Tag Archives: writing

The Needle Issue #8

24 Jun
Juan-Carlos-Lopez
Juan Carlos Lopez		 Andy-Marshall
Andy Marshall

Around 1 in 5000 people live with a maternally inherited mitochondrial disease like MELAS, Leber’s Hereditary Optical Neuropathy (LHON) or MIDD, for which there are limited or no treatment options. Gene- and base-editing therapies for mitochondrial DNA (mtDNA) have lagged behind CRISPR–Cas9-based approaches targeting nuclear genes. Whereas there is already a CRISPR–Cas9-based product on the market and >150 different active trials of investigational therapies, the company closest to the clinic with an I-CreI (mitoARCUS) meganuclease targeting a mtDNA point mutation in MELAS/mitochondrial myopathy (Precision Biosciences) announced last month that it was pausing development for commercial reasons.

Despite this disparity, there is reason for optimism as a flurry of different types of optimized cytidine and adenine base editors for mtDNA are now available, with base conversion efficiencies of 50% now achievable, and some newer formats reaching efficiencies as high as 82%.

The development of mtDNA editors is not without challenges. First, editors must dispense with the targeting guide RNA, as mitochondria possess a double membrane that lacks any RNA transport system, effectively thwarting CRISPR-based gene or base editors (instead, a mitochondrial targeting sequence is used to ferry-in editor proteins). Second, unlike nuclear DNA with two copies of a gene, every human cell contains thousands of mitochondria — oocytes contain a whopping 193,000 mitochondria on average — and each organelle contains an average 10 mitochondrial genomes. Those ~10,000 genomes per cell may not all have the same sequence, with mutations existing in a state known as heteroplasmy, in which both mutant and wild-type genomes co-exist in the same organelle. Disease only occurs when the percentage of mutant mtDNA exceeds a particular threshold, typically between 70% and 95%.

Heteroplasmic mitochondrial diseases, like MELAS and MIDD, could be treated using I-Crel/FokI meganucleases or restriction enzymes linked to either transcription activator-like effector (TALE) domains or zinc fingers (which introduce double-strand DNA breaks into target sequences, leading to elimination of mutant mtDNA and repopulation of wild-type mtDNA); other conditions like LHON are predominantly mutant homoplasmic, which means they can only be treated using base editors or supplemental gene therapy.

One key concern with base-editing technology has been its propensity for off-target and bystander changes. This has led to various strategies to increase specificity, such as engineering the deaminases to narrow the editing window or use of nuclear exclusion sequences to stop nuclear sequence editing. Now, two papers in Nature Biotechnology represent important advances that could speed up translational studies of mitochondrial diseases.

Liang Chen, Dali Li and their colleagues of ShanghaiTech University, China report the engineering of highly efficient mitochondrial adenine base editors (eTd-mtABEs) by introducing mutations into the TALE TadA-8e deaminase for greater activity and specificity. These editors achieved up to 87% editing efficiency in human cells and over 50% in vivo, with reduced off-target effects compared to earlier tools.

In the first study, the researchers used eTd-mtABEs to introduce mutations in the human ND6 gene, encoding a subunit of the oxidative phosphorylation (OXPHOS) system linked to LHON and Leigh syndrome. They found reduced levels of ATP and more reactive oxygen species in the edited cells compared with controls, consistent with disease phenotypes. Next, the team used this adenine TALE base editor to introduce two pathogenic T-to-C mutations in the mitochondrial TRNS1 gene of rat zygotes, a gene linked to childhood-onset sensorineural hearing loss. The resulting offspring showed sensorineural hearing loss, which was transmitted to the F1 generation, providing proof of concept that eTd-mtABEs can be used to create animal models of disease.

In the companion paper, Chen, Li and their colleagues used the adenine TALE base editor to model Leigh disease in rats using a similar strategy. The resulting rats showed reduced motor coordination and muscle strength, defects that were obtained with editing efficiencies of only 54% on average. To test if the abnormalities could be reversed, the authors then used a cytosine TALE base editor in zygotes from the mutant rats. On average, the editing efficiency was only 53%, but this was enough to rescue the disease phenotypes.

