressed, only 2 had a 2.0–2.8-fold difference in signal value between the clone and the controls. These differences were unlikely to be real due to a high false discovery rate in those clones. Evaluation of these potential changes by real time RT-PCR is underway. Both microarray and real time RT-PCR demonstrated that expression of N-ras was unchanged. These data suggest that integrated SIN globin lentiviral vectors have a low rate of altering transcriptional activity in target cells and therefore may be inherently less genotoxic than vectors containing a viral LTR.

Expression of c-abl in Philadelphia-Positive Acute Myelogenous Leukemia

Abstract The identical cytogenetic marker, t(9;22)(q34;q11) (Philadelphia [Ph] translocation), is found in approximately 90%, 20%, and 2% of adult patients with chronic myelogenous leukemia (CML), acute lymphoblastic leukemia (ALL), and acute myelogenous leukemia (AML), respectively. In CML, the molecular events resulting from the Ph translocation include a break within the bcr locus on chromosome 22, transfer of the c-abl protooncogene from chromosome 9 to 22, and formation of an aberrant 210- kD bcr-abl fusion protein (p210bcr-abl). Recently, the absence of bcr rearrangement and expression of a distinct aberrant 190-kd abl protein (p190c-abl) has been described in Ph-positive ALL, with the suggestion that the two abl variants may be pathogenetically associated with myeloid v lymphoid leukemogenesis. Here we report that the genomic configuration and translation product of Ph-positive AML can be similar to that of Ph-positive ALL: the break at 22q11 may occur outside the 5.8 kb bcr region and result in expression of a 190-kD abl protein lacking these bcr sequences. Phosphokinase enzymatic activity, a fundamental property of p210bcr-abl, was also associated with AML- derived p190c-abl. Our current observations indicate that p190c-abl can be found in cells of lymphoid or myeloid lineage and is therefore unlikely to play a specific role in the development of lymphoid leukemias. Formation of p190c-abl instead of p210bcr-abl appears to be a characteristic of the acute rather than the chronic Ph-positive leukemic state.

Expression of c-abl in Philadelphia-positive acute myelogenous leukemia

The identical cytogenetic marker, t(9;22)(q34;q11) (Philadelphia [Ph] translocation), is found in approximately 90%, 20%, and 2% of adult patients with chronic myelogenous leukemia (CML), acute lymphoblastic leukemia (ALL), and acute myelogenous leukemia (AML), respectively. In CML, the molecular events resulting from the Ph translocation include a break within the bcr locus on chromosome 22, transfer of the c-abl protooncogene from chromosome 9 to 22, and formation of an aberrant 210-kD bcr-abl fusion protein (p210bcr-abl). Recently, the absence of bcr rearrangement and expression of a distinct aberrant 190-kd abl protein (p190c-abl) has been described in Ph-positive ALL, with the suggestion that the two abl variants may be pathogenetically associated with myeloid v lymphoid leukemogenesis. Here we report that the genomic configuration and translation product of Ph-positive AML can be similar to that of Ph-positive ALL: the break at 22q11 may occur outside the 5.8 kb bcr region and result in expression of a 190-kD abl protein lacking these bcr sequences. Phosphokinase enzymatic activity, a fundamental property of p210bcr-abl, was also associated with AML-derived p190c-abl. Our current observations indicate that p190c-abl can be found in cells of lymphoid or myeloid lineage and is therefore unlikely to play a specific role in the development of lymphoid leukemias. Formation of p190c-abl instead of p210bcr-abl appears to be a characteristic of the acute rather than the chronic Ph-positive leukemic state.