The future of human gene therapy

Human gene therapy (HGT) is defined as the transfer of nucleic acids (DNA) to somatic cells of a patient which results in a therapeutic effect, by either correcting genetic defects or by overexpressing proteins that are therapeutically useful. In the past, both the professional and the lay community had high (sometimes unreasonably high) expectations from HGT because of the early promise of treating or preventing diseases effectively and safely by this new technology. Although the theoretical advantages of HGT are undisputable, so far HGT has not delivered the promised results: convincing clinical efficacy could not be demonstrated yet in most of the trials conducted so far, while safety concerns were raised recently as the consequence of the "Gelsinger Case" in Philadelphia. This situation resulted from the by now well-recognized disparity between theory and practice. In other words, the existing technologies could not meet the practical needs of clinically successful HGT so far. However, over the past years, significant progress was made in various enabling technologies, in the molecular understanding of diseases and the manufacturing of vectors. HGT is a complex process, involving multiple steps in the human body (delivery to organs, tissue targeting, cellular trafficking, regulation of gene expression level and duration, biological activity of therapeutic protein, safety of the vector and gene product, to name just a few) most of which are not completely understood. The prerequisite of successful HGT include therapeutically suitable genes (with a proven role in pathophysiology of the disease), appropriate gene delivery systems (e.g., viral and non-viral vectors), proof of principle of efficacy and safety in appropriate preclinical models and suitable manufacturing and analytical processes to provide well-defined HGT products for clinical investigations. The most promising areas for gene therapy today are hemophilias, for monogenic diseases, and cardiovascular diseases (more specifically, therapeutic angiogenesis for myocardial ischemia and peripheral vascular disease, restenosis, stent stenosis and bypass graft failure) among multigenic diseases. This is based on the relative ease of access of blood vessels for HGT, and also because existing gene delivery technologies may be sufficient to achieve effective and safe therapeutic benefits for some of these indications (transient gene expression in some but not all affected cells is required to achieve a therapeutic effect at relatively low [safe] dose of vectors). For other diseases (including cancer) further developments in gene delivery vectors and gene expression systems will be required. It is important to note, that there will not be a "universal vector" and each clinical indication may require a specific set of technical hurdles to overcome. These will include modification of viral vectors (to reduce immunogenicity, change tropism and increase cloning capacity), engineering of non-viral vectors by mimicking the beneficial properties of viruses, cell-based gene delivery technologies, and development of innovative gene expression regulation systems. The technical advances together with the ever increasing knowledge and experience in the field will undoubtedly lead to the realization of the full potential of HGT in the future.     

A likelihood framework to measure horizontal gene transfer

We suggest a likelihood-based approach to estimate an overall rate of horizontal gene transfer (HGT) in a simplified setting. To this end, we assume that the number of occurring HGT events within a given time interval follows a Poisson process. To obtain estimates for the rate of HGT, we simulate the distribution of tree topologies for different numbers of HGT events on a clocklike species tree. Using these simulated distributions, we estimate an HGT rate for a collection of gene trees representing a set of taxa. As an illustrative example, we use the "Clusters of Orthologous Groups of proteins" (COGs). We also perform a correction of the estimated rate taking into account the inaccuracies due to gene tree reconstructions. The results suggest a corrected HGT rate of about 0.36 per gene and unit time, in other words 11 HGT events have occurred on average among the 44 taxa of the COG species tree. A software package to estimate an HGT rate is available online (http://www.cibiv.at/software/hgt/).     

Advances in retroviral transduction of hematopoietic stem cells for the gene therapy of atherosclerosis