This is the first report of direct correction of mtDNA mutations via a TALE base editor in an animal model. The next step will be to show feasibility in a model after disease onset (only the UK and Australia allow maternal spindle transfer therapy for mitochondrial diseases; no country has permitted mitochondrial base editing in human zygotes).

Achieving effective therapeutic mitochondrial base editing in affected target tissues will thus require efficient AAV delivery. For LHON, an already approved FDA AAV-2 product transduces the optic nerve and retinal ganglion cells, providing a translational path; ​GenSight Biologics also recently published 5-year outcome data for its AAV-2 therapy Lumevoq (lenadogene nolparvec) in LHON. But AAV delivery in other mitochondrial conditions will not be as simple: MELAS patients, for example, require efficient transduction of the CNS, kidney, skeletal muscle and cardiac muscle; MIDD patients need AAV delivery to the pancreas, inner ear, retina and kidney. Although a commercial AAV vector (AAVrh74) is available for muscle (Sarepta’s Elvidys), vectors that reach many of these other tissues have yet to be commercialized and may require next-generation AAV capsids and/or refinement of machine-guided design of cell type-specific synthetic promoters to reach target organs.

It is encouraging that the roughly 50% base conversion rate achieved in these new studies exceeded the heteroplasmy threshold required for disease manifestation and therapeutic rescue. At the same time, despite this remarkable success, concerns remain about off-target effects — both in mitochondrial and nuclear genomes — and narrow therapeutic windows. And with base editing approaches so far behind conventional gene therapies like Lumevoq in development, compelling commercial and clinical advantages benchmarked against best-in-class gene therapy will be needed to convince investors to back these approaches.

One parting thought: the past year has seen a noticeable uptick in publications on mitochondrial base editing technology from labs outside of the US. TALEN specialist Cellectis, headquartered in Paris, France, acquired 19% of equity in the mitochondrial base editing company Primera Therapeutics in 2022, ostensibly for its rapid TALE assembly platform (FusX System), which streamlines TALE repeat construction. In South Korea, Jin-Soo Kim at the Korea Advanced Institute of Science and Technology (KAIST) recently co-founded startup Edgene with Myriad Partners to develop mitochondrial base editors based on his seminal work on TALE-linked deaminases (TALEDs) enabling A to G conversion, which he has continued to optimize. According to Biocentury, 8 out of 13 base editing studies published in 27 translational journals over the past year came from labs in China. Wensheng Wei’s group at Peking University, a founder of Edigene in Beijing, continues to work on mitobase editors, with two recent patents on strand-selective mitochondrial editing. And Jia Chen of ShanghaiTech University, China, and his collaborators Li Yang and Bei Yang, are scientific advisors to Correctseq in Shanghai, which is developing transformer base editors for ex vivo and in vivo applications. It seems that mitochondrial base editing may be another area where US biotech may soon be finding itself chasing the dragon. David Liu and Beam Therapeutics may have something to say about that.

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The Needle Issue #7

10 Jun
Juan-Carlos-Lopez
Juan Carlos Lopez		 Andy-Marshall
Andy Marshall

Ex vivo HSC lentiviral gene therapies have been on the market for nearly a decade, with six products approved and at least 55 now in clinical testing for rare inherited diseases, HIV infection or cancer. And yet, their commercial success remains in question. Bluebird Bio—which was valued at $10 billion only a few years ago and successfully shepherded to market Zynteglo against transfusion-dependent β-thalassemia, Skysona for early cerebral adrenoleukodystrophy, and Lyfgenia for sickle-cell disease (SCD)—was sold earlier this year to private-equity firms Carlyle and SK Capital for a measly $29 million. Last November, the company had treated only 57 patients (35 for Zynteglo; 17 for Lyfgenia and 5 for Skysona), with just 28 of 70 medical centers across the US ready to treat patients due to delays in accreditation and training of personnel. In Europe, Orchard Therapeutics halted marketing and production of a treatment for severe combined immunodeficiency caused by adenosine deaminase mutations (Strimvelis) after six years, forcing Fondazione Telethon to take over production. Even market uptake of Vertex’s much-heralded CRISPR/Cas9 BCL11a SCD therapy Casgevy has been sluggish.

