Intact Long Terminal Repeat Research Articles

DANTE and DANTE_LTR: lineage-centric annotation pipelines for long terminal repeat retrotransposons in plant genomes.

Long terminal repeat (LTR) retrotransposons constitute a predominant class of repetitive DNA elements in most plant genomes. With the increasing number of sequenced plant genomes, there is an ongoing demand for computational tools facilitating efficient annotation and classification of LTR retrotransposons in plant genome assemblies. Herein, we introduce DANTE, a computational pipeline for Domain-based ANnotation of Transposable Elements, designed for sensitive detection of these elements via their conserved protein domain sequences. The identified protein domains are subsequently inputted into the DANTE_LTR pipeline to annotate complete element sequences by detecting their structural features, such as LTRs, in adjacent genomic regions. Leveraging domain sequences allows for precise classification of elements into phylogenetic lineages, offering a more granular annotation compared with coarser conventional superfamily-based classification methods. The efficiency and accuracy of this approach were evidenced via annotation of LTR retrotransposons in 93 plant genomes. Results were benchmarked against several established pipelines, showing that DANTE_LTR is capable of identifying significantly more intact LTR retrotransposons. DANTE and DANTE_LTR are provided as user-friendly Galaxy tools accessible via a public server (https://repeatexplorer-elixir.cerit-sc.cz), installable on local Galaxy instances from the Galaxy tool shed or executable from the command line.

Identification and characterization of genome-wide long terminal repeat retrotransposons provide an insight into elucidating the trait evolution of five Rhododendron species.

Rhododendron is well-known for its beauty and colourful corolla. Although some high-quality whole-genome sequencing of it has been completed, there are few studies on long terminal repeat (LTR) retrotransposons in Rhododendron, which limits our ability to elucidate the causes of genetic variations in Rhododendron species. Properties of the intact Rhododendron LTR retrotransposons were investigated at a genome-wide level. Based on available data, the high-quality genomes from five species, i.e. R. griersonianum, R. simsii, R. henanense subsp. lingbaoense, R. mucronatum var. ripense and R. ovatum, were selected as targets with good assembly continuity. A total of 17,936 intact LTR retrotransposons were identified; these belong to superfamilies Copia and Gypsy, with 17 clades. The insertion time of these transposons was later than 120 million years ago (Mya), and the outbreak period was concentrated more recently than 30 Mya. Phylogenetic analysis revealed that many LTR retrotransposons might originate from intraspecific duplication. Current evidence also suggests that most LTR retrotransposons were inserted in the interstitial part of genes in R. griersonianum, R. simsii, R. henanense, and R. ovatum, and the functions of the inserted genes mainly involve starch metabolism, proteolysis, etc. The effect of the LTR retrotransposon on gene expression depends on its insertion site and activation. Highly expressed LTR retrotransposons tend to be younger. The results herein improve our knowledge of LTR retrotransposons in Rhododendron genomes and facilitate further study of genetic variation and trait evolution in Rhododendron.

Characterization of genome‐wide long terminal repeat retrotransposons provide insights into trait evolution of four grapevine species

AbstractGrapevine is one of the most economically important crops in the world. Although long terminal repeat (LTR) retrotransposons are thought to have played an important role in plants, its distribution in grapevine is not clear. Here, we identified genome‐wide intact LTR retrotransposons in a total of six high‐quality grapevine genomes from Vitis vinifera L., Vitis sylvestris C.C. Gmel., Vitis riparia Michx. and Vitis amurensis Rupr. with an average of 2938 per genome. Among them, the Copia superfamily (particularly for Ale) is a major component of the LTR retrotransposon in grapevine. Insertion time and copy number analysis revealed that the expansion of 70% LTR retrotransposons concentrating on approximately 2.5 Ma was able to drive genome size variation. Phylogenetic tree and syntenic analyses showed that most LTR retrotransposons in these genomes formed and evolved after species divergence. Furthermore, the function and expression of genes inserted by LTR retrotransposons in V. vinifera (Pinot noir) and V. riparia were explored. The length and expression of genes related to starch metabolism and quinone synthesis pathway in Pinot noir and environmental adaptation pathway in V. riparia were significantly affected by LTR retrotransposon insertion. The results improve the understanding of LTR retrotransposons in grapevine genomes and provide insights for its potential contribution to grapevine trait evolution.