PURPOSE OF REVIEW: Atherosclerosis is a chronic inflammatory disease that is the primary cause of morbidity and mortality in the developed world. Many studies have shown that macrophages and T-cells play critical roles in multiple aspects of the pathogenesis of the disease. Given that these cells are ultimately derived from bone marrow precursors, the concept of performing gene therapy for atherosclerosis through the retroviral transduction of hematopoietic stem cells has received much attention. This review will highlight recent advances that will help bring this goal closer. RECENT FINDINGS: The clinical application of retroviral gene transfer into hematopoietic stem cells has been hampered, in part, by the absence of vectors that can direct long-lasting, cell-type specific gene expression. In this review we will detail recent developments in the design of novel retroviral and lentiviral vectors that appear to overcome these problems, offering approaches to express therapeutic genes in specific cell-types within atherosclerotic lesions. We will also highlight advances in our understanding of the pathogenesis of atherosclerosis that may offer new gene therapeutic targets. SUMMARY: The use of retroviral transduction of hematopoietic stem cells for treatment of patients with atherosclerosis still remains a long-term goal. However, the recent development of retroviral vectors capable of directing expression to specific cell types within the lesion will allow more targeted therapeutic strategies to be devised. In addition, these vectors will provide powerful experimental tools to further our understanding of the pathogenesis of the disease.     

Phylogenetic detection of horizontal gene transfer during the step-wise genesis of Mycobacterium tuberculosis

Background  

In the past decade, the availability of complete genome sequence data has greatly facilitated comparative genomic research aimed at addressing genetic variability within species. More recently, analysis across species has become feasible, especially in genera where genome sequencing projects of multiple species have been initiated. To understand the genesis of the pathogen Mycobacterium tuberculosis within a genus where the majority of species are harmless environmental organisms, we have used genome sequence data from 16 mycobacteria to look for evidence of horizontal gene transfer (HGT) associated with the emergence of pathogenesis. First, using multi-locus sequence analysis (MLSA) of 20 housekeeping genes across these species, we derived a phylogeny that serves as the basis for HGT assignments. Next, we performed alignment searches for the 3989 proteins of M. tuberculosis H37Rv against 15 other mycobacterial genomes, generating a matrix of 59835 comparisons, to look for genetic elements that were uniquely found in M. tuberculosis and closely-related pathogenic mycobacteria. To assign when foreign genes were likely acquired, we designed a bioinformatic program called mycoHIT (mycobacterial homologue investigation tool) to analyze these data in conjunction with the MLSA-based phylogeny.     

The Evolution of Fungal Metabolic Pathways

Fungi contain a remarkable range of metabolic pathways, sometimes encoded by gene clusters, enabling them to digest most organic matter and synthesize an array of potent small molecules. Although metabolism is fundamental to the fungal lifestyle, we still know little about how major evolutionary processes, such as gene duplication (GD) and horizontal gene transfer (HGT), have interacted with clustered and non-clustered fungal metabolic pathways to give rise to this metabolic versatility. We examined the synteny and evolutionary history of 247,202 fungal genes encoding enzymes that catalyze 875 distinct metabolic reactions from 130 pathways in 208 diverse genomes. We found that gene clustering varied greatly with respect to metabolic category and lineage; for example, clustered genes in Saccharomycotina yeasts were overrepresented in nucleotide metabolism, whereas clustered genes in Pezizomycotina were more common in lipid and amino acid metabolism. The effects of both GD and HGT were more pronounced in clustered genes than in their non-clustered counterparts and were differentially distributed across fungal lineages; specifically, GD, which was an order of magnitude more abundant than HGT, was most frequently observed in Agaricomycetes, whereas HGT was much more prevalent in Pezizomycotina. The effect of HGT in some Pezizomycotina was particularly strong; for example, we identified 111 HGT events associated with the 15 Aspergillus genomes, which sharply contrasts with the 60 HGT events detected for the 48 genomes from the entire Saccharomycotina subphylum. Finally, the impact of GD within a metabolic category was typically consistent across all fungal lineages, whereas the impact of HGT was variable. These results indicate that GD is the dominant process underlying fungal metabolic diversity, whereas HGT is episodic and acts in a category- or lineage-specific manner. Both processes have a greater impact on clustered genes, suggesting that metabolic gene clusters represent hotspots for the generation of fungal metabolic diversity.     

