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Haig H Kazazian

Publications and source records attributed to Haig H Kazazian.

At least 19 recordsLinked to original sources
PubMed2006

L1 retrotransposition is suppressed by endogenously encoded small interfering RNAs in human cultured cells.

LINE-1s, or L1s, are highly abundant retrotransposons comprising 17% of the human genome. Most L1s are retrotransposition defective; nonetheless, there are approximately 100 full-length L1s potentially capable of retrotransposition in the diploid genome. L1 retrotransposition may be detrimental to the host and thus needs to be controlled. Previous studies have identified sense and antisense promoters in the 5' UTR of full-length human L1. Here we show that the resulting bidirectional transcripts can be processed to small interfering RNAs (siRNAs) that suppress retrotransposition by an RNA interference (RNAi) mechanism. We thus provide evidence that RNAi triggered by antisense transcripts may modulate human L1 retrotransposition efficiently and economically. L1-specific siRNAs are among the first natural siRNAs reported in mammalian systems. This work may contribute to understanding the regulatory role of abundant antisense transcripts in eukaryotic genomes.

5' Untranslated Regions↗
PubMed2006

Propionyl-CoA and adenosylcobalamin metabolism in Caenorhabditis elegans: evidence for a role of methylmalonyl-CoA epimerase in intermediary metabolism.

We have utilized Caenorhabditis elegans to study human methylmalonic acidemia. Using bioinformatics, a full complement of mammalian homologues for the conversion of propionyl-CoA to succinyl-CoA in the genome of C. elegans, including propionyl-CoA carboxylase subunits A and B (pcca-1, pccb-1), methylmalonic acidemia cobalamin A complementation group (mmaa-1), co(I)balamin adenosyltransferase (mmab-1), MMACHC (cblc-1), methylmalonyl-CoA epimerase (mce-1) and methylmalonyl-CoA mutase (mmcm-1) were identified. To verify predictions that the entire intracellular adenosylcobalamin metabolic pathway existed and was functional, the kinetic properties of the C. elegans mmcm-1 were examined. RNA interference against mmcm-1, mmab-1, mmaa-1 in the presence of propionic acid revealed a chemical phenotype of increased methylmalonic acid; deletion mutants of mmcm-1, mmab-1 and mce-1 displayed reduced 1-[(14)C]-propionate incorporation into macromolecules. The mutants produced increased amounts of methylmalonic acid in the culture medium, proving that a functional block in the pathway caused metabolite accumulation. Lentiviral delivery of the C. elegans mmcm-1 into fibroblasts derived from a patient with mut(o) class methylmalonic acidemia could partially restore propionate flux. The C. elegans mce-1 deletion mutant demonstrates for the first time that a lesion at the epimerase step of methylmalonyl-CoA metabolism can functionally impair flux through the methylmalonyl-CoA mutase pathway and suggests that malfunction of MCEE may cause methylmalonic acidemia in humans. The C. elegans system we describe represents the first lower metazoan model organism of mammalian propionate spectrum disorders and demonstrates that mass spectrometry can be employed to study a small molecule chemical phenotype in C. elegans RNAi and deletion mutants.

Acidosis↗
PubMed2006

L1 retrotransposition in nondividing and primary human somatic cells.

Whether long interspersed element-1 (L1 or LINE-1) retrotransposition can occur in quiescent, nondividing, and/or terminally differentiated somatic cells has remained an unanswered fundamental question in human genetics. Here, we used a ubiquitously active phosphoglycerate kinase-1 promoter to drive the expression of a highly active human L1 element from an adenovirus-L1 hybrid vector. This vector system achieved retrotransposition in up to 91% of actively growing immortalized cells, and we demonstrated that L1 retrotransposition can be suppressed by the reverse transcriptase inhibitor 3'-azido-3'-deoxythymidine. This adenovirus vector enabled efficient delivery of the L1 element into differentiated primary human somatic cells and G1/S-arrested cells, resulting in retrotransposition in both cases; however, it was not detected in G0-arrested cells. Thus, these data indicate that L1 retrotransposition can occur in nondividing somatic cells.