Genome Sequencing of the Endangered Kingdonia uniflora (Circaeasteraceae, Ranunculales) Reveals Potential Mechanisms of Evolutionary Specialization.

SummaryKingdonia uniflora, an alpine herb, has an extremely narrow distribution and represents a model for studying evolutionary mechanisms of species that have adapted to undisturbed environments for evolutionarily long periods of time. We assembled a 1,004.7-Mb draft genome (encoding 43,301 genes) of K. uniflora and found significant overrepresentation in gene families associated with DNA repair, underrepresentation in gene families associated with stress response, and loss of most plastid ndh genes. During the evolutionary process, the overrepresentation of gene families involved in DNA repair could help asexual K. uniflora reduce the accumulation of deleterious mutations, while reducing genetic diversity, which is important in responding to environment fluctuations. The underrepresentation of gene families related to stress response and functional loss of ndh genes could be due to lack or loss of ability to respond to environmental changes caused by long-term adaptation to a relatively stable ecological environment.

Development and Deployment of High-Throughput Retrotransposon-Based Markers Reveal Genetic Diversity and Population Structure of Asian Bamboo

Bamboo, a non-timber grass species, known for exceptionally fast growth is a commercially viable crop. Long terminal repeat (LTR) retrotransposons, the main class I mobile genetic elements in plant genomes, are highly abundant (46%) in bamboo, contributing to genome diversity. They play significant roles in the regulation of gene expression, chromosome size and structure as well as in genome integrity. Due to their random insertion behavior, interspaces of retrotransposons can vary significantly among bamboo genotypes. Capitalizing this feature, inter-retrotransposon amplified polymorphism (IRAP) is a high-throughput marker system to study the genetic diversity of plant species. To date, there are no transposon based markers reported from the bamboo genome and particularly using IRAP markers on genetic diversity. Phyllostachys genus of Asian bamboo is the largest of the Bambusoideae subfamily, with great economic importance. We report structure-based analysis of bamboo genome for the LTR-retrotransposon superfamilies, Ty3-gypsy and Ty1-copia, which revealed a total of 98,850 retrotransposons with intact LTR sequences at both the ends. Grouped into 64,281 clusters/scaffold using CD-HIT-EST software, only 13 clusters of retroelements were found with more than 30 LTR sequences and with at least one copy having all intact protein domains such as gag and polyprotein. A total of 16 IRAP primers were synthesized, based on the high copy numbers of conserved LTR sequences. A study using these IRAP markers on genetic diversity and population structure of 58 Asian bamboo accessions belonging to the genus Phyllostachys revealed 3340 amplicons with an average of 98% polymorphism. The bamboo accessions were collected from nine different provinces of China, as well as from Italy and America. A three phased approach using hierarchical clustering, principal components and a model based population structure divided the bamboo accessions into four sub-populations, PhSP1, PhSP2, PhSP3 and PhSP4. All the three analyses produced significant sub-population wise consensus. Further, all the sub-populations revealed admixture of alleles. The analysis of molecular variance (AMOVA) among the sub-populations revealed high intra-population genetic variation (75%) than inter-population. The results suggest that Phyllostachys bamboos are not well evolutionarily diversified, although geographic speciation could have occurred at a limited level. This study highlights the usability of IRAP markers in determining the inter-species variability of Asian bamboos.

Inhibition of NF-κB-dependent HIV-1 replication by the marine natural product bengamide A

HIV-1 inhibitors that act by mechanisms distinct from existing antiretrovirals can provide novel insights into viral replication and potentially inform development of new therapeutics. Using a multi-cycle HIV-1 replication assay, we screened 252 pure compounds derived from marine invertebrates and microorganisms and identified 6 (actinomycin Z2, bastadin 6, bengamide A, haliclonacyclamine A + B, keramamine C, neopetrosiamide B) that inhibited HIV-1 with 50% effective concentrations (EC50s) of 3.8 μM or less. The most potent inhibitor, bengamide A, blocked HIV-1 in a T cell line with an EC50 of 0.015 μM and in peripheral blood mononuclear cells with an EC50 of 0.032 μM. Bengamide A was previously described to inhibit NF-κB signaling. Consistent with this mechanism, bengamide A suppressed reporter expression from an NF-κB-driven minimal promoter and an HIV-1 long terminal repeat (LTR) with conserved NF-κB response elements, but lacked activity against an LTR construct with mutation of these elements. In single-cycle HIV-1 infection assays, bengamide A also suppressed viral protein expression when viruses encoded an intact LTR but exhibited minimal activity against those with mutated NF-κB elements. Finally, bengamide A did not inhibit viral DNA accumulation, indicating that it likely acts downstream of this step in HIV-1 replication. Our study identifies multiple new antiviral compounds including an unusually potent inhibitor of HIV-1 gene expression.

