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Saturday, 22 September 2018

Zika virus: a whistle-stop tour

Zika virus (false colour transmission
electron microscope image)
In a recent piece on mRNA vaccines[mRNA vaccine technology: industry is getting the message] , I mentioned Zika virus development as an indication for this emerging technology. only to realise my ignorance of this high profile pathogen (shameful, since I spent my post-doc years working in a school of tropical medicine)

First stop in filling the knowledge gap was a visit to the ever-useful World Health Organisation website which features a comprehensive “Zika timeline”. First discovered in 1947 in monkeys living in a Ugandan forest (which gave its name to the virus), and shortly after in species of Aedes mosquito, epidemiological studies conducted in the 60s and 80s indicated widespread human exposure to the Zika virus in Africa and Asia, with infection largely associated with no, or only mild, symptoms.

The more sinister nature of Zika infection emerged in 2007 and 2008, with the first confirmed large scale outbreak on the Micronesian island of Yap, and evidence that infection could be sexually transmitted. Further outbreaks occurred on various Pacific Islands during 2013 and 2014 and pointed to a link between Zika infection and birth defects and with Guillain–Barré  syndrome, a rare autoimmune disease which affects the nervous system. A year later, an epidemic characterized by a skin rash bit otherwise mild symptoms was reported in north-eastern Brazil, but was not recognised as being due to Zika virus.

By October 2015, an increasing numbers of microcephaly cases (newborns with small heads, indicative of abnormal brain development) were being reported. Further Zika outbreaks occurred in several South American and Caribbean countries over the following year, with the first cases (via sexual transmission) being identified in the continental United States in 2016.

Being generally asymptomatic, the prevalence of Zika infection is not easy to quantify, but mosquito-transmitted infection has been reported in over 80 countries (with transmission ongoing in over 60 of these). Over 1.3 million people are thought to have been infected in Brazil alone during the 2015 outbreak. In common with other vector-borne diseases, the spread of Zika owes something to human mobility, although social factors, principally the inability to afford protection against mosquitoes and high population density have been identified as key drivers.

The Zika virus has several properties that contribute to its ready transmission and to its devastating effect on foetal development. The virus is highly persistent in whole blood (up to 100 days) and in the male reproductive tract, allowing sexual transmission. Zika has a preference for certain cell types that facilitate the passage of infection through the placenta; animal studies suggest that viral preference extends to neural progenitor cells essential for normal cortical development. Whether Zika infection is a direct cause of Guillain–Barré  syndrome  has not been established.

As with malaria, Zika’s vulnerability lies in its dependence on mosquito vectors: and targeted insecticide use, management of standing water and the conscientious use of bed nets and repellents can significantly reduce transmission. Like malaria, control initiatives are vulnerable to political and economic factors, including climate change-related changes in mosquito distribution and abundance. Unlike malaria, Zika has a simple lifecycle; as with other flaviviruses (including the causative agents of yellow fever and Japanese encephalitis), infection should, in theory, be preventable through vaccination.

Vaccine development efforts are almost contemporary with the Zika outbreak itself, beginning in the second half of 2015 with the genetic analysis of Brazilian Zika isolates, with the first clinical study of a Zika vaccine being reported in late 2017. Around 45 candidate vaccines have been developed through academic, governmental and industrial efforts, with nine of these reaching the clinic, representing both established (inactivated virus) and experimental approaches (DNA and mRNA vaccines). The WHO’s initial requirement is for a vaccine that can be deployed in response to outbreaks with the primary goal of preventing congenital Zika syndrome through minimizing virus carriage in the immediate population.

Early clinical studies have established that vaccination can elicit aneutralizing antibody responses, although, while an accepted hallmark of flavivirus vaccine efficacy, the importance of neutralising antibodies, and the minimum levels needed to establish protection have still to be established in the context of Zika infection. Other important unknowns include the duration of effective vaccine-induced immunity and whether the reproductive tract can be protected from infection.
Perversely, given the untold misery arising from Zika outbreaks around the globe, the virus’s propensity for neural progenitor cells may offer a new means of treating the most aggressive and intractable form of brain tumour, glioblastoma.

Chinese researchers have found that an experimental live attenuated Zika vaccine functions as an oncolytic virotherapy [Going viral] in an animal model of glioblastoma, specifically infecting and destroying glioma stem cells thought to be responsible for the inevitable recurrence of the tumour. Elimination was also observed using glioma stem cells isolated from individual patients.

Photo credit: Credit: NIH/NIAID

Thursday, 6 September 2018

Gene editing: off the bench and into the clinic

As the first sponsored CRISPR-Cas9 clinical study gets underway,
industry has high hopes that genome editing will eventually make gene
therapy a mainstream treatment for both inherited and non-inherited
Recent progress in gene therapy, with successful proof of concept being reported in a range of otherwise untreatable conditions and the grant of marketing authorisations for commercially-developed treatments makes it easy to forget that these successes were built on almost three decades of endeavour and regular disappointment.