Adenoviridae↗
PubMed2006

Extensive individual variation in L1 retrotransposition capability contributes to human genetic diversity.

Despite being scarce in the human genome, active L1 retrotransposons continue to play a significant role in its evolution. Because of their recent expansion, many L1s are not fixed in humans, and, when present, their mobilization potential can vary among individuals. Previously, we showed that the great majority of retrotransposition events in humans are caused by highly active, or hot, L1s. Here, in four populations of diverse geographic origins (160 haploid genomes), we investigated the degree of sequence polymorphism of three hot L1s and the extent of individual variation in mobilization capability of their allelic variants. For each locus, we found one previously uncharacterized allele in every three to five genomes, including some with nonsense and insertion/deletion mutations. Single or multiple nucleotide substitutions drastically affected the retrotransposition efficiency of some alleles. One-third of elements were no longer hot, and these so-called cool alleles substantially increased the range of individual susceptibility to retrotransposition events. Adding the activity of the three elements in each individual resulted in a surprising degree of variation in mobilization capability, ranging from 0% to 390% of a reference L1. These data suggest that individual variation in retrotransposition potential makes an important contribution to human genetic diversity.

Alleles↗
PubMed2006

Long-term efficacy of adeno-associated virus serotypes 8 and 9 in hemophilia a dogs and mice.

We reported total correction of blood coagulation plasma factor VIII (FVIII) activity, using adeno-associated virus serotype 8 (AAV8) vectors for liver-specific gene transfer in hemophilia A mice. We now show, irrespective of immunosuppression or route of administration, total long-term correction of hemophilia A mice with pseudotyped AAV8 and AAV9 vectors. We delivered two FVIII vectors, one expressing canine heavy chain and the other expressing canine light chain. Interestingly, when these vectors were given by hepatic portal vein to hemophilia A dogs, only modest FVIII levels were seen despite the species-specific transgene. No dogs treated developed FVIII inhibitors. However, of three dogs treated with AAV8 vector, the single male, given 1.25 x 10(13) genome copies per vector per kilogram (GC/vector/kg), maintained a level of >4.5% for more than 2 years. In contrast, the two female dogs expressed only 2% FVIII activity despite receiving higher doses of 1.52 x 10(13) and 3 x 10(13) GC/vector/kg, respectively. On the other hand, a male dog treated with AAV9 vector at a low dose (6 x 10(12) GC/vector/kg) maintained FVIII levels of 2-2.5% of normal without bleeding for 200 days (observation ongoing). Although hemophilia A mice were not predictive of vector efficacy in dogs, the two treated male dogs became symptom-free for long periods. Even so, translation of these robust vectors either in appropriate large animals or human beings remains challenging.

Animals↗
PubMed2005

L1 integration in a transgenic mouse model.

To study integration of the human LINE-1 retrotransposon (L1) in vivo, we developed a transgenic mouse model of L1 retrotransposition that displays de novo somatic L1 insertions at a high frequency, occasionally several insertions per mouse. We mapped 3' integration sites of 51 insertions by Thermal Asymmetric Interlaced PCR (TAIL-PCR). Analysis of integration locations revealed a broad genomic distribution with a modest preference for intergenic regions. We characterized the complete structures of 33 de novo retrotransposition events. Our results highlight the large number of highly truncated L1s, as over 52% (27/51) of total integrants were <1/3 the length of a full-length element. New integrants carry all structural characteristics typical of genomic L1s, including a number with inversions, deletions, and 5'-end microhomologies to the target DNA sequence. Notably, at least 13% (7/51) of all insertions contain a short stretch of extra nucleotides at their 5' end, which we postulate result from template-jumping by the L1-encoded reverse transcriptase. We propose a unified model of L1 integration that explains all of the characteristic features of L1 retrotransposition, such as 5' truncations, inversions, extra nucleotide additions, and 5' boundary and inversion point microhomologies.

