Fusion Sequence Research Articles

345. Sensitive Molecular Detection of Various Stable Episomal Concatemeric and Integrated Forms of rAAV2 after Long-Term Expression In Vivo

rAAV vectors can lead to the long term expression of their double stranded DNA transgenes and are considered a promising vector for gene therapy. The recombinant adeno-associated virus serotype 2 (rAAV2) has been demonstrated to confer long-term gene expression as well as a lack of pathogenicity and toxicity. Successful gene transfer and expression of human coagulation factor IX were shown in hemophilia B patients after muscle-directed gene transfer of an AAV2 vector. Trials are ongoing for muscular dystrophy and cystic fibrosis. AAV has been known for a long time to persist in the form of episomal concatemers (circular and linear). The possibility of in vivo concatemerization of rAAV genomes may have interesting consequences and applications. Therapeutic vector designs with split expression cassettes across two separate vectors have been described, which expressed the combined gene product after concatemerization in the cell nucleus (Nakai H et al. Nat. Biotechnol. 2000, Duan D et al. Nat. Med. 2000, Sun L et al. Nat. Med. 2000). The specific relations and interactions leading to concatimerization are not completely understood. The existence of episomal concatemer forms has so far also hampered precise AAV- integration analysis. To detect existing concatemeric and integrated forms, we have used a combination of a Multiplex- PCR and LAM-PCR for the analysis of rAAV2 transduced long-term cell lines and long-term in vivo murine tissue samples. Interestingly, in an assay set up to detect all possible permutations of head-to-head, head-to-tail and tail-to-tail concatemeric sequences in a cell line, we found a defined number and configuration of partial deletions of 5'-, 3'- ITRs, complete deletions of both ITRs and additional deletions of the ori- region of the transgene, presumably in episomal forms of the vector. We detected more than 20 additional variant amplicons of rAAV2 up to 15 month after systemic in vivo delivery into liver and muscle tissue of mice. No complete HT-, TT-, or HT form could be observed at any time point after injection. In the presence of this defined abundance of concatemeric forms, a high resolution integration site analysis by LAM- PCR of AAV2 transduced murine liver and muscle tissue could both confirm the presence of more than 20 similar concatemeric forms as detected by Multiplex- PCR, and furthermore identify 9 genomically integrated forms with partial vector ITR deletions and exact mapping to the murine genome. We are currently analyzing how completely all present sequences were represented by this analysis. We conclude that highly sensitive LAM PCR for the detection of AAV integration can be performed successfully on in vitro and in vivo samples to detect concatemeric and genomic rAAV fusion sequences. The combination of Multiplex- and LAM- PCR can provide essential precise information on the molecular status of AAV vectors, their insertions and deletions in target cells of gene therapy.

708. Retroviral Vector Integration into the Genome of Rhesus Macaque Long-Term Repopulating Cells

Replication defective retroviruses are very attractive tools for hematopoietic stem cell (HSC) gene therapy largely because of their ability to integrate into genomic DNA. While the risk of insertional mutagenesis has long been recognized, reports of neoplasia consecutive to proto-oncogenes' activation in mice and humans have refocused attention on the safety limitations of these vectors. Mapping studies of retroviral integration sites (RIS) in human cell lines have suggested that retroviral vectors favor integration near transcriptional start sites. Medical applications using these vectors are aimed at HSC, and thus large-scale analysis of RIS in a relevant pre-clinical situation is essential to assess the safety of HSC gene therapy. Here we report the mapping of 423 RIS in a long-term model of autologous retrovirally-transduced HSC transplantation in the non-human primate (Macaca mulatta). Circulating granulocytes and mononuclear cells from 22 rhesus macaques, transplanted with G-CSF + SCF mobilized CD34+ peripheral blood stem cells transduced with an amphotropic Moloney murine leukemia-derived vector containing a neomycin-resistance marker gene, have been analyzed between one and five years after transplantation. Genomic regions adjacent to RIS have been retrieved using linear amplification-mediated PCR followed by shotgun TA cloning, and mapped to the NCBI build 34 of the human genome sequence. Out of the 423 RIS retrieved, 37% landed between the transcriptional start and stop codons of a member of the well-defined RefSeq set of genes, 55% were located in non-coding regions of the genome and 8% were found to be in interspersed repeats and therefore not mappable. Analysis of the targeted genomic regions provides evidence for non-random insertion: 8 independent RIS, recovered from 6 animals, have been mapped within the 2 first introns of the myelodysplasia syndrome 1 gene (MDS1), and seven other genes have 2 independent insertions. Among these 8 genes, it is noteworthy that three are known to be involved in chromosomal rearrangements observed in spontaneous acute human leukemias: the proto-oncogene runt-related transcription factor 1 (RUNX1), the fms-related tyrosine kinase 3 (FLT3) and MDS1. The presence of the fusion sequence between the genomic MDS1 region and the 5'-LTR of the vector has been confirmed using insertion-specific primers for 6 out of the 8 events, and clonal evolution as well as expression of the targeted gene are being analyzed. These observations suggest that retroviral integration in long-term repopulating HSC is highly non-random and is favored at specific loci. Systematic mapping of RIS after HSC gene transfer in the non-human primate will provide critical pre-clinical data necessary for risk assessment of such vectors, and offers also a powerful means for identifying genes involved in the biology of HSC.

