Virus genetic treatment

Differences among single- and double-strand DNA viruses may also be explained in terms of their access to post-replicative repair. Dam methylation of GATC sequence motifs is used to differentiate the template and daughter DNA strands and is thus required to perform mismatch correction [ 22 ]. Mismatches are recognized by MutS, which interacts with MutL and leads to the activation of the MutH endonuclease, which excises the daughter strand. Avoidance of GATC motifs may be a consequence of selection acting on mutation rate, but also of other selective factors.

For instance, inefficient methylation of the phage DNA may render it susceptible to cleavage by MutH, therefore imposing a selection pressure against GATC sequence motifs [ 24 ]. Numerous studies have shown that viruses interact with DNA damage response DDR pathways by altering the localization or promoting the degradation of DDR components [ 25 , 26 ].

DDR activation can occur as an indirect consequence of cellular stress due to the infection per se or as a part of an antiviral response, which would be in turn counteracted by viruses. A general inverse correlation between genome size and mutation rate applies to DNA-based microorganisms including viruses, bacteria and unicellular eukaryotes [ 28 ]. According to this rule, the per-genome mutation rate stays relatively constant at a value of approximately 0.

A similar negative relationship seems to exist in RNA viruses, but their smaller genome size range of variation makes it more difficult to detect such trend Fig. Supporting this correlation, however, coronaviruses have the largest genomes among RNA viruses 30—33 kb and have evolved proofreading capacity, as opposed to all other RNA viruses known [ 11 ].

Therefore, there appears to be a general negative correlation between mutation rates and genome size in microorganisms. However, the underlying causes remain unclear, both at the mechanistic and evolutionary levels. First, there are no known differences in intrinsic replication fidelity among the polymerases of different RNA viruses excepting coronavirus exonuclease activity. Estimates for small double-strand DNA viruses would be needed to clarify which of these two factors contributes more to elevating mutation rates.

The observation that most highly variable and rapidly evolving DNA viruses have small genomes including double-strand viruses indirectly supports an effect of genome size [ 3 ].

Candidate mechanisms that might account for mutation rate differences between large and small DNA viruses may involve virus—DDR interactions. Whereas many viruses appear to evade DDR, others seem to use it for their own benefit [ 25 , 26 ]. Polyomaviruses, papillomaviruses and parvoviruses induce and depend on DDR signaling pathways for efficient replication [ 30 — 32 ].

These viruses share the property of having small, circular DNA genomes which do not encode a polymerase. As such, they depend directly on the cellular replication machinery, as opposed to larger DNA viruses. It is possible that some small viruses promote the DDR to prolong the S cell-cycle phase, which offers a more favorable environment for replication. By adopting circular genomes, these viruses would also avoid the formation of genome concatemers, a typical effect of DDR in linear viral genomes such as, for instance, adenoviruses [ 33 ].

The DDR comprises error-prone DNA polymerases for re-synthesis of excised strands [ 34 ], and involvement of these polymerases in viral replication may lead to higher mutation rates.

Intrinsic polymerase fidelity i. Polymerase variants with altered fidelity have been artificially selected in a number of RNA viruses by subjecting laboratory populations to mutagenic treatments [ 35 ]. For instance, serial passaging of poliovirus in the presence of the base analog ribavirin led to the selection of a polymerase variant G64S with threefold increased fidelity [ 36 ]. This same mutation also confers increased fidelity in the related human enterovirus 71 [ 37 ], and other amino acid replacements such as LF have also been shown to modify the replication fidelity of this virus [ 38 ].

Passaging of coxsackievirus B3 also a member of the enterovirus genus in the picornavirus family in the presence of ribavirin or 5-azacytidine selected for another fidelity variant in the viral polymerase AV [ 39 ].

Outside picornaviruses, fidelity variants have been more recently obtained by serial mutagen treatment in chikungunya virus [ 40 ], influenza A virus [ 41 ], and West Nile virus [ 42 ]. Several antivirals and notably many antiretroviral drugs are base analogs. Resistance to these treatments is well documented in the HIV-1 RT and some of these variants modify replication fidelity, as determined in vitro or in cell cultures [ 13 ]. Intrinsic fidelity can be determined by residues located inside or outside the catalytic domain [ 43 , 44 ].

