Landing therapeutic genes safely in the human genome

Date:
January 24, 2022
Source:
Wyss Institute for Biologically Inspired Engineering at Harvard
Summary:
Researchers have developed a computational approach to identify GSH
sites with significantly higher potential for the safe insertion
of therapeutic genes and their durable expression across many
cell types. For two out of 2,000 predicted GSH sites, the team
provided an in-depth validation with adoptive T cell therapies and
in vivo gene therapies for skin diseases in mind. By engineering
the identified GSH sites to carry a reporter gene in T cells, and
a therapeutic gene in skin cells, respectively, they demonstrated
safe and long-lasting expression of the newly introduced genes.



FULL STORY ==========================================================================
Many future gene and cell therapies to treat diseases like cancer,
rare genetic and other conditions could be enhanced in their efficacy, persistence, and predictability by so-called "genomic safe harbors
(GSHs)." These are landing sites in the human genome able to safely
accommodate new therapeutic genes without causing other, unintended
changes in a cell's genome that could pose a risk to patients.


========================================================================== However, finding GSHs with potential for clinical translation has
been as difficult as finding a lunar landing site for a spacecraft --
which has to be in smooth and approachable territory, not too steep
and surrounded by large hills or cliffs, provide good visibility, and
enable a safe return. A GSH, similarly, needs to be accessible by genome editing technologies, free of physical obstacles like genes and other functional sequences, and allow high, stable, and safe expression of a
"landed" therapeutic gene.

Thus far, only few candidate GSHs have been explored and they all come
with certain caveats. Either they are located in genomic regions that
are relatively dense with genes, which means that one or several of them
could be compromised in their function by a therapeutic gene inserted in
their vicinity, or they contain genes with roles in cancer development
that could be inadvertently activated. In addition, candidate GSHs have
not been analyzed for the presence of regulatory elements that, although
not being genes themselves, can regulate the expression of genes from
afar, nor whether inserted genes change global gene expression patterns
in cells across the entire genome.

Now, a collaboration of researchers at Harvard's Wyss Institute for Biologically Inspired Engineering, Harvard Medical School (HMS), and
the ETH Zurich in Switzerland, has developed a computational approach
to identify GSH sites with significantly higher potential for the safe insertion of therapeutic genes and their durable expression across many
cell types. For two out of 2,000 predicted GSH sites, the team provided
an in-depth validation with adoptive T cell therapies and in vivo gene therapies for skin diseases in mind. By engineering the identified
GSH sites to carry a reporter gene in T cells, and a therapeutic gene
in skin cells, respectively, they demonstrated safe and long- lasting expression of the newly introduced genes. The study is published in Cell Reports Methods.

"While GSHs could be utilized as universal landing platforms for gene targeting, and thus expedite the clinical development of gene and cell therapies, so far no site of the human genome has been fully validated and
all of them are only acceptable for research applications," said Wyss Core Faculty member George Church, Ph.D., a senior author on the study. "This
makes the collaborative approach that we took toward highly-validated GSHs
an important step forward. Together with more effective targeted gene integration tools that we develop in the lab, these GSHs could empower
a variety of future clinical translation efforts." Church is a leader
of the Wyss Institute's Synthetic Biology Platform, and also the Robert Winthrop Professor of Genetics at HMS and Professor of Health Sciences
and Technology at Harvard University and the Massachusetts Institute of Technology (MIT).

Sifting the genome for GSHs The researchers first set up a computational pipeline that allowed them to predict regions in the genome with potential
for use as GSHs by harnessing the wealth of available sequencing data
from human cell lines and tissues. "In this step-by-step whole-genome
scan we computationally excluded regions encoding proteins, including
proteins that have been involved in the formation of tumors, and regions encoding certain types of RNAs with functions in gene expression and
other cellular processes. We also eliminated regions that contain
so-called enhancer elements, which activate the expression of genes,
often from afar, and regions that comprise the centers and ends of
chromosomes to avoid mistakes in the replication and segregation of
chromosomes during cell division," said first-author Erik Aznauryan,
Ph.D. "This left us with around 2,000 candidate loci all to be further investigated for clinical and biotechnological purposes."


========================================================================== Aznauryan started the project as a graduate student with other members
of Sai Reddy's lab at ETH Zurich's Department of Biosystems Science and Engineering before he visited the Church lab as part of his graduate
work, where he teamed up with Wyss Technology Development Fellow Denitsa Milanova, Ph.D. He since has joined Church's group as a Postdoctoral
Fellow. Reddy, senior and lead author of the collaborative study, is an Associate Professor of Systems and Synthetic Immunology at ETH Zurich
and focuses on developing new methods in systems and synthetic biology
to engineer immune cells for diverse research and clinical applications.