Effective primary isolation of wild-type Canine distemper virus in MDCK, MV1 Lu and Vero cells without nucleotide sequence changes within the entire haemagglutinin protein gene and in subgenomic sections of the fusion and phospho protein genes

Canine distemper virus (CDV) is an important pathogen of many carnivores. We are developing a field-based model of morbillivirus virulence and pathogenesis through a study of distemper in naturally infected free-ranging raccoons. The isolation of CDV from raccoon tissues is essential for this work. CDV has often been isolated from animals only after co-cultivation of infected tissues with peripheral blood mononuclear cells derived from specific pathogen-free dogs or similar methods. We explored the utility and consequences of a simpler and cheaper alternative: CDV isolation in Vero, MDCK, and MV1 Lu cells. Virus growth was detected first in MDCK cells, whereas viral cytopathic effects were most obvious in Vero cells. CDV growth in MV1 Lu cells was relatively protracted and occurred without the formation of cytopathic effects. In primary CDV isolates, the entire nucleotide sequence of the receptorbinding haemagglutinin ( H) gene, and subgenomic fusion ( F) and phospho ( P) protein gene sequences corresponding to nt 5399–5733 and 2132–2563 of CDV reference strain Onderstepoort, respectively, were identical to those in matched infected tissues. Virus isolation confirmed the presence of CDV in instances where RT-PCR failed to detect CDV in infected tissues. Different viral phenotypes and genotypes were detected. The conservation of H gene sequences in primary CDV isolates suggests that MDCK, MV1 Lu, and Vero cells express proper receptors for wild-type CDV.

Divergence of Genbank and human tumor Bcl-2 sequences and implications for binding affinity to key apoptotic proteins

Heterodimerization of antiapoptotic and pro-apoptotic Bcl-2 family of proteins provides an important mechanism for apoptosis regulation. Knowledge about key amino acids in the binding groove of native Bcl-2 contributing to this interaction will greatly facilitate the design of Bcl-2-specific inhibitors. There are two different Bcl-2 sequences, M13994 and M14745, in Genbank. Chimeric proteins Bcl-2(1) and Bcl-2(2) derived from the above sequences, although similar in structure, showed different binding affinities to Bak and Bad BH3 peptides (Petros et al., 2001). In this study, we show that the Bcl-2(1) sequence in normal and tumor human tissue samples differs from M13994 and M14745, and contains P59, T96, R110, S117 and G237. The actual sequence in the binding pocket matches the Bcl-2-Ig fusion sequence X06487, originally identified in a t(14:18) translocation of the Bcl-2 gene, associated with follicular lymphoma. The possible effects of the observed amino acid differences compared to M13994 and M14745 were investigated by combining structural data with fluorescence anisotropy. G110R substitution confers on Bcl-2(1) substantially increased binding affinity to Bak, Bad and Bax BH3 peptides, demonstrating that R110 is a key contributor to the BH3 binding affinity of Bcl-2. Although NMR structure did not predict R110 involvement in binding to these BH3 peptides, fluorescence anisotropy data clearly points to a critical role for this residue in binding to pro-apoptotic Bcl-2 family members.