For instance, reorientation of the triphosphate moiety of the incoming nucleotide is a fidelity checkpoint in poliovirus polymerase [ 45 ]. Interestingly, recent work has shown that replication fidelity can also be determined by proteins of the replication complex other than the viral polymerase. Serial passages of chikungunya virus in the presence of nucleoside analogs favored the appearance of substitution GD in the RNA helicase nsP2 [ 40 ].

This variant increased replication fidelity through mechanisms linked to reduced helicase activity, increased replication kinetics, and resistance to low nucleotide concentrations [ 46 ].

Fidelity variants demonstrate the ability of RNA viruses to evolutionarily adjust mutation rates in response to selection acting on mutation rate or other traits. DNA virus mutation rates also respond to selection, as shown in earlier work with bacteriophage T4 in which a series of polymerase variants were identified following chemical mutagenesis [ 47 ].

T4 polymerase variants showing strongly increased fidelity have been described as opposed to more modest effects in RNA viruses and tend to map to the central palm and the carboxyl-terminal thumb subdomain of the viral polymerase. Mutator phenotypes have also been described in T4.

This phenotype can be conferred by changes in replication factors such as single stranded DNA-binding proteins or helicase proteins [ 48 ]. Similar results were obtained with herpes simplex virus type 1 HSV-1 , for which mutations in the conserved regions of the polymerase domain were found to modify replication fidelity.

However, this variant rapidly evolved a compensatory substitution LF that restored DNA replication fidelity in this genetic background [ 49 , 50 ]. Since RNA virus polymerases typically lack this activity, no such mutators can be produced, except for coronaviruses [ 51 ]. Furthermore, the genetic diversity of RNA viruses is probably closer to an upper tolerability limit beyond which the population genetic load increases to levels incompatible with virus survival [ 3 , 52 ].

Therefore, both biochemical and population-genetic factors limit the appearance of strong mutators in RNA viruses. Whereas post-replicative repair probably plays a role in determining DNA virus mutation rates as discussed above , RNA virus mutation rates are strongly influenced by other host-encoded factors. Apolipoprotein B mRNA-editing catalytic polypeptide-like enzymes APOBEC are a family of cellular cytidine deaminases that function as an innate cellular defense against retroviruses [ 53 ].

There are seven APOBEC3 paralogs in the human genome A—D and F—H which have been shown to also edit retroelements and other viruses, including hepatitis B virus [ 59 ], papillomaviruses [ 60 ], and herpesviruses [ 61 ]. Editing is strongly dependent on sequence-context. DNA editing hotspots have been identified and depend both on sequence context and DNA secondary structure [ 62 ].

In many cases, hyper-mutation leads to loss of infectivity and hence effectively exerts its antiviral action. However, APOBECs can also produce moderately mutated, viable viruses, thus raising the question whether these deaminases may contribute to viral diversity and evolution, immune escape, and drug resistance [ 64 — 66 ].

Double-strand RNA-dependent adenosine deaminases ADARs are another type of host enzymes that edit viral genomes by deaminating adenosines in long double-stranded RNA and converting them to inosines.

The latter base-pair with guanosines, resulting in A-to-G base substitutions [ 67 ]. ADAR-driven hyper-mutation was first demonstrated in measles virus [ 69 ] and has since been suggested for a variety of RNA viruses including human parainfluenza virus [ 70 ], respiratory syncytial virus [ 71 ], lymphocytic choriomeningitis virus [ 72 ], Rift Valley fever virus [ 73 ], and noroviruses [ 74 ].

Uracil can be found in DNA abnormally due to spontaneous or enzymatically induced cytidine deamination, leading to G-to-A mutations. Failure to incorporate UNG produces a fourfold increase of the HIV-1 mutation rate in actively dividing cells, and of fold in macrophages [ 75 , 76 ].

