Proof-of-concept studies advance potential new avenues for gene therapy

Researchers at the Johns Hopkins School of Medicine say they have successfully harnessed the cell's natural process of making proteins, "slipping" genetic instructions into the cell and producing key proteins
that are missing in the cell.
If further research confirms their proof-of-concept results, scientists may have a new approach to specific cell types
for a variety of diseases that can be treated with gene therapy.
Such diseases include neurodegenerative diseases that affect the brain, including Alzheimer's disease, various forms of blindness, and some cancers
.

Dr.
Seth Blackshaw, professor of neuroscience in Saul Snyder's Department of Neuroscience and a member of the Institute of Cell Engineering at Johns Hopkins University School of Medicine, said that for those looking to develop treatments for diseases in which cells lack specific proteins, the key is to precisely target disease-causing cells in each structure, such as the brain, to safely kick-start the protein-making process
for specific genes.
He added that therapies that don't precisely target diseased cells could have unexpected effects
on other healthy cells.

There are currently two ways to deliver protein fabrication packets to cells, and the results in animal models and humans vary widely
.
"We wanted to develop a gene expression delivery tool that could be widely useful
in both preclinical and clinical models," Blackshaw said.

One current method of sending biochemical packages involves so-called "mini-promoters," which can direct the expression of certain segments of DNA or the protein-making process
.
Blackshaw says this approach often fails to express genes
in the right cell type.

Another approach, called serotype-mediated gene expression, involves delivery tools that lock in proteins
attached to the surface of certain types of cells.
However, Blackshaw said, the method's ability to target only one type of cell is accidental, and even after successful tests on animal models, they often fail
in humans.

The study, published Oct.
1 in the journal Nature Communications, stems from previous research by Jonathan Ling, Ph.
D.
, assistant professor of pathology at Johns Hopkins University, who published "Maps," describing how various types of cells use alternative splicing of messenger RNA, a close relative of DNA, to construct genetic templates that produce a changing set of proteins
in cells.
These changes depend on the type and location
of the cells.
Cells often use alternative splicing to change the type of
protein the cell can make.

Ling's map maps a pattern
in which cells cut out introns (exons of messenger RNA), leaving only the informational part of the genetic material that actually expresses or makes proteins (exons).

However, introns are typically very large—sometimes millions of base pairs—and too large to encapsulate
in currently available gene expression delivery systems.
Ling found that about 20 percent of the optional splicing patterns contained intron DNA fragments small enough to be packaged into the gene expression delivery system
Blackshaw wanted to test.

Fortunately, for its purposes, the alternative splicing patterns are similar in both mouse and human DNA, so they have the potential to be suitable for preclinical research and clinical applications
.

Blackshaw and Ling, along with then-postdoc Alexei Bygrave, made packets of sheared messenger RNA that could be delivered into cells by benign viruses
.
Grave is now an assistant professor
at Tufts University.
They named these packages "sledge" and used them to stitch link expression designs
.

When the package slides into the cell, it opens
there.
Since the Sledge system does not naturally integrate into the genome, the research team added genetic "promoters" to stimulate the production
of proteins in packaged Sledge products.

Researchers at the Johns Hopkins School of Medicine built the SLED system for lab-grown excitatory neurons and photoreceptors and were able to produce proteins
in only these cell types about half the time.
Current small promoter systems typically put proteins in the right place
5% of the time.

The team also injected SLED packs
into photoreceptor mice that lacked the PRPH2 gene on their retinas.
The PRPH2 gene causes retinitis pigmentosa, a disease
that affects the retina.
The team found evidence that SLED packs help produce the PRPH2 protein
in photoreceptors in treated mice.

In lab-grown human eye melanoma, the scientists injected only SLED packets into melanoma cells
that lacked the SF3B1 gene.
The SLEDGE package releases RNA-producing proteins that cause melanoma cells to die
.

Blackshaw said the biggest potential of the sledge system may be in combination with other gene delivery systems, and his lab is working on ways to miniaturize introns to accommodate larger introns in the sledge system
.

Blackshaw and Ling have filed patents
involving SLED technology.

The study was supported
by the National Institutes of Health (RF1MH123237, R24EY027283, K08EY027093, R01EY033103, 2T32EY007143), the Stein Innovation Award for Blindness Prevention Research, the Wilmer Eye Institute, the National Science Foundation, the Johns Hopkins Kavli NDI Scholarship, and the Johns Hopkins IDIES Seed Fund.