Challenges and opportunities for LINE-1 inhibition

LINE-1 is unique as a target for new medicines. Inhibiting has potential to help slow the progression of many diseases with limited side effects. This is because it is an ancient virus-like element inside our genomes. Normally in healthy cells it is suppressed, but it activates in cancer, autoimmune and neurodegenerative disorders

There are three different approaches scientists and biotechnology companies have used to inhibit LINE-1. Short interfering RNAs (siRNAs), antisense oligonucleotides (ASOs), and nucleoside reverse transcriptase inhibitors (NRTIs). These can slow disease progression and even cause life extension in certain disease models. Hundreds of millions of dollars have been spent attempting to commercialize them, particularly NRTIs

Below we describe the current limitations of approaches to LINE-1 inhibition, the challenges faced and opportunities ahead

Blocking or degrading RNA

In theory you can stop LINE-1 by either blocking or degrading its RNA. There are two ways to do this, short interfering RNAs (siRNA) or antisense oligonucleotides (ASOs). However, LINE-1 exists in 500,000 copies and makes up 17% of the genome. This may make it infeasible to design RNA-based medicines without off-target effects

Short interfering RNAs

siRNAs can be designed to bind to LINE-1 mRNA sequence and signals to the cell to degrade it. siRNA leverages a pathway the cell normally uses to regulate RNA levels. The siRNA sequence itself has a length of 20, which can (somewhat) uniquely bind to LINE-1 messenger RNA and not to other sequences. However, the first few RNA bases of siRNA can behave like a microRNA (miRNA) when bound to the 3' end of mRNA, which sends a signal to remove the polyA tail from the end of mRNA, rendering it unable to be translated into protein. The siRNA can be modified at one position in an unnatural way that reduces the miRNA effect, but it does not eliminate it. For LINE-1 this is unlikely to be sufficient due to the high number of off target sequences. There are also challenges with delivery, as siRNAs are double stranded, bulky molecules. They have only been approved when they have been modified to target the liver

Antisense oligonucleotides

ASOs leverage a pathway the cell uses to degrade foreign DNA. They can be designed as a combination of DNA and RNA, which binds to LINE-1 sequence and creates a DNA:RNA hybrid. The cell recognizes these hybrids as foreign and degrades them. Another type of ASO is known as a steric blocker, it sits on LINE-1 RNA and prevents it from being converted into protein

Unlike processed mRNA that siRNA targets, ASOs bind RNA containing introns, which can contain LINE-1 sequence. All possible ASO designs for LINE-1 contain hundreds of off target effects in introns. This leads to either degradation of RNA the cell needs, or it acts as a sink for LINE-1 blocking ASOs, diluting their effect

There are pre-clinical studies that have successfully used ASOs to knock-down LINE-1 activity. Then there are challenges with delivery. ASOs can be delivered to the brain through injection into cerebral spinal fluid. However, delivery in solid tumors is a major challenge. Even if delivery problems are solved, there is no escape from the risk of off-target effects for LINE-1

Nucleoside reverse transcriptase inhibitors

Although ASOs can suppress LINE-1 more effectively than NRTIs, off-target effects and delivery limit their potential application. NRTIs have received the most attention to date as a therapeutic strategy for inhibiting LINE-1. NRTIs have two advantages over ASOs, delivery and safety. They are small molecules that can either passively diffuse into cells or move in via nucleoside transporters on the cell surface. They can also cross the blood-brain barrier and get into neuronal cell types. Furthermore, they are repurposed HIV medicines, and many have long-term safety data and are prescribed to young children. The reason they also inhibit LINE-1 is owing to the shared evolutionary history of retrotransposons such as LINE-1, and retroviruses such as HIV. Both use reverse transcriptase to copy their RNA into DNA

NRTIs mimic building blocks of DNA causing chain-termination and preventing the virus from completing copying of its genome. For HIV suppression, NRTIs are extremely effective because the virus is a chain of ~9,700 nucleotides, and you only need one NRTI to incorporate to stop viral replication. LINE-1 is smaller at ~6,000 nucleotides. Unlike HIV, the genome already contains 100+ potentially active copies. So stopping the completion of insertions is not enough

LINE-1's ORF2p protein causes damage to DNA by cutting it. Furthermore partial insertions can negatively impact gene expression. LINE-1 reverse transcriptase can also produce small fragments of DNA that trigger an innate immune response as the cell thinks it is infected by a virus. Suppression of completed insertions of LINE-1 is necessary, but not sufficient

In spite of the limitations of NRTIs, academic proof of concept studies have shown they can slow disease progression and even extend lifespan in certain disease models. However, achieving this feat in petri dishes or animal models is not the same as long term use in humans

The NRTI Lamivudine (3TC) inhibits LINE-1, but at standard HIV dosing it can only reduce completed LINE-1 insertions by an estimated ~4% in the human brain, and ~8% outside of it. There are more potent LINE-1 inhibitors. However, each has its own challenge

For example, Censavudine is a thymidine inhibitor with up to 9x greater potency than 3TC in dividing cells. In theory this means it could have up to 40% LINE-1 inhibition at safe doses. However, it is being tested in neurological diseases and the enzyme thymidine kinase necessary to activate Censavudine is expressed at very low levels in neurons

EdA has similar potency to Censavudine, and it mimics adenosine instead of thymidine, which potentially enables neuron activation as adenosine is needed in non-dividing cells. Islatravir is a modified version of EdA with a fluorine atom that increases its potency by slowing its breakdown by enzymes. Unfortunately, the dose then has to be lowered as accumulation can potentially lead to toxicity. At HIV dosing, Islatravir might inhibit completed LINE-1 insertions by ~75% in the brain. Other attempts to create novel NRTIs that are more potent against LINE-1 have also run up against toxicity concerns

Outside the brain, up to 90-95% inhibition could be achieved safely for LINE-1 for existing compounds. With combinations of existing NRTIs we could achieve 99% outside the brain, and perhaps ~85% within it. These are back of the envelope calculations based on real data from HIV studies and cell models. However, even if we could hold this level of inhibition throughout the dosing period, this may not be sufficient in most disease settings. Completed insertions are 6,000 base pairs, but the average insertion size is much smaller than this. Partial insertions inside introns can still cause damaging changes to gene expression. Partial or failed insertions can also trigger structural rearrangements to genomes that are even more damaging. Furthermore, NRTIs do not prevent DNA cutting. Nor do they prevent production of even smaller LINE-1 cytoplasmic DNA that triggers an immune response. NRTIs may reduce these effects, but are unlikely to be effective at safe doses for all but the most serious disease contexts. Even then the benefits may be statistically significant, but not necessarily meaningful to the patient

ORF2p inhibitors

siRNAs, ASOs and NRTIs have helped show what contexts LINE-1 inhibition could be applied, but they are unlikely to achieve widespread use outside of research contexts. More potent therapies are required. The challenge is the inhibitor would have to block both ORF2p DNA cutting (endonuclease) and conversion of RNA to DNA (reverse transcriptase). There has only been one preliminary attempt by an academic group to make an inhibitor, and there is no record in the public domain of research groups developing one. Part of the challenge is that the 3D structure of ORF2p was only figured out a couple of years ago. Developing a potent, selective inhibitor will be extremely challenging, successfully achieving this requires a dedicated team and the formation of a biotechnology company