Variations in the concentration and balance of dNTPs among cell types may also influence viral mutation rates [ 77 ]. Although analysis of HIV-1 mutations in various cell lines revealed no obvious mutation rate differences, it nevertheless showed differences in the type of mutations produced [ 78 ]. In contrast to cells, viruses can adopt a variety of replication modes. Under this theoretical model, there is only one round of copying per cell. In practice, this means that each infecting genome is used to synthesize a single reverse-complementary intermediate which in turn is used as template for synthesizing all progeny genomes.

This contrasts with semi-conservative replication, in which each strand is copied once to produce progeny molecules that are, in turn, used as templates in the next round of copying.

Since under semi-conservative replication the number of strands doubles in each cycle, the virus necessarily has to undergo multiple replication cycles within each cell to produce enough progeny.

Under stamping machine replication the mutation frequency observed after one cell infection equals the mutation rate, but under semi-conservative replication this frequency is also determined by the number of replication cycles, as mutants become amplified.

This means that a given viral polymerase will produce more mutations per cell if replication is semi-conservative than if replication is stamping machine-like. These two models are indeed two extremes of a continuum of possible replication modes. For instance, a virus can produce multiple progeny molecules per round of copying which then undergo a second replication cycle in the same cell to end up producing hundreds or thousands of progeny molecules.

Viral replication modes and mutation accumulation. As opposed to cells, which use only semi-conservative replication, viruses can adopt a variety of replication modes. In the stamping machine model, a single template strand is used to synthesize all progeny genomes within a given cell. Under this model, the mutation frequency after one cell infection cycle will equal the mutation rate except if mutations occur during the first round of copying from genome to anti-genome , in which case they will be present in all of the viral progeny.

Under semi-conservative replication, multiple rounds of copying are required to produce enough progeny, thus allowing for the intra-cellular accumulation of mutations. Longer cell infection cycles late burst can allow for the production of more progeny viruses.

Under semi-conservative replication, this will require more rounds of copying but, if replication follows the stamping machine model, the number of rounds of copying will not change more progeny genomes will be produced from the same template. Hence under this model, a late-burst virus variant will undergo fewer total rounds of copying at the population scale than early-burst variants and will tend to accumulate fewer mutations. It has been suggested that the stamping machine model has been selectively favored in RNA viruses because it compensates for the extremely high error rate of their polymerases [ 79 — 81 ].

However, empirically-informed modeling of the poliovirus replication cycle indicated multiple rounds of copying per cell [ 85 ]. Similarly, single-cell analysis of the genetic diversity produced by vesicular stomatitis virus revealed that some mutations are amplified within cells, implying that multiple rounds of copying take place per cell [ 86 ]. However, it remains unknown whether a given virus can modify its replication mode in response to specific selective pressures in order to promote or down-regulate mutational output.

To a large extent, the replication mode of most viruses should be dictated by the molecular mechanisms of replication and, hence, should be subjected to strong functional constraints.

In contrast, semi-conservative replication is probably the only mechanistically feasible replication model for viruses with large DNA genomes. Changes in lysis time can be thought of as another mechanism for regulating the production of mutations in viral populations. Lysis is a tightly regulated process and, in theory, viral fitness is maximized for some intermediate lysis time [ 89 — 91 ].

If lysis occurs before this optimum, the infected cell will release a small amount viral progeny and hence few cells will be infected in the next infection cycle, retarding population growth. Yet if lysis occurs after the optimum, a large amount of progeny will be produced per cell but cell-to-cell transmission will be delayed.

However, the optimum can also vary according to mutation rate. Interestingly, 5-fluorouracil selected for an amino acid replacement in the N-terminal region of the phage lysis protein V2A. This change conferred partial resistance to the drug, but also delayed lysis [ 93 ]. In turn, delayed lysis was concomitant with an increase in the viral yield per cell, since progeny virions had more time to accumulate intracellularly.

Therefore, at the population level, growth of the V2A variant occurred through longer infection cycles with increased per-cell productivity.

However, because the virus replicates following a stamping machine model, each infection cycle should involve only one round of copying regardless of lysis time. As a result, population growth required fewer total rounds of copying in the delayed lysis variants than in the wild-type, meaning that mutations had fewer opportunities to accumulate Fig.

