Patrick Arbuthnot

Gene Therapy for Respiratory Viral Infections

n Abstractn n Pulmonary infections by viruses may result in serious diseases of public health importance. The problems of the infections are exacerbated by rapid transmission of the pathogenic agents, which occur through inhalation and direct contact with contaminated surfaces. Moreover, cross-species transmission resulting from changes to viral genetic makeup poses a risk for emergence of pathogens with new characteristics, which in some cases may be responsible for causing different diseases. With the advent of efficient sequencing and nucleic acid-based virus-disabling technologies, gene therapy is well placed to advance new treatments to counter respiratory infections. Most studies aimed at using nucleic acids to treat respiratory viral infections have used RNA interference (RNAi) to silence viral gene targets. A few studies have used silencing of host factors required by the viruses as a means of inhibiting viral replication and preventing emergence of escape mutants. By administering antivirals to the airways, studies performed inxa0vivo have taken advantage of the anatomy of the respiratory system to deliver therapeutic nucleic acids. Reported data have shown proof of principle of efficacy of gene therapy in models of respiratory syncytial virus (RSV), severe acute respiratory syndrome coronavirus, influenza virus A, and measles virus, among others. RNAi-based gene therapy has been advanced to clinical trial for treatment of RSV infection. Although the primary endpoint was not met in an intent-to-treat analysis, the investigation has provided useful information for the advancement of gene therapy for current and emergent respiratory infections.n n

Essentials of Viruses and their Suitability for Treatment Using Gene Therapy

Viruses constitute the simplest life forms and are a major cause of disease. They are also very abundant and make up a reservoir of enormous genetic diversity. As obligate intracellular parasites, viruses orchestrate intricate sets of reactions that recruit and usurp cellular functions in such a way as to facilitate their replication. Highly efficient host antiviral mechanisms, particularly innate and adaptive immune responses, are triggered in response to an infection. To control these effects, viruses have evolved ways of evading or partly disabling host antiviral responses. Treatment of patients with small-molecule antivirals, immunomodulators, and immunoprophylaxis by vaccination are currently licensed therapeutic antiviral measures. Harnessing gene therapy offers the potential for developing versatile and effective new intervention strategies. A rational design approach, which only requires basic knowledge of the viral nucleic acid sequences, is particularly advantageous for antiviral gene therapies. Strategies have generally entailed inactivation of viral sequences and host factors. Gene transfer may also be used to induce antiviral immunity. Several approaches to disabling gene function have been used; the use of RNA interference activators and engineering designer nucleases are particularly promising. Because candidate gene therapy drugs are complex, successful implementation of antiviral gene therapy faces significant challenges. Ensuring specificity, efficient delivery to target tissue, and adequate therapeutic effects without toxicity are crucial for the advancement of antiviral gene therapy to clinical use.

Harnessing RNAi to Silence Viral Gene Expression

Since discovery of the RNA interference (RNAi) pathway, there has been rapid progress in research aimed at harnessing this gene silencing mechanism for antiviral therapeutic application. Micro RNAs (miRs) are the prototype natural activators of RNAi. Their maturation entails a regulated stepwise process during which transcripts with hairpin motifs within primary miRs (pri-miRs) are cleaved by the nuclear microprocessor complex to generate precursor miRs (pre-miRs). pre-miRs are exported to the cytoplasm before further processing by Dicer, an RNase III, to generate the mature miR comprising a short duplex RNA sequence. One of the strands is selected for incorporation into the RNA induced silencing complex (RISC), where it naturally serves as a guide to direct translational suppression of mRNA targets. Cellular and viral mechanisms operate to control miR maturation and thereby modulate gene silencing by these short RNA sequences.

Delivery of Antiviral Nucleic Acids with Nonviral Vectors

Nucleic acids are large and highly negatively charged, which make use of carriers to transport the therapeutic sequences to virus-infected cells an important requirement. Because use of gene therapy to treat viral infections has enormous potential, advancement of technology using nonviral vectors (NVVs) to deliver antiviral sequences is fundamental to widespread implementation of the antiviral strategy. NVVs are suited to therapeutic application for several reasons. They are amenable to large-scale synthesis, which will be necessary for treatment of commonly occurring infections. NVVs have lower immunogenicity than viral vectors, and they may be adapted for delivery of various nucleic acids. The modular nature of NVVs enables convenient modification of their components so that biological properties may be altered to match particular applications. NVVs include lipid-based vectors, complexes, various polymer conjugates, formulations containing cell-penetrating peptides, and aptamers. Research into developing NVVs is a highly active field, and insights into the relationship between the physicochemical properties of the vectors and their behavior as carriers of antiviral nucleic acids are rapidly improving. Ingenious methods of ensuring protection from degradation by nucleases, stability of the formulations, attenuation of immunostimulation, targeting of antivirals to specific cells, and efficient escape from maturing endosomes have been developed. NVVs are currently undergoing evaluation in clinical trials for treatment of various diseases that include hepatitis B virus infection. Outcomes from these studies will be very important to inform further development and broader use of antiviral gene therapy.