Autosomal rearrangements in Graomys griseoflavus (Rodentia): a model of non-random Robertsonian divergence.

The south American rodent Graomys griseoflavus exhibits a remarkable chromosome polymorphism as a consequence of four Robertsonian fusions. Focusing on the genetic analysis of the taxon, genome organization of all karyomorphs was studied at chromosome and molecular organization level. Cytogenetic (G, NOR and Re banding) and molecular (satellite and mitochondrial DNAs) events accompanying chromosome divergence allowed tracing a phylogenetic relationship among all karyomorphs. Available data led to propose that chromosome evolution of G. griseoflavus occurred in a non-random sequence of centric fusions, supporting the hypothesis of single origin for Robertsonian karyomorphs.

The Prenatal Origin of Childhood Acute Lymphoblastic Leukemia

Until recently, the etiology of childhood acute lymphoblastic leukemia (ALL) has remained relatively elusive. Several studies have established a time frame for the development of ALL which could lead to the identification of specific exposures linked to leukemogenesis from the generation of the initial leukemic clone until clinical diagnosis. Utilizing newborn screening (‘Guthrie’) cards, leukemic clones have been detected retrospectively in dried blood spots using two different PCR-based approaches: (i) the amplification of patient/leukemia-specific breakpoint fusion sequences of rearranged oncogenes; and (ii) the amplification of clonal immunoglobulin heavy chain gene (IgH) or T cell receptor (TcR) gene rearrangements. These studies support the hypothesis that a large proportion of childhood ALL cases arise in utero. In several studies, a long latency period from the generation in utero of the initial ALL clone to clinical diagnosis, indicates that additional genetic events are required for the full development of the leukemia phenotype, potentially from postnatal exposures (e.g. infections). The identification of leukemia-associated translocations in umbilical cord blood samples of healthy newborns, suggest that in the future children may be identified prospectively who have an increased risk of developing leukemia.

TaqMan RT-PCR assay coupled with capillary electrophoresis for quantification and identification of bcr-abl transcript type

Chronic myelogenous leukemia is characterized by the presence of the reciprocal t(9;22)(q34;q11) in which c-abl located on chromosome 9, and the bcr locus located on chromosome 22, are disrupted and translocated creating a novel bcr-abl fusion gene residing on the derivative chromosome 22. In most cases, the breakpoint in abl occurs within intron 1. Depending on the breakpoint in bcr, exon 2 of abl (a2) joins with exons 1 (e1), 13 (b2), or 14 (b3), or rarely to exon 19 (e19) of bcr resulting in chimeric proteins of p190, p210 and p230, respectively. Currently, several multiplex real-time reverse transcriptase-polymerase chain reaction (RT-PCR)-based assays for detecting bcr-abl are available to assess the levels of the three common fusion transcripts, b2a2, b3a2 and e1a2. Although these assays circumvent the requirement for individual fusion sequence quantitative polymerase chain reaction-based assays, they do not identify the specific fusion transcript. Knowledge of the latter is useful to rule out false-positive results and to compare clones before and after therapy. We designed a novel multiplex real-time RT-PCR assay to detect bcr-abl that allows accurate quantification and determination of the specific fusion transcript. In this assay, abl primer labeled at its 5′ end with the fluorescent dye NED (Applied Biosystems) is incorporated into the bcr-abl fusion product during amplification. The NED fluorescent dye in abl primer, without interfering with fluorescent TaqMan probe signal, allows subsequent identification of the fusion transcript by semiautomated high-resolution capillary electrophoresis and GeneScan analysis.

Metopic synostosis: Defining the temporal sequence of normal suture fusion and differentiating it from synostosis on the basis of computed tomography images.