Animals↗
PubMed2005

L1 retrotransposon-mediated stable gene silencing.

RNA interference (RNAi) is widely used for functional studies and has been proposed as a potential therapeutic agent. Current RNAi systems are largely efficient, but have limitations including transient effect, the need for viral handling and potential insertional mutations. Here, we describe a simple L1 retrotransposon-based system for the delivery of small interfering RNA (siRNA) and stable silencing in human cells. This system demonstrated long-term siRNA expression and significant reduction in both exogenous and endogenous gene expression by up to 90%. Further characterization indicated that retrotransposition occurred in a controlled manner such that essentially only one RNAi-cassette was integrated into the host genome and was sufficient for strong interference. Our system provides a novel strategy for stable gene silencing that is easy and efficient, and it may have potential applications for ex vivo and in vivo molecular therapy.

Genetic Techniques↗
PubMed2005

Lower inhibitor development in hemophilia A mice following administration of recombinant factor VIII-O-phospho-L-serine complex.

Factor VIII is a multidomain protein composed of A1, A2, B, A3, C1, and C2 domains. Deficiency or dysfunction of factor VIII causes hemophilia A, a bleeding disorder. Administration of exogenous recombinant factor VIII as a replacement leads to development of inhibitory antibodies against factor VIII in 15-30% of hemophilia A patients. Hence, less immunogenic preparations of factor VIII are highly desirable. Inhibitory antibodies against factor VIII are mainly directed against immunodominant epitopes in C2, A3, and A2 domains. Further, several universal epitopes for CD4+ T-cells have been identified within the C2 domain. The C2 domain is also known to interact specifically with phosphatidylserine-rich lipid vesicles. Here, we have investigated the hypothesis that complexation of O-phospho-l-serine, the head group of phosphatidylserine, with the C2 domain can reduce the overall immunogenicity of factor VIII. The biophysical (circular dichroism and fluorescence) and biochemical studies (ELISA and size exclusion chromatography) showed that O-phospho-l-serine binds to the phospholipid-binding region in the C2 domain, and this interaction causes subtle changes in the tertiary structure of the protein. O-Phospho-l-serine also prevented aggregation of the protein under thermal stress. The immunogenicity of the factor VIII-O-phospho-l-serine complex was evaluated in hemophilia A mice. The total and inhibitory antibody titers were lower for factor VIII-O-phospho-l-serine complex compared with factor VIII alone. Moreover, factor VIII administered as a complex with O-phospho-l-serine retained in vivo activity in hemophilia A mice. Our results suggest that factor VIII-O-phospho-l-serine complex may be beneficial to increase the physical stability and reduce immunogenicity of recombinant factor VIII preparations.

Animals↗
PubMed2005

Two independent retrotransposon insertions at the same site within the coding region of BTK.

Insertion of endogenous retrotransposon sequences accounts for approximately 0.2% of disease causing mutations. These insertions are mediated by the reverse transcriptase and endonuclease activity of long interspersed nucleotide (LINE-1) elements. The factors that control the target site selection in insertional mutagenesis are not well understood. In our analysis of 199 unrelated families with proven mutations in BTK, the gene responsible for X-linked agammaglobulinemia, we identified two families with retrotransposon insertions at exactly the same nucleotide within the coding region of BTK. Both insertions, an SVA element and an AluY sequence, occurred 12 bp before the end of exon 9. Both had the typical hallmarks of a retrotransposon insertion including target site duplication and a long poly A tail. The insertion site is flanked by AluSx sequences 1 kb upstream and 1 kb downstream and an unusual 60 bp sequence consisting of only As and Ts is located in intron 9, 60 bp downstream of the insertion. The occurrence of two retrotransposon sequences at exactly the same site suggests that this site is vulnerable to insertional mutagenesis. A better understanding of the factors that make this site vulnerable will shed light on the mechanisms of LINE-1 mediated insertional mutagenesis.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed2004

A potential role for the nucleolus in L1 retrotransposition.