Systematic Detection of Large-Scale Multigene Horizontal Transfer in Prokaryotes

Horizontal gene transfer (HGT) is central to prokaryotic evolution. However, little is known about the “scale” of individual HGT events. In this work, we introduce the first computational framework to help answer the following fundamental question: How often does more than one gene get horizontally transferred in a single HGT event? Our method, called HoMer, uses phylogenetic reconciliation to infer single-gene HGT events across a given set of species/strains, employs several techniques to account for inference error and uncertainty, combines that information with gene order information from extant genomes, and uses statistical analysis to identify candidate horizontal multigene transfers (HMGTs) in both extant and ancestral species/strains. HoMer is highly scalable and can be easily used to infer HMGTs across hundreds of genomes. We apply HoMer to a genome-scale data set of over 22,000 gene families from 103 Aeromonas genomes and identify a large number of plausible HMGTs of various scales at both small and large phylogenetic distances. Analysis of these HMGTs reveals interesting relationships between gene function, phylogenetic distance, and frequency of multigene transfer. Among other insights, we find that 1) the observed relative frequency of HMGT increases as divergence between genomes increases, 2) HMGTs often have conserved gene functions, and 3) rare genes are frequently acquired through HMGT. We also analyze in detail HMGTs involving the zonula occludens toxin and type III secretion systems. By enabling the systematic inference of HMGTs on a large scale, HoMer will facilitate a more accurate and more complete understanding of HGT and microbial evolution.     

Defence systems and horizontal gene transfer in bacteria

Horizontal gene transfer (HGT) is a fundamental process in prokaryotic evolution, contributing significantly to diversification and adaptation. HGT is typically facilitated by mobile genetic elements (MGEs), such as conjugative plasmids and phages, which often impose fitness costs on their hosts. However, a considerable number of bacterial genes are involved in defence mechanisms that limit the propagation of MGEs, suggesting they may actively restrict HGT. In our study, we investigated whether defence systems limit HGT by examining the relationship between the HGT rate and the presence of 73 defence systems across 12 bacterial species. We discovered that only six defence systems, three of which were different CRISPR-Cas subtypes, were associated with a reduced gene gain rate at the species evolution scale. Hosts of these defence systems tend to have a smaller pangenome size and fewer phage-related genes compared to genomes without these systems. This suggests that these defence mechanisms inhibit HGT by limiting prophage integration. We hypothesize that the restriction of HGT by defence systems is species-specific and depends on various ecological and genetic factors, including the burden of MGEs and the fitness effect of HGT in bacterial populations.     

Bartonella gene transfer agent: Evolution,function, and proposed role in host adaptation

The processes underlying host adaptation by bacterial pathogens remain a fundamental question with relevant clinical, ecological, and evolutionary implications. Zoonotic pathogens of the genus Bartonella constitute an exceptional model to study these aspects. Bartonellae have undergone a spectacular diversification into multiple species resulting from adaptive radiation. Specific adaptations of a complex facultative intracellular lifestyle have enabled the colonisation of distinct mammalian reservoir hosts. This remarkable host adaptability has a multifactorial basis and is thought to be driven by horizontal gene transfer (HGT) and recombination among a limited genus‐specific pan genome. Recent functional and evolutionary studies revealed that the conserved Bartonella gene transfer agent (BaGTA) mediates highly efficient HGT and could thus drive this evolution. Here, we review the recent progress made towards understanding BaGTA evolution, function, and its role in the evolution and pathogenesis of Bartonella spp. We notably discuss how BaGTA could have contributed to genome diversification through recombination of beneficial traits that underlie host adaptability. We further address how BaGTA may counter the accumulation of deleterious mutations in clonal populations (Muller's ratchet), which are expected to occur through the recurrent transmission bottlenecks during the complex infection cycle of these pathogens in their mammalian reservoir hosts and arthropod vectors.     

Switching on the lights for gene therapy

Strategies for non-invasive and quantitative imaging of gene expression in vivo have been developed over the past decade. Non-invasive assessment of the dynamics of gene regulation is of interest for the detection of endogenous disease-specific biological alterations (e.g., signal transduction) and for monitoring the induction and regulation of therapeutic genes (e.g., gene therapy). To demonstrate that non-invasive imaging of regulated expression of any type of gene after in vivo transduction by versatile vectors is feasible, we generated regulatable herpes simplex virus type 1 (HSV-1) amplicon vectors carrying hormone (mifepristone) or antibiotic (tetracycline) regulated promoters driving the proportional co-expression of two marker genes. Regulated gene expression was monitored by fluorescence microscopy in culture and by positron emission tomography (PET) or bioluminescence (BLI) in vivo. The induction levels evaluated in glioma models varied depending on the dose of inductor. With fluorescence microscopy and BLI being the tools for assessing gene expression in culture and animal models, and with PET being the technology for possible application in humans, the generated vectors may serve to non-invasively monitor the dynamics of any gene of interest which is proportionally co-expressed with the respective imaging marker gene in research applications aiming towards translation into clinical application.     