Out of the 2,000 identified GSH sites, the team randomly selected five
and investigated them in common human cell lines by inserting reporter
genes into each of them using a rapid and efficient CRISPR-Cas9-based
genome editing strategy. "Two of the GSH sites allowed particularly
high expression of the inserted reporter gene -- in fact, significantly
higher than expression levels achieved by the team with the same reporter
gene engineered into two earlier- generation GSHs. Importantly, the
reporter genes harbored by the two GSH sites did not upregulate any cancer-related genes," said Aznauryan. This also can become possible
because regions in the genome distant from one another in the linear
DNA sequence of chromosomes, but near in the three-dimensional genome,
in which different regions of folded chromosomes touch each other,
can become jointly affected when an additional gene is inserted.

Eying clinical translation To evaluate the two most compelling GSH
sites in human cell types with interest for cell and gene therapies,
the team investigated them in immune T cells and skin cells,
respectively. T cells are used in a number of adoptive cell therapies
for the treatment of cancer and autoimmune diseases that could be safer
if the receptor-encoding gene was stably inserted into a GSH. Also,
skin diseases caused by harmful mutations in genes controlling the
function of cells in different skin layers could potentially be cured
by insertion and long-term expression of a healthy copy of the mutated
gene into a GSH of dividing skin cells that replenish those layers.

"We introduced a fluorescent reporter gene into two new GSHs in primary
human T cells obtained from blood, and a fully functional LAMB3 gene,
an extracellular protein in the skin, into the same GSHs in primary
human dermal fibroblasts, and observed long-lasting activity," said
Milanova. "While these GSHs are uniquely positioned to improve on levels
and persistence of gene expression in parent and daughter cells for therapeutics, I am particularly excited about emerging 'gain-of-function' cellular enhancements that could augment the normal function of cells
and organs. The safety aspect is then of paramount importance." With an entrepreneurial team at the Wyss, Milanova is developing a platform for
genetic rejuvenation and enhancements with a focus on skin rejuvenation.

"An extensive sequencing analysis that we undertook in GSH-engineered
primary human T cells clearly demonstrated that the insertion has minimal potential for causing tumor-promoting effects, which always is a main
concern when genetically modifying cells for therapeutic use," said
Reddy. "The identification of multiple GSH sites, as we have done here,
also supports the potential to build more advanced cellular therapies that
use multiple transgenes to program sophisticated cellular responses, this
is especially relevant in T cell engineering for cancer immunotherapy."
"This collaborative interdisciplinary effort demonstrates the power
of integrating computational approaches with genome engineering while maintaining a focus on clinical translation. The identification of GSHs in
the human genome will greatly augment future developmental therapeutics
efforts focused on the engineering of more effective and safer gene and cellular therapies," said Wyss Founding Director Donald Ingber, M.D.,
Ph.D., who is also the Judah Folkman Professor of Vascular Biology at
HMS and Boston Children's Hospital, and Professor of Bioengineering at
the Harvard John A. Paulson School of Engineering and Applied Sciences.

Additional authors on the study are Alexander Yermanos, Ph.D, and Edo Kapetanovic, members of Reddy's group; Anna Devaux at the University of
Basel, Switzerland; and, Elvira Kinzina at the McGovern Institute for
Brain Research at MIT. The study was supported by ETH Research Grants,
the Helmut Horten Stiftung and Aging and Longevity-Related Research Fund
at HMS, as well as a Genome Engineer Innovation Grant 2019 from Synthego
to Aznauryan.

========================================================================== Story Source: Materials provided
by Wyss_Institute_for_Biologically_Inspired_Engineering_at
Harvard. Original written by Benjamin Boettner. Note: Content may be
edited for style and length.


========================================================================== Journal Reference:
1. Erik Aznauryan, Alexander Yermanos, Elvira Kinzina, Anna Devaux, Edo
Kapetanovic, Denitsa Milanova, George M. Church, Sai
T. Reddy. Discovery and validation of human genomic safe harbor
sites for gene and cell therapies. Cell Reports Methods, 2022;
100154 DOI: 10.1016/ j.crmeth.2021.100154 ==========================================================================

Link to news story: https://www.sciencedaily.com/releases/2022/01/220124090501.htm

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