Determining the subcellular localization of the L1 ORF2 protein (ORF2p) has been impossible to date because of technical limitations in detecting either endogenous or overexpressed forms of the protein. Here we report visualization of the full-length ORF2p in cultured human cells following expression in a modified vaccinia virus/T7 RNA polymerase (MVA/T7RP) system. The MVA/T7RP system was used to ascertain subcellular localization of L1 ORF1p and ORF2p both as fusions with green fluorescent protein and by immunocytochemistry. Full-length ORF2p was predominantly cytoplasmic, while carboxy-terminal-deleted ORF2p localized additionally to the nucleolus. We mapped a functional nucleolar localization signal in ORF2p. ORF1p appeared in the cytoplasm with a speckled pattern and colocalized with ORF2p in nucleoli in a subset of cells. These findings help explain the presence of chimeras between L1s and small RNA gene sequences recently discovered in the human genome.

Cell Culture Techniques↗
PubMed2004

Mobile elements: drivers of genome evolution.

Mobile elements within genomes have driven genome evolution in diverse ways. Particularly in plants and mammals, retrotransposons have accumulated to constitute a large fraction of the genome and have shaped both genes and the entire genome. Although the host can often control their numbers, massive expansions of retrotransposons have been tolerated during evolution. Now mobile elements are becoming useful tools for learning more about genome evolution and gene function.

Alu Elements↗
PubMed2004

More active human L1 retrotransposons produce longer insertions.

The vast majority of L1 insertions are 5' truncated and thus inactive. Yet, the mechanism of 5' truncation is unknown. To examine whether the frequency of L1 retrotransposition is directly correlated with the length of genomic L1 insertions, we used a cell culture assay to measure retrotransposition frequency and a PCR-based assay to measure L1 insertion length. We tested five full-length human L1 elements that retrotranspose at different frequencies: LRE3, L1(RP), L1.3, L1.2A and L1.2B. Our data suggest that L1 insertion length correlates with L1 retrotransposition frequency for insertions >1 kb in length. For two elements, L1(RP) and L1.2A, we found that swapping the reverse transcriptase domains had little effect. Instead, we found that genomic insertion length and retrotransposition frequency are substantially affected by amino acid substitutions at positions 363, 1220 and 1259 in ORF2. We suggest that the region containing residues 1220 and 1259 may be important in the binding of ORF2p to L1 RNA to facilitate reverse transcription.

Amino Acid Sequence↗
PubMed2003

SVA elements are nonautonomous retrotransposons that cause disease in humans.

L1 elements are the only active autonomous retrotransposons in the human genome. The nonautonomous Alu elements, as well as processed pseudogenes, are retrotransposed by the L1 retrotransposition proteins working in trans. Here, we describe another repetitive sequence in the human genome, the SVA element. Our analysis reveals that SVA elements are currently active in the human genome. SVA elements, like Alus and L1s, occasionally insert into genes and cause disease. Furthermore, SVA elements are probably mobilized in trans by active L1 elements.

Base Sequence↗
PubMed2003

Allelic heterogeneity in LINE-1 retrotransposition activity.

De novo LINE-1 (long interspersed element-1, or L1) retrotransposition events are responsible for approximately 1/1,000 disease-causing mutations in humans. Previously, L1.2 was identified as the likely progenitor of a mutagenic insertion in the factor VIII gene in a patient with hemophilia A. It subsequently was shown to be one of a small number of active L1s in the human genome. Here, we demonstrate that L1.2 is present at an intermediate insertion allele frequency in worldwide human populations and that common alleles (L1.2A and L1.2B) exhibit an approximately 16-fold difference in their ability to retrotranspose in cultured human HeLa cells. Chimera analysis revealed that two amino acid substitutions (S1259L and I1220M) downstream of the conserved cysteine-rich motif in L1 open reading frame 2 are largely responsible for the observed reduction in L1.2A retrotransposition efficiency. Thus, common L1 alleles can vary widely in their retrotransposition potential. We propose that such allelic heterogeneity can influence the potential L1 mutational load present in an individual genome.