Phoenix rising: gene therapy makes a comeback

Despite the first application of gene therapy in 1990, gene therapy has until recently failed to meet the huge expectations set forth by researchers, clinicians, and patients, thus dampening enthusiasm for an imminent cure for many life-threatening genetic diseases. Nonetheless, in recent years we have witnessed a strong comeback for gene therapy, with clinical successes in young and adult subjects suffering from inherited forms of blindness or from X-linked severe combined immunodeficiency disease. In this review, various gene therapy vectors progressing into clinical development and pivotal advances in gene therapy trials will be discussed.     

Gene therapy: progress and challenges.

Gene therapy is the delivery of new genetic material into a patient's somatic cells for the treatment of disease and is made possible through the development of viral and non-viral gene transfer vectors. In the first five years of gene therapy, clinical studies failed to yield efficacy data with the vectors available at that time. The lack of consistent clinical benefit prompted the United States National Institute of Health Recombinant DNA Advisory Committee to evaluate gene therapy research and conclude that substantial improvements in gene transfer vectors were needed in the areas of vector safety and control of the level and duration of gene expression, and to increase the understanding of the biological interaction of gene transfer vectors with the host. We will describe the progress in development of gene delivery technology, focusing on improvements in vector safety, analysis of vector biodistribution and GMP manufacturing of viral and non-viral gene transfer systems over the last six years since the report. Whereas 5 years ago, investigators tested every vector for every potential disease indication, the accumulated database now enables investigators to select a single vector based upon it's known performance in a wide number of animal models and human clinical studies. We will also highlight several directions investigators have taken to improve the safety and efficacy of gene therapy vectors.     

Confounding factors in HGT detection: statistical error, coalescent effects, and multiple solutions.

Prokaryotic organisms share genetic material across species boundaries by means of a process known as horizontal gene transfer (HGT). This process has great significance for understanding prokaryotic genome diversification and unraveling their complexities. Phylogeny-based detection of HGT is one of the most commonly used methods for this task, and is based on the fundamental fact that HGT may cause gene trees to disagree with one another, as well as with the species phylogeny. Using these methods, we can compare gene and species trees, and infer a set of HGT events to reconcile the differences among these trees. In this paper, we address three factors that confound the detection of the true HGT events, including the donors and recipients of horizontally transferred genes. First, we study experimentally the effects of error in the estimated gene trees (statistical error) on the accuracy of inferred HGT events. Our results indicate that statistical error leads to overestimation of the number of HGT events, and that HGT detection methods should be designed with unresolved gene trees in mind. Second, we demonstrate, both theoretically and empirically, that based on topological comparison alone, the number of HGT scenarios that reconcile a pair of species/gene trees may be exponential. This number may be reduced when branch lengths in both trees are estimated correctly. This set of results implies that in the absence of additional biological information, and/or a biological model of how HGT occurs, multiple HGT scenarios must be sought, and efficient strategies for how to enumerate such solutions must be developed. Third, we address the issue of lineage sorting, how it confounds HGT detection, and how to incorporate it with HGT into a single stochastic framework that distinguishes between the two events by extending population genetics theories. This result is very important, particularly when analyzing closely related organisms, where coalescent effects may not be ignored when reconciling gene trees. In addition to these three confounding factors, we consider the problem of enumerating all valid coalescent scenarios that constitute plausible species/gene tree reconciliations, and develop a polynomial-time dynamic programming algorithm for solving it. This result bears great significance on reducing the search space for heuristics that seek reconciliation scenarios. Finally, we show, empirically, that the locality of incongruence between a pair of trees has an impact on the numbers of HGT and coalescent reconciliation scenarios.     