Gene Therapy for HIV-1 Infection

Human immunodeficiency virus (HIV)-1 infection of humans, which originally resulted from zoonotic transmission of a simian retrovirus from chimpanzees in Africa, emerged as a serious public health problem during the early 1980s. The infection was lethal and rapidly spread to become a global pandemic. This drove a major research effort that led to dramatically improved treatment of people infected with HIV-1. Currently available combination antiretroviral therapy (cART) limits progression to the acquired immune deficiency syndrome, effectively suppresses viral replication, and improves hosts immune function. Although mortality and morbidity have improved since the commencement of cART, the infection remains a significant disease burden. Failures of vaccine trials and difficulties with developing a cure have been particularly disappointing. Interest in gene therapy has recently been spurred because of the potential of this technology to achieve durable suppression of HIV-1 replication and a functional cure from the infection. In addition, elimination of HIV-1 from an infected individual who received a bone marrow transplant from a donor who had a homozygous deficiency in the CCR5 viral co-receptor, the “Berlin patient,” has encouraged harnessing of gene therapy to simulate the CCR5-deficient phenotype in autologous cells. Different approaches to inhibiting HIV-1 replication have been developed, and use of the technology to modify hematopoietic progenitor cells exxa0vivo has shown promise. Using RNA interference-based silencing and gene editing technology to disable viral sequences or host factors have been especially encouraging. In addition to making cells resistant to HIV-1 infection by disrupting ccr5 , gene editing could be used to eliminate the provirus from reservoirs of latently infected cells. However, before gene therapy can be expected to make an effect on the disease burden caused by HIV-1, issues such as ensuring specificity, optimizing delivery to target cells, and devising simple and affordable regimens will be important.

Gene Therapy for Hepatitis C Virus Infection

Hepatitis C virus (HCV) infection remains an important global health problem. Persistence of the virus is associated with high risk for hepatocellular carcinoma and cirrhosis. Since the discovery of the virus in 1989, progress in understanding the molecular biology of replication of HCV has been impressive. The improved insights have been accompanied by rapid advances in the development of new therapies. There are now several new licensed therapeutics or drug candidates, which include anti-HCV nucleic acids, at advanced stages of development. Reliance of HCV on miR-122 for replication has led to the development of miravirsen, an antisense-locked nucleic acid, for HCV treatment. Results from phase IIa clinical trials are encouraging and indicate that this antisense sequence has promising therapeutic potential. As an RNA virus that replicates in the hepatocyte cytoplasm, HCV is suitable for RNA-targeting strategies. RNA interference (RNAi), aptamers, ribozymes, and antisense technologies have all been harnessed but with varying success. Many studies have shown that activating RNAi is effective against HCV. Synthetic and expressed RNAi activators have been used to inhibit markers of HCV replication in various models. Detailed analysis indicates that the plus, and not minus, RNA strand of the virus is susceptible to RNAi-based inactivation. Because HCV is prone to mutation, combinatorial RNAi methodologies have also been developed to counter the emergence of escape mutations. In addition to viral sequences, host dependency factors have been the target for silencing. Gene therapy strategies against HCV show promise and are likely to be useful when used alone or in combination with small-molecule antiviral drugs.

Engineering Sequence-Specific DNA Binding Proteins for Antiviral Gene Editing

Customizing DNA-binding proteins to inactivate viral gene expression has advanced at a rapid pace and the technology has considerable potential for treating viral infections. Viruses that cause serious public health problems, and which are amenable to therapeutic effects of DNA-binding proteins, include human immunodeficiency virus (HIV)-1, hepatitis B virus, herpes simplex virus, and human papilloma virus. The four main classes of DNA-targeting proteins are derivatives of zinc finger proteins, transcription activator-like effectors (TALEs), homing endonuclease (HEs), and clustered regularly interspaced short palindromic repeats (CRISPR) with CRISPR-associated (Cas) systems. Engineered proteins may be functionalized by addition of nuclease moieties or through the coupling of transcriptional suppressing domains, such as the Kruppel-associated box. Site-specific cleavage of target DNA may induce targeted mutation as a result of error-prone repair by nonhomologous end joining or through homology-directed repair. The CRISPR/Cas system has the significant advantage of being dependent on the use of an RNA guide to direct the Cas protein to cleave targets at specific sites. The RNA-guided nucleases are easier to engineer than are the proteins that interact directly with DNA sequences, and this has been the main reason for the enormous popularity of CRISPR/Cas systems. Concerns about off-target effects of engineered CRISPR/Cas nucleases are currently being addressed by ingenious methods that improve guide RNA specificity and also through engineering of two “nickases” that only cleave both DNA strands when juxtaposed on a larger cognate. Zinc finger nucleases (ZFNs) have been studied thoroughly and are now at an advanced stage of clinical development for the disabling of CCR5 to treat HIV-1 infection. However, a difficulty with engineering ZFNs has been the poor efficacy that results from context-dependent positioning of the finger modules. TALENs do not suffer from this problem, they are easier to generate than ZFNs, and they have been reported to act with greater target specificity than ZFNs. Advances with tailoring HEs to achieve target-specific cleavage have also been significant, but the coupled sequence-binding and enzymatic cleavage functions restrict the ease with which HEs may be engineered. The small size of HEs is a distinct advantage for their delivery in a therapeutic context. The gene editing function of designer DNA binding proteins is powerful and will no doubt have therapeutic application for viral infections that have DNA replication intermediates.