Insertionally polymorphic sites of human endogenous retrovirus-K (HML-2) with long target site duplications

BackgroundHuman endogenous retroviruses (HERVs) belong to the LTR-retrotransposon family, where the complete HERV sequence contains two long terminal repeats (LTRs) located at each end. Intact LTRs possess highly conserved transcriptional promoter and enhancer sequences, so analyses of HERV insertional polymorphisms are expected to provide greater insights into human genomic variation compared with the conventional analysis of single nucleotide variations. High-throughput sequencing technology is developing but genome-wide investigations of HERVs are methodically challenging, and thus a comprehensive understanding of HERV insertional polymorphisms and target site duplications (TSDs) remains elusive.ResultsWe identified five human-specific insertionally polymorphic sites in HERVK (HML-2), one of the HERV subgroups, by extracting HML-2-deleted sequences from the genomic structural variation database, which we successfully characterized and then updated the existing catalogue of HML-2 insertional polymorphisms. The insertionally polymorphic states were confirmed in a small Japanese population by genomic PCR analysis for four of the five sites identified. Sequencing of the preintegration sites clearly showed that the HML-2 site located at 7p21.2 had 250-base pair (bp) TSDs, which is one of the longest TSDs in HML-2. In addition to these five sites, another insertionally polymorphic site for a non-human-specific HML-2 site was also identified at 6p25.2, which was flanked by 111-bp TSDs and the corresponding ERV locus was also annotated in the genome of non-human primates.ConclusionsOur analysis demonstrated the existence of HERV insertions flanked by unconventionally long TSDs, including those with lengths as high as 250 bp. This suggests that the length range of retroviral TSDs is larger than considered previously, which might help to understand how retroviral integration occurs in the host genome.

Rapid and Recent Evolution of LTR Retrotransposons Drives Rice Genome Evolution During the Speciation of AA-Genome Oryza Species.

The dynamics of long terminal repeat (LTR) retrotransposons and their contribution to genome evolution during plant speciation have remained largely unanswered. Here, we perform a genome-wide comparison of all eight Oryza AA-genome species, and identify 3911 intact LTR retrotransposons classified into 790 families. The top 44 most abundant LTR retrotransposon families show patterns of rapid and distinct diversification since the species split over the last ∼4.8 MY (million years). Phylogenetic and read depth analyses of 11 representative retrotransposon families further provide a comprehensive evolutionary landscape of these changes. Compared with Ty1-copia, independent bursts of Ty3-gypsy retrotransposon expansions have occurred with the three largest showing signatures of lineage-specific evolution. The estimated insertion times of 2213 complete retrotransposons from the top 23 most abundant families reveal divergent life histories marked by speedy accumulation, decline, and extinction that differed radically between species. We hypothesize that this rapid evolution of LTR retrotransposons not only divergently shaped the architecture of rice genomes but also contributed to the process of speciation and diversification of rice.

Comprehensive definition of genome features in Spirodela polyrhiza by high-depth physical mapping and short-read DNA sequencing strategies.

Spirodela polyrhiza is a fast-growing aquatic monocot with highly reduced morphology, genome size and number of protein-coding genes. Considering these biological features of Spirodela and its basal position in the monocot lineage, understanding its genome architecture could shed light on plant adaptation and genome evolution. Like many draft genomes, however, the 158-Mb Spirodela genome sequence has not been resolved to chromosomes, and important genome characteristics have not been defined. Here we deployed rapid genome-wide physical maps combined with high-coverage short-read sequencing to resolve the 20 chromosomes of Spirodela and to empirically delineate its genome features. Our data revealed a dramatic reduction in the number of the rDNA repeat units in Spirodela to fewer than 100, which is even fewer than that reported for yeast. Consistent with its unique phylogenetic position, small RNA sequencing revealed 29 Spirodela-specific microRNA, with only two being shared with Elaeis guineensis (oil palm) and Musa balbisiana (banana). Combining DNA methylation data and small RNA sequencing enabled the accurate prediction of 20.5% long terminal repeats (LTRs) that doubled the previous estimate, and revealed a high Solo:Intact LTR ratio of 8.2. Interestingly, we found that Spirodela has the lowest global DNA methylation levels (9%) of any plant species tested. Taken together our results reveal a genome that has undergone reduction, likely through eliminating non-essential protein coding genes, rDNA and LTRs. In addition to delineating the genome features of this unique plant, the methodologies described and large-scale genome resources from this work will enable future evolutionary and functional studies of this basal monocot family.