Inferring phylogenetic networks by the maximum parsimony criterion: a case study

Horizontal gene transfer (HGT) may result in genes whose evolutionary histories disagree with each other, as well as with the species tree. In this case, reconciling the species and gene trees results in a network of relationships, known as the "phylogenetic network" of the set of species. A phylogenetic network that incorporates HGT consists of an underlying species tree that captures vertical inheritance and a set of edges which model the "horizontal" transfer of genetic material. In a series of papers, Nakhleh and colleagues have recently formulated a maximum parsimony (MP) criterion for phylogenetic networks, provided an array of computationally efficient algorithms and heuristics for computing it, and demonstrated its plausibility on simulated data. In this article, we study the performance and robustness of this criterion on biological data. Our findings indicate that MP is very promising when its application is extended to the domain of phylogenetic network reconstruction and HGT detection. In all cases we investigated, the MP criterion detected the correct number of HGT events required to map the evolutionary history of a gene data set onto the species phylogeny. Furthermore, our results indicate that the criterion is robust with respect to both incomplete taxon sampling and the use of different site substitution matrices. Finally, our results show that the MP criterion is very promising in detecting HGT in chimeric genes, whose evolutionary histories are a mix of vertical and horizontal evolution. Besides the performance analysis of MP, our findings offer new insights into the evolution of 4 biological data sets and new possible explanations of HGT scenarios in their evolutionary history.     

A model of horizontal gene transfer and the bacterial phylogeny problem

How much horizontal gene transfer (HGT) between species influences bacterial phylogenomics is a controversial issue. This debate, however, lacks any quantitative assessment of the impact of HGT on phylogenies and of the ability of tree-building methods to cope with such events. I introduce a Markov model of genome evolution with HGT, accounting for the constraints on time -- an HGT event can only occur between concomitantly living species. This model is used to simulate multigene sequence data sets with or without HGT. The consequences of HGT on phylogenomic inference are analyzed and compared to other well-known phylogenetic artefacts. It is found that supertree methods are quite robust to HGT, keeping high levels of performance even when gene trees are largely incongruent with each other. Gene tree incongruence per se is not indicative of HGT. HGT, however, removes the (otherwise observed) positive relationship between sequence length and gene tree congruence to the estimated species tree. Surprisingly, when applied to a bacterial and a eukaryotic multigene data set, this criterion rejects the HGT hypothesis for the former, but not the latter data set.     

The interplay of restriction-modification systems with mobile genetic elements and their prokaryotic hosts

The roles of restriction-modification (R-M) systems in providing immunity against horizontal gene transfer (HGT) and in stabilizing mobile genetic elements (MGEs) have been much debated. However, few studies have precisely addressed the distribution of these systems in light of HGT, its mechanisms and its vectors. We analyzed the distribution of R-M systems in 2261 prokaryote genomes and found their frequency to be strongly dependent on the presence of MGEs, CRISPR-Cas systems, integrons and natural transformation. Yet R-M systems are rare in plasmids, in prophages and nearly absent from other phages. Their abundance depends on genome size for small genomes where it relates with HGT but saturates at two occurrences per genome. Chromosomal R-M systems might evolve under cycles of purifying and relaxed selection, where sequence conservation depends on the biochemical activity and complexity of the system and total gene loss is frequent. Surprisingly, analysis of 43 pan-genomes suggests that solitary R-M genes rarely arise from the degradation of R-M systems. Solitary genes are transferred by large MGEs, whereas complete systems are more frequently transferred autonomously or in small MGEs. Our results suggest means of testing the roles for R-M systems and their associations with MGEs.     

Association between translation efficiency and horizontal gene transfer within microbial communities

Horizontal gene transfer (HGT) is a major force in microbial evolution. Previous studies have suggested that a variety of factors, including restricted recombination and toxicity of foreign gene products, may act as barriers to the successful integration of horizontally transferred genes. This study identifies an additional central barrier to HGT-the lack of co-adaptation between the codon usage of the transferred gene and the tRNA pool of the recipient organism. Analyzing the genomic sequences of more than 190 microorganisms and the HGT events that have occurred between them, we show that the number of genes that were horizontally transferred between organisms is positively correlated with the similarity between their tRNA pools. Those genes that are better adapted to the tRNA pools of the target genomes tend to undergo more frequent HGT. At the community (or environment) level, organisms that share a common ecological niche tend to have similar tRNA pools. These results remain significant after controlling for diverse ecological and evolutionary parameters. Our analysis demonstrates that there are bi-directional associations between the similarity in the tRNA pools of organisms and the number of HGT events occurring between them. Similar tRNA pools between a donor and a host tend to increase the probability that a horizontally acquired gene will become fixed in its new genome. Our results also suggest that frequent HGT may be a homogenizing force that increases the similarity in the tRNA pools of organisms within the same community.     