Base Sequence↗
PubMed2003

Total correction of hemophilia A mice with canine FVIII using an AAV 8 serotype.

Despite the popularity of adeno-associated virus 2 (AAV2) as a vehicle for gene transfer, its efficacy for liver-directed gene therapy in hemophilia A or B has been suboptimal. Here we evaluated AAV serotypes 2, 5, 7, and 8 in gene therapy of factor VIII (FVIII) deficiency in a hemophilia A mouse model and found that AAV8 was superior to the other 3 serotypes. We expressed canine B domain-deleted FVIII cDNA either in a single vector or in 2 separate AAV vectors containing the heavy- and light-chain cDNAs. We also evaluated AAV8 against AAV2 in intraportal and tail vein injections. AAV8 gave 100% correction of plasma FVIII activity irrespective of the vector type or route of administration.

Adenoviridae↗
PubMed2003

An important role for RUNX3 in human L1 transcription and retrotransposition.

LINE-1s (long interspersed nuclear elements-1) are abundant non-LTR retrotransposons that comprise 17% of the human genome. The 5' untranslated region (5'UTR) of human L1 (L1Hs) houses a poorly understood internal promoter. Here we report that mutations at a putative runt-domain transcription factor (RUNX) site (+83 to +101) in the 5'UTR decreased L1Hs transcription and retrotransposition in cell culture-based assays. Exogenous expression of RUNX3, but not the other two RUNX family members, RUNX1 and RUNX2, increased L1Hs transcription and retrotransposition, which were otherwise decreased by siRNAs targeting RUNX3 and a dominant negative RUNX. Further more, the specific interaction between RUNX3 and its binding site was demonstrated by an electrophoretic mobility shift assay (EMSA) using an anti-RUNX3 antibody. Interestingly, RUNX3 may also regulate the antisense promoter activity of L1Hs 5'UTR via another putative RUNX site (+526 to +508), as revealed by site-directed mutations and exogenous expression of RUNX factors. Our results indicate an important role for RUNX3 in L1Hs retrotransposition as well as transcription from its 5'UTR in both sense and antisense directions, and they should contribute to our understanding of the mechanism underlying L1Hs retrotransposition and its impact on the expression of adjacent cellular genes.

5' Flanking Region↗
PubMed2003

Phenotypic correction of a mouse model of hemophilia A using AAV2 vectors encoding the heavy and light chains of FVIII.

Using separate adeno-associated viral 2 (AAV2) vectors to deliver the heavy and light chains of factor VIII (FVIII) we have overcome the packaging limitations of AAV, achieving phenotypic correction of hemophilia A in mice. AAV vectors were constructed that use a liver-specific promoter and the cDNA sequences of either the human or canine heavy and light chains of FVIII. After intraportal vein injection of these vectors in hemophilia-A mice, therapeutic to superphysiologic levels of active FVIII were achieved in plasma in a dose-dependent manner. Phenotypic correction of the bleeding diathesis was demonstrated by survival of all treated mice after tail clipping. Biochemical analysis demonstrated lower levels of heavy-chain (25- to 100-fold) compared with light-chain protein in the plasma of treated animals. Differences in gene transfer and transcription did not account for the differences in protein expression. We hypothesize that improvements in FVIII activity could be achieved by improvements in FVIII heavy-chain expression. This work demonstrates that cotransduction of liver with AAV vectors expressing the heavy and light chains of FVIII corrects hemophilia A in vivo, providing an alternative approach to the use of a single vector. This strategy may potentially be useful for other large therapeutic proteins that contain functionally distinct domains.

Adenoviridae↗