Abstract 2966A: A comprehensive investigation of insertional variations of human endogenous retrovirus elements in tumor tissues

Abstract Human endogenous retrovirus (HERV) is a member of LTR-retrotransposon family. Retroviral invasions are thought to have occurred in the germ cell lines of human or human ancestors, and the fixed retroviral elements are recognized as HERV. A complete HERV sequence contains two long terminal repeats (LTRs) located at both ends. Because intact LTRs possess the highly conserved transcriptional promoter- and enhancer-sequences, to investigate insertional variations of HERV is a key process for understanding the etiology of human genetic diseases that remain unclear with the analyses targeting single nucleotide variations. However, genome-wide investigation of repetitive sequences including HERV is methodologically difficult, and comprehensive understanding of insertional variations of HERV, including a somatic retrotransposition of HERV, remains poor. In the previous study, we have identified two novel insertional polymorphisms of solo LTRs using the inverse PCR and cloning methods, and one of them was involved with the susceptibility to lung adenocarcinoma. Here, we detected multiple novel LTR loci in the human cancer tissues by developing the high-throughput sequencing method combined with inverse PCR. In this system, to preferentially detect insertional variations, primer sequences were designed based on the conserved region of the LTRs which have an identical sequence to the mate within the same HML-2 proviruse, because almost of known HERV insertional polymorphisms are found in HML-2, which is thought to be the most recently endogenized element of HERV, and the presence of an identical LTR sequence at both ends is necessary for the retrotransposition of exogenous retrovirus. The sequence reads generated from the high-throughput sequencer were trimmed, and the fragments consistent with the inverse PCR process were selected. Following the mapping to the reference genome, the sites not corresponding to known LTR loci were sorted out and subjected to the filtration to exclude the artificial fragments. As the result, some candidates of novel LTR loci were detected, and several of them were verified to be actually present both in tumor and non-tumor genomes using the Sanger method, suggesting they are insertional polymorphisms. By using this detection system, we are now challenging to investigate a novel LTR site specifically detected in tumor genome. Citation Format: Tomoaki Kahyo, Hidetaka Yamada, Hong Tao, Yusuke Inoue, Nobuya Kurabe, Haruhiko Sugimura. A comprehensive investigation of insertional variations of human endogenous retrovirus elements in tumor tissues. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2966A. doi:10.1158/1538-7445.AM2015-2966A

Alpharetroviral Self-inactivating Vectors: Long-term Transgene Expression in Murine Hematopoietic Cells and Low Genotoxicity

Comparative integrome analyses have highlighted alpharetroviral vectors with a relatively neutral, and thus favorable, integration spectrum. However, previous studies used alpharetroviral vectors harboring viral coding sequences and intact long-terminal repeats (LTRs). We recently developed self-inactivating (SIN) alpharetroviral vectors with an advanced split-packaging design. In a murine bone marrow (BM) transplantation model we now compared alpharetroviral, gammaretroviral, and lentiviral SIN vectors and showed that all vectors transduced hematopoietic stem cells (HSCs), leading to comparable, sustained multilineage transgene expression in primary and secondary transplanted mice. Alpharetroviral integrations were decreased near transcription start sites, CpG islands, and potential cancer genes compared with gammaretroviral, and decreased in genes compared with lentiviral integrations. Analyzing the transcriptome and intragenic integrations in engrafting cells, we observed stronger correlations between in-gene integration targeting and transcriptional activity for gammaretroviral and lentiviral vectors than for alpharetroviral vectors. Importantly, the relatively "extragenic" alpharetroviral integration pattern still supported long-term transgene expression upon serial transplantation. Furthermore, sensitive genotoxicity studies revealed a decreased immortalization incidence compared with gammaretroviral and lentiviral SIN vectors. We conclude that alpharetroviral SIN vectors have a favorable integration pattern which lowers the risk of insertional mutagenesis while supporting long-term transgene expression in the progeny of transplanted HSCs.