Gene Therapy for Chronic Hepatitis B Virus Infection

Chronic infection with hepatitis B virus (HBV) occurs in 6% of the worlds population. These carriers of the virus are at high risk for the life-threatening complications of cirrhosis and liver cancer. Although effective vaccines are available to prevent HBV infection, they are of no use to individuals who are already infected with the virus. Therefore, complications of chronic HBV infection are likely to remain a significant public health problem for some time. Stability of the HBV covalently closed circular DNA (cccDNA), together with resistance of this replication intermediate to licensed therapies, are the main reasons for the limited success of currently available treatment interventions. Gene therapy approaches, particularly using exogenous RNA interference (RNAi) activators and derivatives of DNA sequence-specific proteins, have shown potential as HBV therapeutics. Synthetic and expressed RNAi activators have been used successfully to inhibit HBV replication in cell culture and murine models of the infection. A candidate drug comprising a short interfering RNA conjugate is currently in phase II of clinical trial. Although efficacy against the virus is impressive, it remains to be established whether gene silencing will be curative of persistent HBV infection. In one of the first studies using sequence-specific DNA binding proteins, zinc finger proteins (ZFPs) were shown to inhibit transcription from duck HBV cccDNA. Subsequent investigations showed that coupling FokI endonuclease elements to ZFPs or transcription activator-like effectors achieved specific cleavage of HBV DNA, with resultant disabling of viral gene expression. This was an important development in HBV gene therapy because it demonstrated that inactivation of the problematic viral cccDNA replication intermediate is feasible.

Gene Therapy for Infection with Hemorrhagic Fever Viruses

Available treatment and vaccination for viral infections that cause hemorrhagic fevers are largely ineffective. Some infections, such as are caused by Ebola virus and Marburg virus, are associated with a particularly grave prognosis and a high rate of mortality. The viruses of the group have genomes that comprise RNA and replicate in the cytoplasm of infected cells. Therefore, developing RNA interference -based gene therapies has been a popular strategy against hemorrhagic fever viruses, and good efficacy has been reported in preclinical studies. Formulations comprising complexes of cationic lipids with synthetic small interfering RNAs have demonstrated protection of nonhuman primates from lethal exposure to Ebola virus and Marburg virus. These are the most advanced gene therapy-based regimens for hemorrhagic fever virus infection, and their effectiveness augurs well for use in clinical settings. Aptamer- and antisense-based technologies are also under development, and chemically modified antisense oligonucleotides have demonstrated efficacy against Ebola virus in nonhuman primates. In addition to introducing antiviral sequences directly into the human host, strategies that aim to interrupt transmission by insect vectors may also be feasible when arboviruses are the cause of hemorrhagic fevers. The serious humanitarian threats of hemorrhagic fever viruses, as shown by the recent outbreak of Ebola virus infection in West Africa, make developing new and effective means of treating the infections a global priority. Although faced with technical challenges and difficulties with performing research on hazardous viruses, strategies that use rational design of gene therapy are particularly appealing and show promise.

Viral Vectors for Delivery of Antiviral Sequences

As nucleic acid parasites, viruses have evolved efficient mechanisms for the delivery of DNA and RNA to cells. This feature has been exploited to develop recombinant viruses as vectors for various gene therapy-based treatments of viral infections. The generic approach to producing recombinant viral vectors entails separation of the engineered transgene from sequences encoding structural, enzymatic, regulatory, and accessory proteins. Typically, the transgene is coupled to the packaging signal whereas the other components required to generate the intact recombinant virions are provided in trans. Introducing all of the necessary constituents into packaging cells is then used to produce recombinant replication-defective vectors. To address concerns about toxicity and improve vectors efficiency, several interesting approaches have been used to diminish interaction of viral vectors with host proteins and to alter vector tropism. To date, the main types of viral vectors used for antiviral gene therapy have been derived from adeno-associated viruses (AAVs), adenoviruses (Ads), and lentiviruses. AAVs are safe and efficient, but they are incapable of accommodating large antiviral cassettes. By contrast, Ads, particularly helper-dependent Ads, may deliver large cassettes to target cells. However, the toxicity of Ads, consequent to their powerful immunostimulation, is severely limiting. Lentiviruses, derived from human immunodeficiency virus-1, stably transduce cells and have broad tropism. The stable integration of lentivirus-derived proviral sequences into host cells is useful to achieve durable transgene expression, but insertional mutagenesis remains potentially problematic. Although currently available recombinant vectors are efficient, their propagation is very demanding of resources. Together with concerns about safety, this may limit widespread use of recombinant viral vectors for the treatment of globally common viral infections.