Combined Gene Therapy Shows Promise, but Not Synergy, in Mice

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by Steve Bryson, PhD |

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A combined gene therapy that delivered two nerve growth factors — NRG1-I and NRG1-III — to muscle and nerve cells improved motor function and delayed disease onset in a mouse model of amyotrophic lateral sclerosis (ALS), a study demonstrated.

However, the combo therapy did not show a synergistic effect, as hoped, compared with treatment with NRG1-I and NRG1-III alone, “suggesting an overlap between NRG1-I and NRG1-III activated pathways and their beneficial effects,” the researchers noted.

Noting that “very few studies have reported synergistic effects after combinatory therapies,” the researchers said one explanation could be the use of mouse models in testing.

“This might reflect that there is an endogenous [internal] limitation for the beneficial outcomes that can be achieved using the … mouse model,” they wrote.

Further studies investigating the impact of NRG1-based gene therapy will support the development of this therapeutic approach for treating ALS patients, the scientists said.

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The study, “Gene Therapy Overexpressing Neuregulin 1 Type I in Combination With Neuregulin 1 Type III Promotes Functional Improvement in the SOD1G93A ALS Mice,” was published in the journal Frontiers in Neurology.

ALS is a neurodegenerative disorder characterized by the loss of motor neurons, the nerve cells that control voluntary movements. With the loss of these cells, the brain can no longer control muscles, and they become smaller and weaker.

In most patients, the cause of ALS is sporadic, but in some cases, people can inherit faulty disease-causing genes such as SOD1.

Neuregulin 1 or NRG1 is a protein mainly located in motor neurons and at neuromuscular junctions — the place where neurons make contact with the muscles they control. NRG1 is essential for promoting motor neuron survival, and for supporting nerve fibers and neuromuscular development and maintenance.

Studies have found altered production of two forms of NRG1 — NRG1-I and NRG1-III — in the spinal cord in both ALS patients and mice carrying a disease-causing SOD1 mutation called G93A.

To learn more, a team of researchers based at the Autonomous University of Barcelona, in Spain, used a gene therapy approach to deliver a copy of the gene that encodes for NRG1-III to neurons in SOD1-G93A mice. The goal was to make the animals overexpress, or overproduce, the protein. In the lab, the animals showed improved motor function of hindlimb muscles and increased motor neuron survival.

Using the same method, the team overexpressed NRG1-I in muscle cells, which resulted in neuromuscular improvement, maintained muscle-nerve communication (innervation), and increased motor neuron survival in the SOD1-G93A mice.

Given these findings, the team wondered whether a combined gene therapy that delivers both NRG1-I to nerve cells and NRG1-III to muscle cells would generate an additive — or synergistic effect — in the same ALS mouse model.

To find out, the team packaged the genes that encode NRG1-I and NRG1-III in harmless adeno-associated viral vectors and treated ALS mice at about seven weeks of age, just before disease onset.

To ensure NRG1-I reached the muscles, its viral vector was administered into the bloodstream (intravenously), while NRG1-III was delivered to neurons via an infusion into the spinal canal (intrathecally).

Initial analysis at 16 weeks, or about four months, revealed that NRG1-I expression was increased in the muscle compared with control mice treated with an empty viral vector. Likewise, NRG1-III was higher in the spinal cord of treated mice than control mice.

An experiment confirmed that NRG1-I was not overexpressed in the spinal cord, and excess NRG1-III was not seen in muscle.

Motor nerve electrical conduction tests indicated that treated ALS mice had a significant preservation of nerve signals in three different muscle groups, compared with control mice.

Tissue analysis at that 16-week mark also showed a higher number of surviving motor neurons in the spinal cords of treated ALS mice than controls, and more innervated neuromuscular junctions.

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The gene therapy also caused a significant decrease in the activation of microglial cells — specialized immune cells found in the brain and spinal cord — as well as astrocytes,  star-shaped cells that also have detrimental effects in ALS.

Furthermore, treatment delayed disease onset and led to overall functional improvement, as assessed by the rotarod test. This performance test for motor ability is based on the time and endurance of the mice remaining on a rotating rod.

Finally, to determine if the combined treatment led to a synergistic effect, the team compared results from this study to their previous studies in which mice were treated with NRG1-III and NRG1-I separately.

Compared with the single gene therapies, however, the combined treatment did not significantly increase electric signals in the three muscle groups examined, nor did dual gene therapy further delay disease onset or improve rotarod performance.

Additionally, the number of spinal motor neurons, and microglia and astrocyte activation were similar, regardless of treatment. Notably, however, the number of functioning neuromuscular junctions increased in ALS mice treated with the combined gene therapy, whereas NRG1-III treatment alone did not.

“In summary, the combined NRG1-I and III overexpression significantly improved motor function preservation and promoted neuroprotection, accompanied by reduction of neuro-inflammatory reaction,” the researchers wrote. “However, we did not see an improved effect compared to the NRG1-I or III overexpression alone.”