Self-Inactivating Alpharetroviral Vectors with a Split-Packaging Design

Accidental insertional activation of proto-oncogenes and potential vector mobilization pose serious challenges for human gene therapy using retroviral vectors. Comparative analyses of integration sites of different retroviral vectors have elucidated distinct target site preferences, highlighting vectors based on the alpharetrovirus Rous sarcoma virus (RSV) as those with the most neutral integration spectrum. To date, alpharetroviral vector systems are based mainly on single constructs containing viral coding sequences and intact long terminal repeats (LTR). Even though they are considered to be replication incompetent in mammalian cells, the transfer of intact viral genomes is unacceptable for clinical applications, due to the risk of vector mobilization and the potentially immunogenic expression of viral proteins, which we minimized by setting up a split-packaging system expressing the necessary viral proteins in trans. Moreover, intact LTRs containing transcriptional elements are capable of activating cellular genes. By removing most of these transcriptional elements, we were able to generate a self-inactivating (SIN) alpharetroviral vector, whose LTR transcriptional activity is strongly reduced and whose transgene expression can be driven by an internal promoter of choice. Codon optimization of the alpharetroviral Gag/Pol expression construct and further optimization steps allowed the production of high-titer self-inactivating vector particles in human cells. We demonstrate proof of principle for the versatility of alpharetroviral SIN vectors for the genetic modification of murine and human hematopoietic cells at a low multiplicity of infection.

Effect of the Internal Promoter on Insertional Gene Activation by Lentiviral Vectors with an Intact HIV Long Terminal Repeat

Insertional mutagenesis by viral vectors is a problem in gene therapy. We recently reported that lentiviral vectors with an intact HIV long terminal repeat (LTR) caused insertional gene activation by transcripts from the 5' LTR splicing to an adjacent gene. Here we demonstrate that the level of transcription from the 5' LTR, and also insertional gene activation, is dependent on the internal promoter in the vector. We also show that there are more transcripts originating from the 5' LTR than from, or reading through, the 3' LTR. This study will allow the design of safer lentiviral vectors for applications in which an intact HIV LTR is required.

Reduced Genotoxicity of Avian Sarcoma Leukosis Virus Vectors in Rhesus Long-term Repopulating Cells Compared to Standard Murine Retrovirus Vectors

Insertional mutagenesis continues to be a major concern in hematopoietic stem-cell gene therapy. Nonconventional gene transfer vectors with more favorable integration features in comparison with conventional retrovirus and lentivirus vectors are being developed and optimized. In this study, we report for the first time a systematic analysis of 198 avian sarcoma leukosis virus (ASLV) insertion sites identified in rhesus long-term repopulating cells, and a comparison of ASLV insertion patterns to Moloney murine leukemia virus (MLV) (n = 396) and simian immunodeficiency virus (SIV) (n = 289) using the newly released rhesus genome databank. Despite a weak preference toward gene-coding regions, ASLV integration is nonclustered, does not favor gene-rich regions, transcription start sites, or CpG islands. There was no propensity for ASLV insertions within or near proto-oncogenes, and most importantly, no insertions close to or within the Mds1-Evi1 locus, which is in contrast to the significant over-representation of this insertion site for MLV vectors in the same transplantation model. Furthermore, ASLV long terminal repeats (LTRs) do not have detectable promoter and enhancer activity in a quantitative luciferase assay to measure neighboring gene activation. The combination of these features is unique for ASLV and suggests that optimized vectors based on this virus could be useful and safe for gene transfer to hematopoietic stem cells and progenitor cells.

An experimental system for the evaluation of retroviral vector design to diminish the risk for proto-oncogene activation

Pathogenic activation of the LMO2 proto-oncogene by an oncoretroviral vector insertion in a clinical trial for X-linked severe combined immunodeficiency (X-SCID) has prompted safety concerns. We used an adeno-associated virus vector to achieve targeted insertion of a gamma-retroviral long terminal repeat (LTR) driving a GFP expression cassette with flanking loxP sites in a human T-cell line at the precise location of vector integration in one of the patients with X-SCID. The LTR-GFP cassette was inserted into the first intron of the LMO2 gene, resulting in strong activation of LMO2. Cre-mediated cassette exchange was used to replace the original LTR-GFP cassette with one flanked by insulator elements leading to a several fold reduction in LMO2 expression. The LTR-GFP cassette was also replaced with a globin gene regulatory cassette that failed to activate the LMO2 gene in lymphoid cells. A gamma-retroviral vector with 2 intact LTRs resulted in activation of the LMO2 gene when inserted into the first intron, but a self-inactivating lentiviral vector with an internal cellular promoter and flanking insulator elements did not activate the LMO2 gene. Thus, this system is useful for comparing the safety profiles of vector cassettes with various regulatory elements for their potential for proto-oncogene activation.