Gene therapy restores aberrant or missing biological functions through the introduction of functional genes carried into the cell using engineered viruses or by zapping with electrical pulses. A more involved process is the alteration of specific nucleotide sequences within a faulty gene to restore or 
modify function- so-called “gene editing”. 

Around the turn of the millennium, certain bacterial genomes were observed to have multiple copies of repeated DNA sequences which read the same in both directions -“clustered regularly interspaced palindromic repeats” (CRISPR), which were subsequently found to be responsible for the exquisitely specific recognition of infecting viruses. In 2012, it was shown that CRISPR could be exploited to edit genes in mammalian cells, prompting an explosion in translational science, patent wrangles and an influx of venture funding. 

Reduced to its bare bones, CRISPR gene editing utilises a short piece of “guide” RNA, designed to bind to a specific sequence of DNA, complexed with an enzyme able to cut double-stranded DNA, with the bacterial enzyme “Cas9” being widely used. Once within the nucleus, the guide RNA latches onto its complementary DNA sequence, directing the enzyme to cut in the right place: the cell’s DNA repair mechanism completes the editing process. 

CRISPR-Cas9 (and other CRISPR based systems) have brought relative simplicity and low cost to gene editing, providing a powerful research tool with application across biological disciplines and a route to significant advances in agriculture and the treatment of both inherited and non-inherited disease.

American and European regulators have been understandably cautious with respect to CRISPR clinical studies. As with other forms of gene therapy, CRISPR-Cas9 editing is not without potential risk, such as unwanted alteration of DNA sequences other than the target gene sequence, or the triggering of immune responses to the Cas9 enzyme (obtained from one or other of two common skin bacteria) or through introduced RNA being recognised as being  “foreign”. 

Chinese investigators have been less troubled by the unknowns surrounding CRISPR clinical development:  first in man studies began in 2010, with around 80 subjects being treated in cancer and HIV gene editing studies by February this year. Several Chinese research groups have explored  gene editing in non-viable, and most recently, viable human embryos.  

CRISPR Therapeutics, in partnership with Vertex,  successfully addressed undisclosed FDA concerns raised in May this year and are now recruiting beta thalassemia patients for a Phase I/II gene editing study. Like sickle cell disease, beta thalassemia arises from an inherited defect in the gene encoding the oxygen-carrying protein, haemoglobin. 

Rather than directly editing the haemoglobin gene, the CRISPR-Cas9 complex (designated CTX001) is designed to alter a DNA sequence responsible for the shutdown of foetal haemoglobin production, in the hope that turning the switch back on will produce sufficient levels of functional haemoglobin. The editing process is performed ex vivo, with blood-forming stem cells being extracted from the patient, transfected with the CRISPR-Cas9 complex, expanded and then returned by infusion. 

CRISPR Therapeutics and its peers, Editas Medicine and Intellia Therapeutics have ambitions to develop gene editing therapies for a range of genetic conditions that result in metabolic dysfunction (Hurler syndrome, glycogen storage disease, alpha-1 antitrypsin deficiency, transthyretin amyloidosis) and visual impairment (Leber congenital amaurosis, Usher syndrome), as well as cystic fibrosis and Duchenne muscular dystrophy (DMD). Exonics Therapeutics recently published data indicating that their “SingleCut” CRISPR technology was able to  restore near normal levels of dystrophin expression in an animal model of DMD. 

CRISPR may prove to be more efficient and flexible means of engineering “off the shelf” (allogeneic) cancer-fighting T cell therapies, reducing the cost and complexity of current patient-specific CAR-T therapy  [ASCO Clinical Cancer Advances 2018. And the winner is...].

Pre-CRISPR era gene editing methodologies may also prove to have therapeutic application. Sangamo Therapeutics has used a zinc finger nuclease-based approach to achieve “in body” editing of a faulty gene in four individuals with Hunter syndrome, an enzyme disorder that prevents the breakdown of complex sugars, leading to harmful accumulation. A decrease in urinary complex sugar level was observed in two individuals, although not an increase in normal enzyme levels in the blood. Full interpretation of the significance of these results will take time but importantly, “in body” editing raised no safety issues. 

Clinical development of  gene editing-based therapies will continue to advance cautiously (at least in the West), but is likely to can to benefit from experience gained in viral and non-viral vector design now being exploited in other forms of gene therapy and growing regulatory familiarity with gene therapy risk-benefit assessment. 

Image credit: Credit: Jill George, NIH.