Development of a Tethered mRNA Amplifier to increase protein expression

Herein, we present a novel method to specifically increase a messenger RNA's (mRNA) expression at the post‐transcriptional level. This is accomplished using what we term a “Tethered mRNA Amplifier.” The Tethered mRNA Amplifier specifically binds an mRNA's 3′ untranslated region and enhances its stability/translation, often doubling protein output. We test this approach on several transcripts associated with haploinsufficiency disorders and increase their steady‐state expression in cell culture. We suggest this approach may be a tenable therapeutic modality with precise activity and broad‐spectrum application.

In these cases, a mutation-agnostic approach that enhances gene expression is desirable.
In the treatment of haploinsufficiencies, directly targeting endogenous transcripts may offer a novel therapeutic window. Though changes in gene expression are commonly considered to reflect programmed transcriptional variability, it is a lesser-appreciated fact that extensive regulation of mRNA expression also occurs in the cytosol. [9] Indeed, mRNA stabilization and translation kinetics are key features defining post-transcriptional regulation. The cell achieves post-transcriptional regulation through sequence and/or structural elements that recruit specific positive or negative acting factors to mRNAs. All transcripts are degraded and translated at unique rates, and these rates can be controlled, dramatically impacting per transcript protein output. [10] Herein, we describe the development of a novel means to enhance the expression of specific mRNAs -we term this a "Tethered mRNA Amplifier." We document that this technology can be used to control both reporter and endogenous transcripts in human cells. Importantly, we show the broad applicability of the technology, stimulating the translation of transcripts associated with haploinsufficiency disorders.
Lastly, we optimize our mRNA Amplifier such that it can be engineered into current AAV gene therapy strategies. We propose the Tethered mRNA Amplifier approach might provide an efficacious therapeutic modality in the treatment of human haploinsufficiencies.