Only the metopic suture normally fuses during early childhood; all other cranial sutures normally fuse much later in life. Despite this, metopic synostosis is one of the least common forms of craniosynostosis. The temporal sequence of normal physiologic metopic suture fusion remains undefined and controversial. Therefore, diagnosis of metopic synostosis on the basis of computed tomography images alone can prove misleading. The present study sought to determine the normal sequence of metopic suture fusion and characterize both endocranial and ectocranial suture morphology. An analysis of computed tomography scans of 76 trauma patients, ranging in age from 10 days to 18 months, provided normative craniofacial data that could be compared to similar data obtained from the preoperative computed tomography scans of 30 patients who had undergone surgical treatment for metopic synostosis. Metopic suture fusion was complete by 6 to 8 months in all nonsynostotic patients, with initiation of suture fusion evident as early as 3 months of age. Fusion was found to commence at the nasion, proceed superiorly in progressive fashion, and conclude at the anterior fontanelle. Although an endocranial ridge was not commonly seen in synostotic patients, an endocranial metopic notch was virtually diagnostic of premature suture fusion and was seen in 93 percent of synostotic patients. A metopic notch was not seen in any nonsynostotic patient. The morphologic and normative craniofacial data presented permit diagnosis of metopic synostosis based on computed tomography images obtained beyond the normal fusion period.

BCL-2 is consistently expressed in hyperplastic marginal zones of the spleen, abdominal lymph nodes, and ileal lymphoid tissue.

BCL-2 is an antiapoptotic protein overexpressed in follicular lymphomas, principally as a result of the t(14;18)(q32;q21), and useful in distinguishing follicular lymphoma (usually BCL-2 positive) from follicular hyperplasia (BCL-2 negative). BCL-2 is also overexpressed in other lymphoma types without the t(14;18), including marginal zone B-cell lymphoma, because of other, poorly understood mechanisms. It has been suggested that BCL-2 immunoreactivity can distinguish between malignant (BCL-2 positive) and reactive (BCL-2 negative) marginal zone B cells. In this study, we evaluated 26 spleen, 10 abdominal lymph node, and 3 ileum specimens with marginal zone B-cell hyperplasia for BCL-2 expression immunohistochemically. We also analyzed these cases using polymerase chain reaction methods to evaluate for the presence of clonal rearrangements of the immunoglobulin heavy chain gene (IgH) using consensus V FRIII and J region primers, and the t(14;18) involving both the major breakpoint and the minor cluster regions of the bcl-2 gene. All (100%) cases of splenic, abdominal lymph node, and ileal marginal zone hyperplasia displayed strong BCL-2 reactivity in the marginal zone B cells. In all cases analyzed, IgH polymerase chain reaction demonstrated a polyclonal pattern, and bcl-2/JH DNA fusion sequences were not detected. Our results indicate that BCL-2 is consistently expressed by reactive marginal zone B cells of the spleen, abdominal lymph nodes, and ileal lymphoid tissue and should not be used as a criterion for discriminating between benign and malignant marginal zone B-cell proliferations involving these sites.

Phagosome maturation: a few bugs in the system.

Cells of the innate immune system ingest and destroy invading microorganisms by initially engulfing them into a specialized vacuole, known as the phagosome. The membrane of the forming phagosome is similar to the plasmalemma and its contents resemble the extracellular milieu. As such, the nascent phagosome is not competent to kill and eliminate the ingested microorganisms. However, shortly after sealing, the phagosome undergoes a series of rapid and extensive changes in its composition, the result of a sophisticated sequence of membrane fusion and fission reactions. Understanding the molecular basis of these events is of particular importance, since they are often the target of disruption by intracellular parasites such as Mycobacterium, Salmonella and Legionella. The objective of this review is to summarize the current knowledge of the molecular mechanisms underlying phagosomal maturation and its subversion by parasitic microorganisms.

Determination of minimal residual disease in leukaemia patients.

Determination of minimal residual disease in leukaemia patients.

Characteristic sequence motifs located at the genomic breakpoints of the translocation t(X;18) in synovial sarcomas.