These subpar commercial launches relate to the complexity of ex vivo lentiviral gene therapy: patient identification and qualification is lengthy; HSC mobilization and sourcing efficiencies vary due to patient heterogeneity; and manufacture and distribution processes remain lengthy and convoluted (sometimes requiring repetition if a poor quality product batch is generated). From first evaluation, patients are required to make several hospital visits over a period (of up to a year) and must undergo punishing conditioning regimes with lymphodepletive bisulfan before infusion, which itself carries infertility and cancer risks. All of these challenges have added impetus to the search for alternative and more efficient approaches for carrying out HSC gene therapy.

​A group led by Alessio Cantore and Luigi Naldini, from the San Raffaele Telethon Institute for Gene Therapy in Milan, Italy, report in Nature that it may be possible to obviate these challenges by delivering recombinant lentiviral vectors in vivo soon after birth, when HSCs continue to circulate in the bloodstream in large numbers and are beginning their transition from the liver (where they are located in the fetus) to bone marrow (where they remain through adulthood).

Cantore, Naldini and their colleagues started by measuring the number of circulating HSCs in neonatal, 1-, 2- and 8-week-old mice, looking at the peripheral blood, spleen, liver and bone marrow. They found that HSCs were present in the circulation right after birth and that their number immediately declined. These cells could be transduced with lentiviruses, successfully engrafted, and persisted in the mice for several months.

To show that these HSCs could be harnessed to treat genetic disorders, the team tried to correct three mouse models of disease — adenosine deaminase deficiency, autosomal recessive osteopetrosis and Fanconi anemia. Although the therapeutic effect of the cells varied depending on the disease, the results provided compelling evidence for the potential for in vivo gene transfer to HSCs.

The authors reported that human neonates also have circulating HSCs in high numbers. And although the therapeutic window in the mouse only existed during the neonatal period, it was possible to lengthen it by mobilizing the HSCs from their niche in two-week-old animals using protocols in clinical use (granulocyte-colony stimulating factor/CXCR4 antagonist Plerixafor) These observations raise the possibility of therapeutically targeting HSCs in newborns, potentially opening the gates to treatment of a variety of inherited conditions.

Compared with the headaches of ex vivo manipulation, the authors’ concept of simply injecting a lentiviral gene therapy into a newborn to bring about a genetic cure is certainly alluring. But reducing this to clinical practice will require optimization of many different factors. How to account for the heterogeneity and fragility of patient HSCs in a particular disease? How to measure the cellular activation/metabolic state of HSCs in newborns and assess the affect on amenability to lentiviral transduction in the hostile milieu of blood? What effect would shear stress in circulation have on lentiviral transduction efficiencies in situ? What would be the selective engraftment advantage provided to HSCs after engraftment of a particular gene? And what would be the potential safety implications of off-target transduction events in cells other than HSCs, given instances of dysplastic syndromes have been reported with ex vivo lentivectors?

Current ex vivo lentiviral gene therapy like Lyfgenia and Zynteglo infuse between 3–5×106 gene-modified CD34+ HSCs/kg in a patient. The challenge for in vivo lentiviral gene therapy will be to achieve transduction efficiencies that transduce as many cells and obtain similar engraftment rates in the rapidly turning over HSC population. Beyond these issues, there are additional practical challenges: can genetic testing of an infant happen fast enough to take advantage of the short therapeutic window for which an in vivo lentiviral HSC therapy could work?

Clearly, the new work raises many intriguing questions for the lentiviral gene therapy space. And for newborns with genetic diseases, such as severe immunodeficiencies or Fanconi anemia, in vivo HSC gene therapy may open up new treatment options.

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