Adeno-associated virus (AAV) vectors are viable tools for delivering therapeutic drugs to treat various diseases. Nevertheless, during their clinical development, it is essential to address impurities present in even highly purified AAV preparations. Impurities in the AAV packaging production include DNA, proteins, and empty capsids. Most of the DNA impurities, consist of viral plasmids and host cell genomes, can be digested by subsequent purification steps. But non-GOI DNA components encapsulated within the AAV cannot be efficiently eliminated. These ITR guided mis-packaging contaminants are commonly referred to as cross-packaging. Consequently, adjacent plasmid backbone sequence can be driven for transcription and potential expression of unknown toxic proteins by ITR, which may both incite immune responses and bring safety risk for AAV products. The polyA signal is a DNA sequence that exerts a transcriptional termination effect. To mitigate the ITR-driven transcripts of cross-packaged AAV, we introduced polyA signal sequences (PolyA SS) proximate to ITR and developed a novel viral plasmid for AAV production. In addition, 3x overlapped stop codons was incorporated prior to the polyA SS to restrict possible translation from the unexpected transcripts, and these modifications are termed translation and transcription termination elements (TTTE). We evaluated the effects of TTTE on the transcription of the plasmid backbone in vitro. HEK293T were transduced with viral plasmids with the additional TTTE at upstream of the 5’ ITR or downstream of the 3’ ITR or both. Compared to parental plasmid, the insertion of TTTE at upstream of the 5’ ITR or downstream of the 3’ ITR reduced the backbone transcription products by 38% or 18%, and by about 87% when both insertions were applied. To confirm the TTTE effects in vivo, AAV were packaged through triple transfection system and administered into the tibialis anterior muscle of B6 mice. The modified and unmodified products were injected in the left and right legs respectively. Two weeks post injection, muscles were collected and analyzed for backbone expression. In vivo experiments demonstrated that TTTE modifications significantly reduced backbone transcription by more than 99%. These findings indicated that the introduction of TTTE into the upstream of 5’ITR and 3’ITR of AAV packaging plasmid can effectively inhibit the ITR-driven backbone transcription, which facilitates to increase the safety of AAV products.