Therefore, delayed lysis increased the ability of the phage to tolerate mutagenesis. The fidelity of a given polymerase varies according to certain template properties. It is well known that misalignments at homopolymeric runs can cause frameshift mutations and base substitutions [ 94 ].

Sequence context may influence the fidelity of HIV-1 RT by modulating enzyme binding and dissociation [ 95 ]. Also, RNA secondary structures have been shown to promote template switching, a process that does not lead to new mutations but produces recombinant viruses [ 96 — 98 ]. Shuttle vectors are systems in which most or all sequences except essential cis-acting elements such as the Rev-responsive element or long terminal repeats have been removed from the viral genome.

Shuttle vectors allow propagating HIV-1 in the absence of selection because all required functions are provided in trans by helper plasmids that are freshly provided in each infection cycle [ ] Fig.

The shuttle vector simply carries forward sequences of interest, which can be reporter genes for selecting and visualizing transduced cells, or transgenes for engineering purposes. However, the vector also accepts HIV-1 sequences.

These will have no role in the infection cycle, as they are not expressed. Because selection is absent, such HIV-1 sequences cloned in a shuttle vector can be used for interrogating the viral mutation rate in cognate templates, which is helpful for testing the effects of sequence context or RNA structure on mutation rate. Cell culture systems for the accumulation of mutations in the absence of selection. A resistance gene RES, red is inserted to allow for the selection of cells containing the vector.

Any short sequence of interest SEQ, blue , including HIV sequences, can be cloned in the shuttle vector and propagated in the absence of selection. The shuttle vector DNA is co-transfected with helper plasmids encoding the Gag capsid and Pol RT, integrase proteins as well as a viral glycoprotein suited for transducing a given cell line here vesicular stomatitis G protein, VSV-G, which has a broad tropism.

Pseudotyped viruses are produced, used for transduction, and cells carrying the retroviral shuttle vector are selected with the appropriate antibiotic. The infection cycle can be restarted at any time by transfecting the two helper plasmids.

Two cistrons are separated by an internal ribosome entry site IRES. The right cistron encodes HCV non-structural NS proteins required for replication, but lacks the envelope proteins and hence does not support viral budding. The left replicon carries a resistance gene to select cells carrying the replicon.

Reporters such as luciferase can be also cloned in this cistron. Replicon RNA is obtained by in vitro transcription and transfected into Huh7 hepatoma cells. Cells are selected using the appropriate antibiotic and passaged before confluence to allow vigorous replication of the viral RNA. Using this system, we recently characterized the distribution of mutations along the HIV-1 envelope, integrase, vif , and vpr genes [ 99 ].

We found that a 1 kb region encompassing the V1—V5 loops of the gp envelope protein accumulated approximately three times fewer mutations than other regions of the HIV-1 genome. This coldspot mapped to the outermost domains of gp, which are preferred targets of circulating antibodies and show extensive glycosylation. Examination of this region revealed two differential properties. To more directly test the effect of RNA structure on HIV-1 RT fidelity, we used in vitro polymerization assays with two different templates: a random sequence and RNA from potato spindle tuber viroid, which shows a marked, stem-like secondary structure [ ].

Using a conceptually similar approach, we recently characterized the accumulation of mutations along the HCV genome under weak or no selection using a bicistronic replicon by cloning HCV sequences at a site commonly used for inserting reporter genes Fig. This revealed extreme mutation rate variations across individual nucleotide sites of the viral genome, with differences of orders of magnitude even between adjacent sites [ ].

In that system, we found little or no effect of RNA structure on mutation rate, but a more significant effect of base identity, such that A and U bases were more prone to mutation than G and C. The finding that HIV-1 has a reduced mutation rate in the genome region encoding the outermost domains of the gp envelope protein reveals an uncoupling between mutation rate and genetic diversity, as these domains are the most variable regions of the HIV-1 genome, mainly as a consequence of immune pressure [ ].

This indicates that HIV-1 has not evolved the ability to target mutation to regions wherein they are more likely to be needed for adaptation. Similarly, strong selection at the protein level may have favored amino acid replacements within this region even at the cost of disrupting pre-existing RNA secondary structures and, as a consequence, these RNA structural changes would have modified replication fidelity [ 99 ].

