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SDS & Science Snapshots (2023-03-25)

In this issue: New proof-of-concept article on RNA Therapeutics and Base/Prime Editing for SDS; And, a review of diagnostic testing for Exocrine Pancreatic Insufficiency (EPI)

Welcome to our timely updates on all things SDS, Science, and Advocacy. We bring you a digest of recent scientific publications, conferences, and other newsworthy content - all relevant to SDS - with links to more details and learning opportunities. Are you interested in anything specific? Did we miss something? Let us know. Email or message us on Facebook! This is all for you!

New proof-of-concept article on RNA Therapeutics and Base/Prime Editing for SDS

Last month, Dr. Balestra and colleagues in Italy published the first of its kind article summarizing the results of several different SDS specific therapeutic strategies. This work was in part supported by the Italian SDS patient advocacy group, AISS. The first half of the article focuses on approaches targeting RNA in SDS cells, while the second half covers DNA targeting approaches.

The American Society of Gene & Cell Therapy has some fantastic resources to help patients understand what cell and gene therapy is. Check out their recourses (videos, graphics, etc) on the Gene Therapy 101 page. We also have many educational resources on our Understanding SDS Science page.

We reached out to Dr. Balestra to bring you an exclusive summary of their work.

Here is the summary of the work they shared:

Counteracting the Common Shwachman-Diamond Syndrome-Causing SBDS c.258+2T>C Mutation by RNA Therapeutics and Base/Prime Editing

Shwachman–Diamond syndrome (SDS) is a rare recessive autosomal disease and one of the most common inherited bone marrow failure syndromes. SDS is caused by mutations in the Shwachman-Bodian-Diamond Syndrome (SBDS) gene, that encodes the homonymous protein SBDS.

Among the over 20 different causative mutations, the c.258+2T>C variant is one of the most common and it originates from gene conversion events with the pseudogene copy (SBDSP), which shares 97% of nucleotide homology with SBDS. This splicing mutation impairs messenger RNA processing, thus leading to synthesis of a dysfunctional shorter SDS protein isoform.

Only supportive treatments are available for SDS patients, with hematopoietic cell transplantation required when bone marrow failure occurs. Therefore, novel therapeutic strategies are highly desirable.

Here, we investigated the molecular mechanisms underlying aberrant SBDS splicing and explored different correction approaches at the DNA and RNA levels, such as engineered U1snRNA, trans-splicing molecules and Base/Prime Editors (BE and PE, respectively).

A little summary on splicing mechanism

Eukaryotic genes consist of coding sequences (“exons”) periodically interrupted by non-coding sequences (“introns”). After transcription of the gene into messenger RNA (mRNA), the pre-mRNA is processed by a huge macromolecular complex (called spliceosome) to remove the introns and thus originate the mature mRNA. Exons and introns are defined by specific nucleotide sequences that dictate where the splicing take place. The 5’ end of the intron (5’ splice site; 5’ss) is characterized by a highly conserved GT sequence and in the earlier splicing step it is recognized by U1snRNP through base pair complementarity with its U1snRNA component.

Since the SBDS c.258+2T>C mutation occurs within the highly conserved 5’ss GT dinucleotide of SBDS exon 2, it is commonly considered a null mutation, with no production of correct SBDS transcripts and thus proteins. However, genetic and animal studies indicate that the complete absence of SBDS is virtually incompatible with life. Therefore, we initially investigated in cells derived from a SDS patient the presence of trace levels of correctly SBDS spliced RNA. Studies at RNA level enabled us to demonstrate the presence of trace levels (~2%) of correctly spliced transcripts, a finding explaining the survival of homozygous patients for this mutation.

Development of RNA therapeutics for SBDS c.258+2T>C mutation

In the attempt to increase total amount of SBDS correctly spliced transcripts, we explored two RNA therapeutics acting on pre-mRNA.

The approach based on engineered U1snRNAs is aimed at restoring proper SBDS exon 2 5’ss recognition and thus correct splicing. The screening of a panel of engineered U1snRNAs identified one U1snRNA variant able to partially restore the usage of the mutated 5’ss, with correctly spliced transcripts increasing from barely detectable to 2,5% of the total transcripts.

On the other hand, since most of the causative SDS mutations are located within the SBDS exon 2 or in the downstream exons, we envisioned to develop a therapeutic strategy aimed at replacing, at RNA level, the coding sequence spanning SBDS exon 2 to exon 4. To this aim, we exploited the trans-splicing process, where the splicing occurs between two different RNA molecules. To trigger this process, we developed 3’ pre-trans splicing molecule (PTM). Worth noting that, due to the high homology between SBDS and SBDSP pseudogene, both transcripts can be targeted with this approach at the same time, thus increasing the possible amount of correctly spliced transcripts. We demonstrated in vitro that our PTMs can trigger the trans-splicing of our therapeutic cassette with either SBDS or SBDSP, but quantification of trans-spliced transcripts by specific qPCR was unfeasible due to the cellular model exploited.

Overall, these data provide the first proof of principle of the U1snRNA/PTM-based correction of SBDS c.258+2T>C mutation and encourage further research aimed at optimizing these approaches.

Development of Base/Prime Editing for SBDS c.258+2T>C mutation

To permanently revert the SBDS c.258+2T>C mutation at DNA level with a single intervention, and thus develop a “hit and run” correction approach, we exploited Base and Prime Editors. These strategies are based on state-of-the-art variants of the famous CRISP/Cas9 protein, largely exploited to make precise genetic manipulations. We tested a panel of BE and PE editors and demonstrated that all of them corrected the mutation, as witnessed by the appearance of correctly spliced transcripts. In particular, one of these editors led to 5% of correct. Overall, this represents the first proof of-principle of the Base/Prime Editors-mediated correction of the c.258+2T>C mutation that would be permanent, transmitted to the daughter’s cells in proliferating tissues such as the bone marrow, and would maintain the physiological SBDS gene regulation. These data encourage further researches aimed at optimizing this approach by exploring viral and non-viral delivery as well as novel generations of DNA editors, combined with the careful evaluation of target specificity. This could eventually lead to an innovative “SDS personalized therapy” based on the autologous transplantation of edited hematopoietic stem cells.

Peretto L, Tonetto E, Maestri I, Bezzerri V, Valli R, Cipolli M, Pinotti M, Balestra D.

Int J Mol Sci. 2023 Feb 16;24(4):4024.

doi: 10.3390/ijms24044024.

PMID: 36835434

New review of diagnostic testing for Exocrine Pancreatic Insufficiency (EPI)

As you all know, Shwachman Diamond syndrome is a rare, autosomal recessive, inherited disorder in ribosomal biogenesis that results in bone marrow failure and Exocrine pancreatic Insufficiency (EPI). Pancreatic issues are thought to be due to acinar cell hypoplasia, that is, damage or displacement of the acinar cells in the pancreas. The pancreatic acinar cell is a highly specialized structure developed for synthesis, storage, and secretion of digestive enzymes. More here.


EPI in SDS patients is typically seen within the first 6 to 12 months of life, with variable severity. In about half of patients, the symptoms improve by age 5, but may return later in life. Patients may also have skeletal defects, such as short stature and poor growth, and frequent infections due to bone marrow failure. SDS is the second most common cause of

EPI in children after Cystic Fibrosis (CF).

This new review article offers an overview of the various testing methods for EPI, with a discussion of the advantages, drawbacks, and recommendations.

Yuzyuk TN, Nelson HA, Johnson LM. Crit Rev Clin Lab Sci. 2023 Mar 6:1-16.

doi: 10.1080/10408363.2023.2179968.

PMID: 36876586

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