The SYT-SSXI and SYT-SSX2 fusion genes, derived by reciprocal translocations t(X;18), are acquired genetic events strongly associated with the tumorigenesis of synovial sarcoma. In approaching the mechanisms underlying the formation of these fusion oncogenes, we have analysed the genomic sequences surrounding the SYT-SSX breakpoints in 10 tumors, two expressing SYT-SSXI and eight expressing SYT-SSX2 fusion transcripts. The breakpoints were found to be clustered in the 5' end of intron 10 of SYT, and in two cluster regions within intron 4 of SSX2, whereas the two breakpoints in SSX1 intron 4 were 0.5 kb apart. SYT intron 10 is abundant in repetitive regions with the interspersed repeats occupying 66% of the whole intron. Nine of the 10 breakpoints in intron 10 of SYT and six of the eight breakpoints in intron 4 of SSX2 were at or near repetitive regions. These findings suggest that repetitive regions may contribute to the distribution of genomic breakpoints. Several of the fusion sequences exhibited characteristic signs of nonhomologous end joining, including microhomologies at the end points as well as deletions. Sequences highly homologous (83-94%) to consensus topoisomerase II cleavage sites were identified at or near the breakpoints in all 10 tumors, suggesting a role of this enzyme in creating staggered ends at the breakpoint. Furthermore, sequences highly homologous to consensus Translin binding sequences were found at the breakpoints in two cases, and an Alu-Alu fusion and an insertion of a 206-bp LINE-1 element were found at the breakpoint in one case each. The demonstration of characteristic sequences at the SYT-SSX breakpoint regions is expected to improve our understanding of the molecular genetic mechanisms behind translocations in general, and of the SYT-SSX fusions in synovial sarcoma in particular.

Prenatal origin of TEL-AML1-positive acute lymphoblastic leukemia in children born in California.

Acute lymphoblastic leukemia (ALL) is the most common form of childhood cancer. The peak incidence of ALL between ages 2 and 5 is accounted for by one subtype, referred to as common acute lymphoblastic leukemia (cALL). About 25% of cALL patients have the TEL-AML1 gene fusion derived from the t(12;21) chromosomal translocation. Recent evidence from retrospective analysis of neonatal blood spots (Guthrie cards) in Europe has demonstrated that this chromosome translocation may arise prenatally. The aim of our study was to determine whether TEL-AML1 fusions arise prenatally in a U.S. population of cALL patients. TEL-AML1-positive cALL cases (n = 14) were identified by fluorescence in situ hybridization, and the genomic breakpoints were identified by a streamlined long-distance PCR approach and sequenced. Clonotypic primers were designed for each patient breakpoint, and a nested PCR assay was used to determine the presence of the TEL-AML1 fusion sequence in neonatal Guthrie cards. Seven of 14 cases demonstrated clonotypic sequences on the archival Guthrie cards. The oldest patient that was positive was 6.7 years old at the time of diagnosis of leukemia. These results confirm previously published findings of a prenatal origin of TEL-AML1 in Europe by demonstrating its occurrence in a California-born population. Secondary changes were also similar to those described previously, with deletion of the second TEL allele being the most common. Other secondary changes included duplication of the fusion gene, trisomy 21, and monosomy X.

Sex- and age-related variations in cranial measurements and suture closure in the Australian sea lion, Neophoca cinerea (Peron, 1816)

A total of 65 skulls of the Australian sealion, Neophoca cinerea, was examined to investigate the extent to which sexual dimorphism is reflected in cranial dimensions (n = 32) and skull growth, and to determine whether cranial sutures (n = 18) can be useful in age determination. All adult skull dimensions studied display significant sexual dimorphism. Skull growth ceases close to 4–7 years of age for females (Suture Fusion Rating, SFR 25–34) but skull growth in males continues until at least 16 years of age. In animals with a SFR ≥� 25, male skulls have a minimum condylobasal length of 272 mm, whereas female skulls have a maximum condylobasal length of 259 mm. The relatively early closure of the cranial vault sutures (cessation of brain growth) is balanced by the continued growth of the bony projections that provide muscle attachment (e.g. mastoid width). The later fusion of the snout and palate sutures corresponds with the continued growth of the snout and palate to match the prolonged growth of the mandibles. The upper sixth postcanine tooth was present in 43% of the adult female skulls, but only 15% of the adult male skulls. The data suggest that it may be possible to determine age(s) from examination of the sequence of fusion of cranial sutures as well as by calculation of an overall suture fusion rating for the skull.