RESULTS AND DISCUSSION
As an entrée toward a disease-modifying technology that could be used to treat haploinsufficiency disorders, we developed a method to specifically enhance an mRNA's expression. We increase mRNA expression by tethering a known translational stimulator, PABPC1, to the 3′UTR of a target mRNA. [11,12] Tethering is achieved by fusing PABPC1 to the RNA binding protein dCas13b and co-expressing a guide RNA (gRNA). The gRNA is critical in that it has anti-sense homology to specific mRNAs and a short hairpin required for dCas13b binding ( Figure 1A). [13] We demonstrate that this gRNA-targeted tethering enhances both reporter and endogenous mRNAs in a gRNAdependent manner. First, using HEK293 cells we co-transfect our Tethered mRNA Amplifier alongside a luciferase reporter construct.
An approximate 1.5-to 2-fold increase in reporter protein amount is seen when gRNAs directed against the 3′UTR of the luciferase reporter are present. No stimulation occurs when the dCas13b-PABPC1 fusion is expressed alone ( Figure 1B; Ctrl). Continuing in HEK293 cells, we stimulate the translation of an endogenous mRNA, MeCP2. An approximate 1.5-fold stimulation of translation is seen using two distinct gRNAs directed against the endogenous MeCP2 transcript's 3′UTR ( Figure 1C). The effect of the mRNA Amplifier on MeCP2 expression is dependent on PABPC1 and not seen when just Cas13b is bound ( Figure 1D). A mild increase of 15% in MeCP2 transcript steady-state levels is observed when in the presence of the mRNA Amplifier ( Figure 1E). These data suggest that the stimulatory role of the Tethered mRNA Amplifier is through both mRNA stability and mRNA translation -known roles for PABPC1 in regulating mRNA metabolism. [12] We further show that the Tethered mRNA Amplifier enhances mRNA expression in multiple cell types; a stimulatory effect on MeCP2 protein expression is seen in SH-SY5Y (a neuronal cell line) and HepG2 (a liver cell line; Figure 1F). Finally, the effect of the Tethered mRNA amplifier appears to be tunable (at least for MeCP2; this remains to be tested for other transcripts) by moving the gRNA to distinct positions within the 3′UTR. In the case of MeCP2, we observed the strongest stimulatory effect as the gRNA is moved closer to the 3′ end of the transcript ( Figure 1G). This most likely reflects the nature of PABPC1, naturally bound at the transcript's 3′ end.  (Figure 2A-D). The loss of function of one allele for each of these genes is associated with autism spectrum disorders. In all cases, the stimulatory effect seen was between 1.2and 2.0-fold for protein expression with an approximately 15%-20% increase in mRNA levels. These data demonstrate that the Tethered mRNA Amplifier is a promising gene therapy candidate for haploinsufficiency and is portable across multiple transcripts of clinical relevance.
Finally, gene therapy vectors such as AAV have payload size limitations of approximately 4.5 kb. [14] To minimize the Tethered mRNA Amplifier (5.2 kb), we made specific truncations of PABPC1 and tested their efficacy on MeCP2 expression. PABPC1 contains four RNArecognition motifs (RRM1-4) at its N-terminus followed by a linker and a Mademoiselle (MLLE) domain at the C-terminus [12] ( Figure 3A). The RRM domains bind to poly(A) tails while the MLLE domain is known to regulate its stimulatory role in translation. [12] Since we are artificially and specifically tethering PABPC1 to mRNAs independent of PABPC1 poly(A)-binding capacity, we reasoned the RRMs would be dispensable for the Tethered mRNA Amplifier's function. We fused, therefore, just the MLLE domain to dCas13b (3.2 kb). As a first test, we analyzed the putative folding pattern of this new fusion with an in silico approach using AlphaFold v2.0. [15] As seen in Figure  Here, we have demonstrated a first-of-its-kind approach to enhancing the mRNA transcript expression in human cells. We call this technology a Tethered mRNA Amplifier. We demonstrate that the Tethered mRNA Amplifier can be used in multiple cell types and can enhance mRNA expression of genes associated with haploinsufficiencies, such as SYNGAP1, [16] SHANK3, [17] CHD2, [18] and PTEN. [19] The Tethered mRNA Amplifier is tunable by the movement of the tethered moiety to distinct regions within the 3′UTR, imparting informed gene-specific stimulatory effect control. Lastly, we have minimized the mRNA Amplifier to a size that is suitable for gene therapy vehicles such as AAV; although it is feasible to consider delivery of the Tethered mRNA Amplifier through other means such as a lipid nanoparticle.
Further development is needed to demonstrate the utility of a Tethered mRNA Amplifier in the treatment of haploinsufficiencies.
Nonetheless, the Tethered mRNA Amplifier has several major advantages over current strategies. First and foremost, the Tethered mRNA Amplifier would be mutation agnostic, amplifying the expression of the normal allele. Mutations associated with haploinsufficiency result in a loss of function for the diseased allele, [4,5] and thus amplification of this protein product would likely be inconsequential; this will have to be empirically determined. Second, the Tethered mRNA Amplifier is potentially disease-modifying, correcting the haploinsufficiency by enhancing protein production. Third, the Tethered mRNA Amplifier is broadly applicable; we show that we can specifically enhance the expression of four mRNAs associated with haploinsufficiency and CNS disease. Since PABPC1 regulates most human mRNAs, [12] we anticipate that the Tethered mRNA Amplifier could be used to enhance to any one of the nearly 300+ known haploinsufficiency disorders. [6,7] The next steps will be to test the Tethered mRNA Amplifier in a haploinsufficiency model system and determine the efficacy of this approach.  Table 3). Briefly, pJC1206

dCas13b in vitro optimization
was site mutated at nucleotide 5606 to make a unique BamHI site; this construct was hereafter referred to as pJC1210. The ADAR2DD sequence was then removed from using BamHI + NotI and replaced with PCR amplified PABPC1. The generated construct was pJC1211, which included the full human PABPC1 sequence (  Multiple sgRNAs targeting the 3′UTR of the genes of interest were designed using the "nygenome" online tool for the prediction dCas13b guide (Cas13design [nygenome.org]) [ 20 ] (Table 2) Table 3). The data were analyzed using the CT value compared to a no sgRNA transfection and normalized to ACTB as a housekeeping gene.

AlphaFold method
Structural predictions of fusion proteins and native Cas13b and PABP were generated using AlphaFold v2.0 as pulled from the GitHub repository https://github.com/deepmind/alphafold from commit "1d43aaff941c84dc56311076b58795797e49107b" (ref15). Both native and customized fusion FASTAs were processed according to the AlphaFold documentation using the provided Docker script with the following parameters: "-max_template_date = 2020-05-14 -preset = reduced_dbs." Relaxed predicted structures with the highest pLDDT scores were used for interpretation of the corresponding input FASTAs.

Quantifications and statistical analysis
All data, shown in figures as bar charts, were quantified as mean ± standard error. Results were considered significant at p < 0.05 as noted

CONFLICT OF INTEREST
Jeff Coller is a founder and holds equity in Tevard Biosciences, also holds a patent for the Tethered mRNA Amplifier technology which is licensed between Tevard Biosciences and Johns Hopkins University. These arrangements have been reviewed and approved by Johns Hopkins University following its conflict-of-interest policies.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.