In HCV, we found no significant differences in mutation rate across genes [ ], as opposed to genetic variation, which concentrates in specific genomes regions including external domains of the E2 envelope protein [ ]. This again supports the view that RNA viruses cannot target mutations to specific genomes regions to improve their adaptability. This contrasts with bacteria and DNA viruses, in which mechanisms of error-prone replication have evolved at specific loci involved in host-pathogen interactions [ — ].

A well-characterized system of mutation targeting, called diversity-generating retro-elements DGRs , is found in large DNA bacteriophages [ ]. DGRs are typically located in genes involved in host attachment, a step of the infection cycle that is subject to rapid changes depending on host species availability. The cDNA is then transferred to the VR, producing a large number of variants of the mtd gene capable of interacting with new host ligands [ ].

DGRs have also been described in plasmids, bacterial and archaeal chromosomes, and archaeal viruses [ — ]. It therefore appears that at least some prokaryotic DNA viruses have evolved the ability to target mutations to specific regions, as opposed to RNA viruses. Diversity-generating retro-elements have not been described in eukaryotic viruses, but these viruses can use other mechanisms of mutational targeting that involve recombination. The inverted terminal repeats of vaccinia virus contain 10— base repeated sequence motifs known to experience frequent unequal crossover events and rapid changes in copy number [ , ].

Recombination has been shown to promote the rapid production of genetic diversity in other genome regions of the vaccinia virus involved in immune escape and the colonization of novel hosts. Protein kinase R PKR is a central effector of innate antiviral immunity that induces translational shutoff, modifies protein phosphorylation status, alters mRNA stability, and induces apoptosis [ ]. Poxvirus proteins K3L and E3L block PKR and have evolved as antagonists of innate immune responses in a host-specific manner [ , ].

Experimental deletion of E3L renders vaccinia virus more susceptible to host antiviral responses, imposing a strong selection pressure in the other PKR suppressor K3L to increase its function [ ].

Serial transfers of E3L-deleted vaccinia virus led to an elevated K3L copy number, a recombination-driven process that allowed the virus to overexpress this gene. This gain-of-function mutation had a direct fitness benefit, but also increased the number of available targets for the appearance of subsequent selectively advantageous point mutations in K3L.

Remarkably, upon selection of these mutants K3L copy numbers were again reduced. Hence, recombination led to an evolutionary process characterized by expansion and contraction of a specific genome region. These so-called genomic accordions have been posited to mediate adaptive duplications in other poxviruses such as myxoma virus [ ].

Interesting interplays between recombination and mutation rates have also been recently found in RNA viruses. These two processes are primarily controlled by the viral polymerase since, in RNA viruses, recombination takes place when the viral polymerase switches between different template genomes present in the same cell [ ]. The estimated recombination rates of different riboviruses and retroviruses correlate positively with estimated mutation rates [ ].

High mutation rates confer viruses the ability to rapidly produce advantageous mutations, but also inflate the genetic load of the population. In turn, frequent recombination allows beneficial mutations to unlink from deleterious genetic backgrounds, as well different beneficial mutations to be combined into the same genome. The gene therapy clinical trials underway in the U. Currently, the only way for you to receive gene therapy is to participate in a clinical trial. Clinical trials are research studies that help doctors determine whether a gene therapy approach is safe for people.

They also help doctors understand the effects of gene therapy on the body. Your specific procedure will depend on the disease you have and the type of gene therapy being used. Viruses aren't the only vectors that can be used to carry altered genes into your body's cells.

Other vectors being studied in clinical trials include:. The possibilities of gene therapy hold much promise. Clinical trials of gene therapy in people have shown some success in treating certain diseases, such as:. But several significant barriers stand in the way of gene therapy becoming a reliable form of treatment, including:. Gene therapy continues to be a very important and active area of research aimed at developing new, effective treatments for a variety of diseases. Explore Mayo Clinic studies of tests and procedures to help prevent, detect, treat or manage conditions.

Mayo Clinic does not endorse companies or products. Advertising revenue supports our not-for-profit mission. Check out these best-sellers and special offers on books and newsletters from Mayo Clinic Press. This content does not have an English version.