Adeno-associated virus integrates site-specifically into human chromosome 19 in either orientation and with equal kinetics and frequency.

Adeno-associated virus type 2 (AAV-2) establishes latency by site-specific integration into a unique locus, AAVS1, on human chromosome 19 (chr19). To study the kinetics and frequency of chr19-specific integration, a rapid, sensitive and quantitative real-time PCR assay specific for AAV inverted terminal repeat (ITR)-chr19 junction sequences was developed. Since the assay only detected right-hand AAV ITR-specific integration events, the development of a complementary left-hand ITR-specific real-time PCR assay is described. The time-course of left-hand ITR-dependent AAV integration at AAVS1 of chr19 was determined in AAV-2-infected HeLa cells. Both the kinetics and frequencies of left-hand ITR-dependent integration were found to be similar to those of the right-hand ITR. In addition, left-hand ITR-specific fusion sequences and chromosomal breakpoints within AAVS1 were variable, yet were the same as those found in right-hand ITR-chr19 junction sequences. Thus, the AAV-2 genome integrates site-specifically into chr19 with similar efficiency in either orientation.

The Influence of Fusion Sequences on the Thermal Stabilities of Coiled-Coil Proteins

Five coiled-coil-containing proteins were synthesized to evaluate the influence of fusion sequences on their thermal stability. All proteins contained the same coiled-coil domain (VSSLESK)6, but differed in the fusion sequence attached. Circular dichroism spectroscopy was used to characterize their secondary structure and thermal stability. The proteins formed a coiled-coil structure, but their melting temperatures were different. The difference in the melting temperatures was not caused by either protein concentration or trace nickel ions. Sedimentation equilibrium experiments suggested an effect of the fusion sequence on the oligomerization state, but did not show a direct relationship between the oligomerization state and the melting temperature. CD spectrum of the coiled-coil-containing proteins (10 × 10−6 M) in 10 × 10−3 M PBS buffer at 25 °C.

Quantification of bcl-2/JH Fusion Sequences and a Control Gene by Multiplex Real-Time PCR Coupled with Automated Amplicon Sizing by Capillary Electrophoresis

Follicular lymphoma is characterized by the presence of the t(14;18)(q32;q21) chromosomal translocation which juxtaposes the bcl-2 gene at 18q21 with the immunoglobulin heavy chain locus at 14q32. Quantification of t(14;18) carrying cells in FL patients can be achieved by real-time PCR, a highly sensitive technique for evaluating treatment efficacy and minimal residual disease. Despite the many advantages of real-time technology for this purpose, one disadvantage is that current real-time t(14;18) PCR assays amplify a control gene as a normalizer in a separate reaction. Since each PCR reaction has its own kinetics, separate PCR assays for target and control sequences can potentially result in inaccurate quantification of t(14;18)-positive cells. In addition, the real-time t(14;18) PCR assays do not determine the size of the amplified fusion sequence, which is helpful for excluding contamination and is commonly used to demonstrate clonal identity between pre- and post-treatment specimens from a patient. To address these limitations, we designed a multiplex real-time PCR protocol that allows amplification of control and target genes in the same reaction and precise size determination of bcl-2/JH fusion sequences by capillary electrophoresis. This multiplex PCR assay is equally sensitive to previous assays, allows more accurate quantification of bcl-2/JH fusion sequences, and is more convenient.