Correction of Clinical Manifestations of Canine Mucopolysaccharidosis I with Neonatal Retroviral Vector Gene Therapy

Mucopolysaccharidosis I (MPS I) (Hurler syndrome) is due to deficient alpha-L-iduronidase (IDUA) activity and is the most common of the MPS disorders. Neonatal MPS I dogs were injected intravenously (IV) with a gamma retroviral vector containing a complete long-terminal repeat (LTR) and an internal human alpha(1)-antitrypsin (hAAT) promoter upstream of the canine IDUA complementary DNA (cDNA). This resulted in stable serum IDUA activity of 366 +/- 344 units (U)/ml (28-fold normal) for up to 1.8 years, which likely derived primarily from secretion of IDUA by transduced liver cells. Retroviral vector (RV)-treated dogs had >18% of normal IDUA activity in organs and had decreased severity and/or incidence of hernias, chest deformities, joint disease, facial dysmorphia, corneal clouding, valvular heart disease, and aortic dilatation as compared with untreated MPS I dogs. The marked reduction that was observed in lysosomal storage in the brain of RV-treated dogs may have been due in part to expression from the LTR of the vector in cells in the brain. This possibility will be explored in future studies, because the potential for insertional mutagenesis has raised concerns about using vectors with an intact LTR. If proven safe, this gene therapy technique may be utilized in treating children with Hurler syndrome.

De novo identification of LTR retrotransposons in eukaryotic genomes

BackgroundLTR retrotransposons are a class of mobile genetic elements containing two similar long terminal repeats (LTRs). Currently, LTR retrotransposons are annotated in eukaryotic genomes mainly through the conventional homology searching approach. Hence, it is limited to annotating known elements.ResultsIn this paper, we report a de novo computational method that can identify new LTR retrotransposons without relying on a library of known elements. Specifically, our method identifies intact LTR retrotransposons by using an approximate string matching technique and protein domain analysis. In addition, it identifies partially deleted or solo LTRs using profile Hidden Markov Models (pHMMs). As a result, this method can de novo identify all types of LTR retrotransposons. We tested this method on the two pairs of eukaryotic genomes, C. elegans vs. C. briggsae and D. melanogaster vs. D. pseudoobscura. LTR retrotransposons in C. elegans and D. melanogaster have been intensively studied using conventional annotation methods. Comparing with previous work, we identified new intact LTR retroelements and new putative families, which may imply that there may still be new retroelements that are left to be discovered even in well-studied organisms. To assess the sensitivity and accuracy of our method, we compared our results with a previously published method, LTR_STRUC, which predominantly identifies full-length LTR retrotransposons. In summary, both methods identified comparable number of intact LTR retroelements. But our method can identify nearly all known elements in C. elegans, while LTR_STRUCT missed about 1/3 of them. Our method also identified more known LTR retroelements than LTR_STRUCT in the D. melanogaster genome. We also identified some LTR retroelements in the other two genomes, C. briggsae and D. pseudoobscura, which have not been completely finished. In contrast, the conventional method failed to identify those elements. Finally, the phylogenetic and chromosomal distributions of the identified elements are discussed.ConclusionWe report a novel method for de novo identification of LTR retrotransposons in eukaryotic genomes with favorable performance over the existing methods.