This content does not have an Arabic version. Overview Gene therapy involves altering the genes inside your body's cells in an effort to treat or stop disease. In support of our mission , we are committed to advancing research on genetic therapies in part through the following ways. We lead or sponsor many studies relevant to genetic therapies using gene transfer or genome editing.

See whether you or someone you know is eligible to participate in our clinical trials. Learn more about participating in a clinical trial. View all trials from ClinicalTrials.

After reading our Genetic Therapies Health Topic, you may be interested in additional information found in the following resources.

Genome editing can do the following: Remove a stretch of DNA that causes a disease Turn off a gene to prevent it from making a harmful protein Turn on a gene or instruct a cell to make more of a needed protein Correct a mutated gene Gene transfer or genome editing treatments can directly modify the cells in your body, or your cells can be collected and treated outside of your body and then returned to you.

How It Works - Genetic Therapies. Gene transfer. These two panels represent a cell with a faulty gene, caused by a mutation, before and after successful gene transfer. The faulty gene in this example makes proteins that are faulty, as shown at left. After gene transfer, the cell makes normal functional proteins from the addition of the treatment gene.

Genome editing. These two panels represent a cell with a faulty gene before and after successful genome editing. In this case, genome editing repairs the gene itself, rather than adding an extra gene. After genome editing, the repaired gene allows the cell to make normal functional proteins. Who May Benefit - Genetic Therapies. Treatments for blood and immune conditions. Cell-based genetic therapy. Treatments for organs or tissues. What Are the Risks? Research for Your Health. Improving health with current research.

The goal is to have these genetic therapies ready to safely use in clinical research in 5 to 10 years. The patient-focused Initiative brings together academic and private sector researchers, patients, providers, advocacy groups, and others as it supports research, education, and community engagement activities. Supporting Safe Manufacturing of Cell-based Therapies. PACT offers education and resources through its five cell processing facilities and a coordinating center that support activities such as safety testing, product shipment, and design of clinical testing protocols.

Addressing Issues in Blood and Marrow Transplantation. In the United States, nearly 21, patients receive blood or marrow transplants annually, mainly for rare blood disorders.

Supporting Development of Genome Editing Tools. These research tools will be made widely available to the research community to reduce the time and cost required to develop new therapies. The researchers then used a virus vector to add a corrective gene to the stem cells. In preliminary laboratory tests, the red blood cells derived from the corrected stem cells produced almost all normal hemoglobin and very little sickle hemoglobin.

This genetic therapy approach may hold promise for sickle cell disease, but more research is needed. Visit Experimental gene-editing approach holds promise for curing sickle cell disease for more information.

Helping scientists prepare gene therapies for clinical trials. It provides a publicly available repository of AAV- and lentiviral plasmids, archiving services for samples generated during pharmacology-toxicology studies, and testing services for vector copy number, replication competency, and insertion site analysis. NGVB also offers a searchable database containing detailed summaries of more than 50 preclinical gene therapy pharmacology-toxicology studies and additional educational resources.

Our Gene Therapy Resource Program GTRP offers a variety of services to researchers to help them advance experimental gene therapies from the laboratory into clinical human trials.

Since , GTRP has provided safety testing services and set up a coordinating center to manage the program. GTRP can also help researchers apply for approvals from federal agencies to start clinical trials. These efforts have helped launch studies across a wide range of genetic, heart, lung, and blood diseases. Learn more about the GTRP and the projects it has supported. Advancing research for improved health. We perform research.

Our Division of Intramural Research , which includes investigators in our Hematology Branch and Sickle Cell Program , is actively engaged in research on genetic therapies. We fund research. The research we fund today will help improve our future health.

Our Division of Blood Diseases and Resources DBDR and Division of Cardiovascular Sciences oversee much of the research on genetic therapies we fund, helping us advance new genetic therapies for noncancerous blood disorders. Our Division of Lung Diseases also supports research that includes genetic therapies for inherited lung diseases such as cystic fibrosis. We stimulate high-impact research. Our Cure Sickle Cell Initiative is a patient-focused effort that engages investigators to develop genetic therapies for patients who have sickle cell disease.