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Developing a second-generation clinical candidate AAV vector for gene therapy of familial hypercholesterolemia.

ABSTRACT: Gene therapy for hypercholesterolemia offers the potential to sustainably ameliorate disease for life with a single dose. In this study, we demonstrate the combinatorial effects of codon and vector optimization, which significantly improve the efficacy of an adeno-associated virus (AAV) vector in the low-density lipoprotein receptor (LDLR)-deficient mouse model (Ldlr ^-/-, Apobec1 ^-/- double knockout [DKO]). This study investigated vector efficacy following the combination of intervening sequence 2 (IVS2) of the human beta-globin gene and codon optimization with the previously developed gain-of-function, human LDLR triple-mutant variant (hLDLR-L318D/K809R/C818A) in the treatment of homozygous familial hypercholesterolemia (HoFH). Vector doses as low as 3 × 10¹¹ genome copies (GC)/kg achieved a robust reduction of serum low-density lipoprotein cholesterol (LDL-C) by 98% in male LDLR-deficient mice. Less efficient LDL-C reduction was observed in female mice, which was attributable to lower gene transfer efficiency in liver. We also observed persistent and stable transgene expression for 120 days, with LDL-C levels being undetectable in male DKO mice treated with the second-generation vector. In conclusion, codon and vector optimization enhanced transgene expression and reduced serum LDL-C levels effectively at a lower dose in LDLR-deficient mice. The second-generation clinical candidate vector we have developed has the potential to achieve therapeutic effects in HoFH patients.

SUBMITTER: Wang L

PROVIDER: S-EPMC8237527 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Internal ribosome entry site (IRES) sequences have become a valuable tool in the construction of gene transfer and therapeutic vectors for multi-cistronic gene expression from a single mRNA transcript. The optimal conditions for effective use of this sequence to construct a functional expression vector are not precisely defined but it is generally assumed that the internal ribosome entry site dependent expression of the second gene in such as cassette is less efficient than the cap-dependent expression of the first gene. Mainly tailoring inter-cistronic sequence significantly enhances IRES dependent second gene expression in bicistronic vector further in construction of optimised cassette for gene therapy of familial hypercholesterolemia. We tailored the size of the inter-cistronic spacer sequence at the 5' region of the internal ribosome entry site sequence using sequential deletions and demonstrated that the expression of the 3' gene can be significantly increased to similar levels as the cap-dependent expression of the 5' gene. Maximum expression efficiency of the downstream gene was obtained when the spacer is composed of 18-141 base pairs. In this case a single mRNA transcriptional unit containing both the first and the second Cistron was detected. Whilst constructs with spacer sequences of 216 bp or longer generate a single transcriptional unit containing only the first Cistron. This suggests that long spacers may affect transcription termination. When the spacer is 188 bp, both transcripts were produced simultaneously in most transfected cells, while a fraction of them expressed only the first but not the second gene. Expression analyses of vectors containing optimised cassettes clearly confirm that efficiency of gene transfer and biological activity of the expressed transgenic proteins in the transduced cells can be achieved. Furthermore, Computational analysis was carried out by molecular dynamics (MD) simulation to determine the most emerges as viable containing specific binding site and bridging of 5' and 3' ends involving direct RNA-RNA contacts and RNA-protein interactions. These results provide a mechanistic basis for translation stimulation and RNA resembling for the synergistic stimulation of cap-dependent translation.

Project description:Familial hypercholesterolemia (FH) is the most common genetic disorder associated with premature atherosclerotic cardiovascular (CV) disease (ASCVD). However, it still is severely underdiagnosed. Initiating lipid-lowering therapy (LLT) in FH patients early in life can substantially reduce their ASCVD risk. As a result, identifying FH is of the utmost importance. The increasing availability of genetic testing may be useful in this regard. We aimed to evaluate the genetic profiles, clinical characteristics, and gender differences between the first consecutive patients referred for genetic testing with FH clinical suspicion in our institution (a Spanish cohort). Clinical information was reviewed, and all participants were sequenced for the main known genes related to FH: LDLR, APOB, PCSK9 (heterozygous FH), LDLRAP1 (autosomal recessive FH), and two other genes related to hyperlipidaemia (APOE and LIPA). The genetic yield was 32%. Their highest recorded LDLc levels were 294 ± 65 SD mg. However, most patients (79%) were under > 1 LLT medication, and their last mean LDLc levels were 135 ± 51 SD. LDLR c.2389+4A>G was one of the most frequent pathogenic/likely pathogenic variants and its carriers had significantly worse LDLc highest recorded levels (348 ± 61 SD vs. 282 ± 60 SD mg/dL, p = 0.002). Moreover, we identified an homozygous carrier of the pathogenic variant LDLRAP1 c.207delC (autosomal recessive FH). Both clinical and genetic hypercholesterolemia diagnosis was significantly established earlier in men than in women (25 years old ± 15 SD vs. 35 years old ± 19 SD, p = 0.02; and 43 ± 17 SD vs. 54 ± 19 SD, p = 0.02, respectively). Other important CV risk factors were found in 44% of the cohort. The prevalence of family history of premature ASCVD was high, whereas personal history was exceptional. Our finding reaffirms the importance of early detection of FH to initiate primary prevention strategies from a young age. Genetic testing can be very useful. As it enables familial cascade genetic testing, early prevention strategies can be extended to all available relatives at concealed high CV risk.

Project description:RationaleFamilial hypercholesterolemia is a genetic disorder that arises because of loss-of-function mutations in the low-density lipoprotein receptor (LDLR) and homozygous familial hypercholesterolemia is a candidate for gene therapy using adeno-associated viral vectors. Proprotein convertase subtilisin/kexin type 9 (PCSK9) and inducible degrader of LDLR (IDOL) negatively regulate LDLR protein and could dampen adeno-associated viral vector encoded LDLR expression.ObjectiveWe sought to create vectors expressing gain-of-function human LDLR variants that are resistant to degradation by human PCSK9 (hPCSK9) and IDOL and thereby enhance hepatic LDLR protein abundance and plasma LDL cholesterol reduction.Methods and resultsAmino acid substitutions were introduced into the coding sequence of human LDLR cDNA to reduce interaction with hPCSK9 and human IDOL. A panel of mutant human LDLRs was initially screened in vitro for escape from PCSK9. The variant human LDLR-L318D was further evaluated using a mouse model of homozygous familial hypercholesterolemia lacking endogenous LDLR and apolipoprotein B mRNA editing enzyme catalytic, APOBEC-1 (double knockout). Administration of wild-type human LDLR to double knockout mice, expressing hPCSK9, led to diminished LDLR activity. However, LDLR-L318D was resistant to hPCSK9-mediated degradation and effectively reduced cholesterol levels. Similarly, the LDLR-K809R\C818A construct avoided human IDOL regulation and achieved stable reductions in serum cholesterol. An adeno-associated viral vector serotype 8.LDLR-L318D\K809R\C818A vector that carried all 3 amino acid substitutions conferred partial resistance to both hPCSK9- and human IDOL-mediated degradation.ConclusionsAmino acid substitutions in the human LDLR confer partial resistance to PCSK9 and IDOL regulatory pathways with improved reduction in cholesterol levels and improve on a potential gene therapeutic approach to treat homozygous familial hypercholesterolemia subjects.

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Developing a second-generation clinical candidate AAV vector for gene therapy of familial hypercholesterolemia.

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