Amelioration of the macrothrombocytopenia associated with the murine Bernard-Soulier syndrome

AbstractAn absent platelet glycoprotein (GP) Ib-IX receptor results in the Bernard-Soulier syndrome and is characterized by severe bleeding and the laboratory presentation of macrothrombocytopenia. Although the macrothrombocytopenic phenotype is directly linked to an absent GP Ib-IX complex, the disrupted molecular mechanisms that produce the macrothrombocytopenia are unknown. We have utilized a mouse model of the Bernard-Soulier syndrome to engineer platelets expressing an α-subunit of GP Ib (GP Ibα) in which most of the extracytoplasmic sequence has been replaced by an isolated domain of the α-subunit of the human interleukin-4 receptor (IL-4Rα). The IL-4Rα/GP Ibα fusion is membrane expressed in Chinese hamster ovary (CHO) cells, and its expression is facilitated by the presence of human GP IX and the β-subunit of GP Ib. Transgenic animals expressing a chimeric receptor were generated and bred into the murine Bernard-Soulier syndrome–producing animals devoid of mouse GP Ibα but expressing the IL-4Rα/GP Ibα fusion sequence. The characterization of these mice revealed a 2-fold increase in circulating platelet count and a 50% reduction in platelet size when compared with platelets from the mouse model of the Bernard-Soulier syndrome. Immunoprecipitation confirmed that the IL-4Rα/GP Ibα subunit interacts with filamin-1 and 14-3-3ζ, known binding proteins to the GP Ibα cytoplasmic tail. Mice expressing the chimeric receptor retain a severe bleeding phenotype, confirming a critical role for the GP Ibα extracytoplasmic domain in hemostasis. These results provide in vivo insights into the structural elements of the GP Ibα subunit that contribute to normal megakaryocyte maturation and thrombopoiesis.

Insight into the potential for DNA idiotypic fusion vaccines designed for patients by analysing xenogeneic anti-idiotypic antibody responses.

DNA vaccines induce immune responses against encoded proteins, and have clear potential for cancer vaccines. For B-cell tumours, idiotypic (Id) immunoglobulin encoded by the variable region genes provides a target antigen. When assembled as single chain Fv (scFv), and fused to an immunoenhancing sequence from tetanus toxin (TT), DNA fusion vaccines induce anti-Id antibodies. In lymphoma models, these antibodies have a critical role in mediating protection. For application to patients with lymphoma, two questions arise: first, whether pre-existing antibody against TT affects induction of anti-scFv antibodies; second, whether individual human scFv fusion sequences are able to fold consistently to generate antibodies able to recognize private conformational Id determinants expressed by tumour cells. Using xenogeneic vaccination with scFv sequences from four patients, we have shown that pre-existing anti-TT immunity slows, but does not prevent, anti-Id antibody responses. To determine folding, we have monitored the ability of nine DNAscFv-FrC patients' vaccines to induce xenogeneic anti-Id antibodies. Antibodies were induced in all cases, and were strikingly specific for each patient's immunoglobulin with little cross-reactivity between patients, even when similar VH or VL genes were involved. Blocking experiments with human serum confirmed reactivity against private determinants in 26-97% of total antibody. Both immunoglobulin G1 (IgG1) and IgG2a subclasses were present at 1.3 : 1-15 : 1 consistent with a T helper 2-dominated response. Xenogeneic vaccination provides a simple route for testing individual patients' DNAscFv-FrC fusion vaccines, and offers a strategy for production of anti-Id antibodies. The findings underpin the approach of DNA idiotypic fusion vaccination for patients with B-cell tumours.

In utero origin of t(8;21) AML1-ETO translocations in childhood acute myeloid leukemia

Recent reports have established the prenatal origin of leukemia translocations and resultant fusion genes in some patients, including MLL-AF4 translocations in infants and TEL-AML1 translocations in children. We now report evidence for the prenatal origin of a translocation in childhood acute myeloid leukemia (AML). The t(8;21) AML1-ETO translocations were sequenced at the genomic level in 10 diagnostic leukemia samples from children with available neonatal Guthrie blood spots. Clonotypic genomic AML1-ETO sequences were detected in the Guthrie spots for 5 individuals, providing unambiguous evidence of prenatal origin in these cases. Two of these patients were older than 10 years of age at diagnosis, indicative of a protracted postnatal latency. Three of the patients were assessed for the persistence of genomic fusion sequences in complete clinical remission samples and were found to be positive. These data indicate that t(8;21) in childhood AML can arise in utero, possibly as an initiating event in childhood AML, and may establish a long-lived or stable parental clone that requires additional secondary genetic alterations to cause leukemia.