Extensive Intra-Kingdom Horizontal Gene Transfer Converging on a Fungal Fructose Transporter Gene

Comparative genomics revealed in the last decade a scenario of rampant horizontal gene transfer (HGT) among prokaryotes, but for fungi a clearly dominant pattern of vertical inheritance still stands, punctuated however by an increasing number of exceptions. In the present work, we studied the phylogenetic distribution and pattern of inheritance of a fungal gene encoding a fructose transporter (FSY1) with unique substrate selectivity. 109 FSY1 homologues were identified in two sub-phyla of the Ascomycota, in a survey that included 241 available fungal genomes. At least 10 independent inter-species instances of horizontal gene transfer (HGT) involving FSY1 were identified, supported by strong phylogenetic evidence and synteny analyses. The acquisition of FSY1 through HGT was sometimes suggestive of xenolog gene displacement, but several cases of pseudoparalogy were also uncovered. Moreover, evidence was found for successive HGT events, possibly including those responsible for transmission of the gene among yeast lineages. These occurrences do not seem to be driven by functional diversification of the Fsy1 proteins because Fsy1 homologues from widely distant lineages, including at least one acquired by HGT, appear to have similar biochemical properties. In summary, retracing the evolutionary path of the FSY1 gene brought to light an unparalleled number of independent HGT events involving a single fungal gene. We propose that the turbulent evolutionary history of the gene may be linked to the unique biochemical properties of the encoded transporter, whose predictable effect on fitness may be highly variable. In general, our results support the most recent views suggesting that inter-species HGT may have contributed much more substantially to shape fungal genomes than heretofore assumed.     

Horizontal gene transfer from extinct and extant lineages: biological innovation and the coral of life

Horizontal gene transfer (HGT) is often considered to be a source of error in phylogenetic reconstruction, causing individual gene trees within an organismal lineage to be incongruent, obfuscating the ‘true’ evolutionary history. However, when identified as such, HGTs between divergent organismal lineages are useful, phylogenetically informative characters that can provide insight into evolutionary history. Here, we discuss several distinct HGT events involving all three domains of life, illustrating the selective advantages that can be conveyed via HGT, and the utility of HGT in aiding phylogenetic reconstruction and in dating the relative sequence of speciation events. We also discuss the role of HGT from extinct lineages, and its impact on our understanding of the evolution of life on Earth. Organismal phylogeny needs to incorporate reticulations; a simple tree does not provide an accurate depiction of the processes that have shaped life''s history.     

Cell-Type-Specific Gene Transfer into Human Cells with Retroviral Vectors That Display Single-Chain Antibodies

The successful application of human gene therapy protocols on a broad clinical basis will depend on the availability of in vivo cell-type-specific gene delivery systems. We have developed retroviral vector particles, derived from spleen necrosis virus (SNV), that display the antigen binding site of an antibody on the viral surface. Using retroviral vectors derived from SNV that displayed single-chain antibodies (scAs) directed against a carcinoembryonic antigen-cross-reacting cell surface protein, we have shown that an efficient, cell-type-specific gene delivery can be obtained. In this study, we tested whether other scAs displayed on SNV vector particles can also lead to cell-type-specific gene delivery. We displayed the following scAs on the retroviral surface: one directed against the human cell surface antigen Her2neu, which belongs to the epidermal growth factor receptor family; one directed against the stem cell-specific antigen CD34; and one directed against the transferrin receptor, which is expressed on liver cells and various other tissues. We show that retroviral vectors displaying these scAs are competent for infection in human cells which express the antigen recognized by the scA. Infectivity was cell type specific, and titers above 10⁵ CFU per ml of tissue culture supernatant medium were obtained. The density of the antigen on the target cell surface does not influence virus titers in vitro. Our data indicate that the SNV vector system is well suited for the development of a large variety of cell-type-specific targeting vectors.