Equal potency of gammaretroviral and lentiviral SIN vectors for expression of O6-methylguanine–DNA methyltransferase in hematopoietic cells

Severe adverse events related to insertional mutagenesis have reinforced interest in self-inactivating (SIN) retroviral vectors lacking enhancer–promoter sequences in the long terminal repeats (LTRs). Here, we have compared the potency of gammaretroviral and lentiviral vectors expressing the P140K mutant of O6-methylguanine–DNA methyltransferase (MGMT). MGMT-P140K is a clinically relevant selection marker that mediates a strong survival advantage in hematopoietic cells exposed to alkylating agents. We designed gammaretroviral and lentiviral vectors that contained identical enhancer–promoter sequences located either in the LTR or downstream of the packaging region, for internal initiation of transcription from SIN backbones. Gammaretroviral vectors with intact LTRs containing enhancer–promoter sequences showed both higher titers and higher expression levels than the lentiviral counterparts, likely a result of suboptimal RNA processing of the lentiviral leader region. In the SIN context, gammaretroviral and lentiviral vectors with comparable internal cassettes had similar expression properties. Interestingly, gammaretroviral SIN vectors pseudotyped with RD114/TR had a higher transduction efficiency on proliferating human CD34+ cells than lentiviral counterparts. These results encourage further investigations into the formation of retroviral hybrid vectors that combine the desired properties of high efficiency and increased biosafety.

Transduction of the Choroid Plexus and Ependyma in Neonatal Mouse Brain by Vesicular Stomatitis Virus Glycoprotein-Pseudotyped Lentivirus and Adeno-Associated Virus Type 5 Vectors

Evaluation of gene transfer into the developing mouse brain has shown that when adeno-associated virus serotype 1 (AAV1) or AAV2 vectors are injected into the cerebral lateral ventricles at birth, widespread parenchymal transduction occurs. Lentiviral vectors have not been tested by this route. In this study, we found that injection of lentiviral vectors pseudotyped with vesicular stomatitis virus glycoprotein (VSV-G) resulted in targeted transduction of the ependymal cells lining the ventricular system and the choroid plexus along the entire rostrocaudal axis of the brain, whereas a Mokola pseudotype transduced only a few cells after injection into the neonatal ventricle. In contrast, when lentiviral vectors pseudotyped with either VSV-G or Mokola glycoprotein are injected into the adult mouse brain, they transduce similar patterns of cells. An Ebola-Zaire-pseudotyped vector did not transduce any neonatal CNS cells, as was also the case for adult parenchymal injections. Long-term gene expression (12 months) occurred with a constitutively active mammalian promoter and a self-inactivating long terminal repeat (LTR), whereas the cytomegalovirus promoter in a vector with an intact LTR was expressed only in short-term experiments. We found that an AAV5 vector also targeted the ependymal and choroid plexus cells throughout the ventricular system. This vector exhibited limited penetration from the ventricle to other structures, which was significantly different from the previously reported patterns of transduction after intraventricular injection of AAV1 and AAV2 vectors.

Liver-Directed Gene Therapy: A Retroviral Vector with a Complete LTR and the ApoE Enhancer-α1-Antitrypsin Promoter Dramatically Increases Expression of Humanα1-AntitrypsinIn Vivo

Hepatic gene therapy could improve the treatment of many inherited disorders. Although retroviral vectors result in long-term expression in hepatocytes in vivo, their low level of expression currently precludes most clinical applications. Four copies of the liver-specific apolipoprotein E (ApoE) enhancer were placed upstream of the human alpha 1-antitrypsin (hAAT) promoter in either orientation into a retroviral vector with a complete long terminal repeat (LTR) and the hAAT cDNA to generate ApoE(+)hAAT-LTR and ApoE(-)hAAT-LTR. In addition, the ApoAI promoter was placed upstream of the hAAT cDNA in a similar retroviral vector backbone. Amphotropic retroviral vectors were transferred into regenerating rat liver cells in vivo by intraportal injection. ApoE(-)hAAT-LTR and ApoE(+)hAAT-LTR led to average hAAT levels of 5 micrograms/ml (0.5% of normal levels of a very abundant protein), and 2.5 micrograms/ml, respectively, which was stable for at least 10 months after transduction. This level of serum hAAT was > 25-fold higher than what was observed from the ApoAI promoter used in this study. Serum levels of hAAT were > 15-fold higher than what was observed from retroviral vectors containing the hAAT cDNA that were analyzed previously by this lab. In some cases, improved expression was due to the promoter chosen. In other cases, the increase in expression was primarily due to the higher titers obtained by using a retroviral backbone with an intact LTR as opposed to a vector with a deletion in the LTR. The increased expression levels observed from this enhancer/promoter combination in an intact retroviral backbone may enable one to achieve therapeutic levels of clinically important genes from a retroviral vector in liver cells of animals.